Pronuclear formation of freeze-dried canine spermatozoa microinjected into mouse oocytes

Purpose

The aim of the present study was to investigate the fertilizing capacity of fresh, frozen-thawed and freeze-dried canine spermatozoa.

Methods

After canine spermatozoa were injected into mouse oocytes, the rates of oocyte activation, male pronuclear formation and chromosomal aberrations were investigated.

Results

The rates of oocyte activation were comparable (90.6–100%), no matter the sperm type injected. The percentage of male pronuclear formation was higher (P?<?0.001) in the freeze-dried spermatozoa (92.3%) than the fresh (61.5%) and frozen-thawed (69.2%) spermatozoa. However, the chromosomal damage in the oocytes injected with freeze-dried spermatozoa was higher (72.9%: P < 0.001) than with fresh (26.9%) and frozen-thawed (21.4%) spermatozoa.

Conclusions

These data indicate using mouse oocytes that freeze-dried canine spermatozoa may potentially fertilize canine oocytes although chromosomal damage is frequently generated.     

A study of meiotic segregation in a fertile human population following ovarian stimulation with recombinant FSH-LH

Objective

The aim of the study is to investigate the meiotic segregation in fresh eggs from anonymous egg donors and to analyze the baseline levels of aneuploidy in this population.

Results

The study includes the largest series of donor eggs so far studied: 203 eggs from donors aged between 20 and 31 years. No diagnosis was obtained in 10.8 % of cases (22/ 203). The biopsy of the first and second polar bodies was completed in a sequential manner on day 0 and day 1 of embryo development. Chromosomes 13, 16, 18, 21 and 22 are analyzed by means of the FISH test. The diagnosable fertilized eggs gave an aneuploidy rate of 19.1 % (31/162), with 83.8 % (26/31) of the errors produced during meiosis I, 12.9 % (4/31) produced during meiosis II, and 3.2 % (1/31) produced during both meiosis I and II. The premature division of sister chromatids is the main source of meiotic error during Meiosis I, resulting in the creation of oocyte aneuploidy.

Conclusions

FISH analysis of the first and second polar body in donor oocytes gave an aneuploidy rate of 19.1 %. This study shows the majority of errors occur during Meiosis I.     

Predictive value of spindle retardance in embryo implantation rate

Purpose

We evaluated the relationship between meiotic spindle characteristics and in vitro fertilization cycle outcome.

Methods

Five hundred sixty-nine oocytes from 86 in vitro fertilization cycles were analyzed for fertilization and subsequent implantation rates. Oocytes were assessed for maturation status. The oocytes and embryos were cultured in sequential and nonsequential media (G Series, Vitrolife, Sweden) and incubated in 6% CO₂, 5% O₂ at 37 °C.Two hours following oocyte decumulation (38–39 h post-hCG/GnRH administration) and prior to microinjection, the structure of the meiotic spindle was assessed using the Oosight Imaging System (CRI, UK).

Results

Four hundred fifty-six oocytes (80.5%) had a visible meiotic spindle, 82 (14.7%) had no meiotic spindle, and 31 (5.5%) were in telophase I. Oocytes exhibiting a meiotic spindle had a significantly higher fertilization rate and a lower rate of abnormal fertilization. Implantation data were obtained for 195 of the embryos transferred. The implantation rate for embryos derived from oocytes with a meiotic spindle was 32.9%, while in embryos originating from oocytes without a meiotic spindle and oocytes in telophase, this value dropped significantly (8.8 and 0%, respectively). To determine the correlation between retardance values and implantation rate for each oocyte, we established four groups, finding a range of retardance values with significantly higher implantation rates (27.5, 21, 29.3, and 53.8%, respectively).

Conclusion

Meiotic spindle imaging may be a valuable tool for prediction of oocyte quality, and retardance values of meiotic spindles, together with classical morphological classification, can be useful to select embryos with a higher implantation potential.

     

The effect of vitrification on maturation and viability capacities of immature human oocytes

Background

15 % of oocytes collected from Assisted Reproductive Technology (ART) cycles are immature. These oocytes may be matured following in vitro maturation (IVM) program. It is possible to cryopreserve the immature oocytes for further use in ART after application of IVM.

Objective

The aim was to determine the maturation rate and viability of human oocytes that were matured in vitro after vitrification program.

Materials and methods

63 women (19–43 years old) who underwent controlled ovarian stimulation for ART were included in this study. 53 immature oocytes were used for fresh group (fIVM) and 50 immature oocytes for vitrification group (vIVM). The maturation medium was Ham’s F₁₀ supplemented with 0.75 IU FSH, 0.75 IU LH and 40 % human follicular fluid (HFF). After 36 h, maturation and morphology of all oocytes were assessed. Also, the oocyte viability was assessed using PI/Hoechst immunostaining technique.

Results

The maturation rates were reduced in vIVM group (56.0 %) in comparison to fIVM group (88.7 %; P < 0.001). Oocyte viability rate were also reduced in vIVM group (56.0 %) in comparison to fIVM (86.8 %, P < 0.007).

Conclusions

Cryopreservation via vitrification reduced both the maturation capacity and viability of human oocytes in IVM technology. It is, therefore, recommended to apply IVM on fresh immature oocytes, instead.     

Embryo developmental capability and pregnancy outcome are related to the mitochondrial DNA copy number and ooplasmic volume

Purpose

To investigate the correlation between the ooplasmic volume and the number of mitochondrial DNA (mtDNA) copies in embryos and how they may affect fecundity.

Method

Using real-time PCR, mtDNA quantification was analyzed in unfertilized oocytes and uncleaved embryos. The size of the ovum was also assessed by calculating the ooplasmic volume at the time of granulosa cell removal for IVF or ICSI. Quantification analysis of the mtDNA in blastomeres was performed by real-time PCR at the 7–8 cell stage of the cleaved embryos at 72 h after oocyte retrieval. We calculated the cytoplasmic volume of the blastomeres.

Result

Our studies showed a significantly lower mtDNA copy number in unfertilized oocytes and uncleaved embryos in women who were older than 40 years of age (p?<?0.05). The larger ooplasmic volume was also associated with earlier and more rapid cleavage (p?<?0.05). The ooplasmic volume was also significantly larger in the group achieving pregnancy. We found a significant positive correlation between blastomere volume and the number of mtDNA copies (r?=?0.76, p?<?0.01, from Pearson product–moment correlation coefficient).

Conclusions

We have shown that blastomere volume is directly proportional to the number of mtDNA copies. Therefore, larger cytoplasmic volume, with earlier cleavage speed, implies more mtDNA copies. Evaluation of mtDNA quantification and the measurement of ooplasmic and blastomere volume may be useful for selection of high quality embryo and pregnancy outcome.     

Do quantitative birefringence characteristics of meiotic spindle and zona pellucida have an impact on implantation in single embryo transfer cycles?

Objective

The aim of this study was to determine whether quantitative PolScope characteristics of meiotic spindle and zona pellucida could be used as a non-invasive marker to predict implantation success in elective single embryo transfer cycles.

Methods

Quantitative birefringence parameters; including mean retardance, area, length and polar body deviation angle of meiotic spindle and mean retardance and width of inner zona pellucida belonging to 53 transfer oocytes from elective single embryo transfer cycles were retrospectively analyzed. The relevant PolScope features were compared between 20 conception and 33 non-conception cycles.

Results

Meiotic spindle mean retardance, area, length and inner zona pellucida mean retardance and width did not reveal a statistically significant difference between transfer oocytes from conception and non-conception cycles. Deviation angle of the polar bodies was also comparable between the groups. Spindle and inner zona PolScope characteristics of transfer oocytes were not correlated with the maternal age.

Conclusion

Quantitative PolScope features of meiotic spindle and inner zona pellucida can not be used as a non-invasive marker to predict assisted reproductive technology success in elective single embryo transfer cycles.     

Expression of G protein estrogen receptor (GPER) on membrane of mouse oocytes during maturation

Purpose

To determine expression of G-protein estrogen receptor (GPER) in mouse oocyte membrane during maturation.

Methods

The expression of GPER from different maturation stages of oocytes, in vivo and in vitro matured oocytes as well as aging oocytes was examined by immune-fluorescence GPR30 antibody and the images were analyzed by laser scanning confocal microscope. Further confirmation was performed by Western blots for cell fractionation.

Results

Significant fluorescent signal was observed on the surface of mouse oocytes. The image expression was lower in germinal vesicle (GV) stage than mature metaphase-II (M-II) stage oocytes. There was high expression in in-vivo matured oocytes compared to in vitro matured oocytes. The highest expression was observed in aging oocytes compared with other oocytes.

Conclusions

The changes of expression of GPER on mouse oocytes plasma membrane confirm oocyte membrane maturation, suggesting that those changes of GPER may be related to the functional role of oocyte maturation.     

A novel method for detection of chromosomal integrity in cryopreserved livestock spermatozoa using artificially fused mouse oocytes

Purpose

The aim of the present study was to investigate the effect of mouse oocyte volume on the efficiency of chromosomal analysis in livestock spermatozoa.

Methods

Oocytes were injected with bull, ram, boar and dog sperm heads, and then fused with enucleated mouse oocytes.

Results

The increment of oocyte volume increased the rates of morphologically normal oocytes after sperm injection, which induced much higher rates of overall chromosome detection in bull, ram and dog spermatozoa. The recipient oocyte volume did not affect the chromosomal integrity. Furthermore, in bull, the chromosomal integrity detected by fused mouse oocytes was similar to that derived from a homologous system. On the other hand, chromosomal plates of boar spermatozoa could not be detected despite the use of fused oocytes.

Conclusion

These data indicate that fused mouse oocytes improved the efficiency of chromosome detection in bull, ram and dog spermatozoa.     

The presence of 1 mM glycine in vitrification solutions protects oocyte mitochondrial homeostasis and improves blastocyst development

Purpose

Embryos generated from oocytes which have been vitrified have lower blastocyst development rates than embryos generated from fresh oocytes. This is indicative of a level of irreversible damage to the oocyte possibly due to exposure to high cryoprotectant levels and osmotic stress. This study aimed to assess the effects of vitrification on the mitochondria of mature mouse oocytes while also examining the ability of the osmolyte glycine, to maintain cell function after vitrification.

Methods

Oocytes were cryopreserved via vitrification with or without 1 mM Glycine and compared to fresh oocyte controls. Oocytes were assessed for mitochondrial distribution and membrane potential as well as their ability to fertilise. Blastocyst development and gene expression was also examined.

Results

Vitrification altered mitochondrial distribution and membrane potential, which did not recover after 2 h of culture. Addition of 1 mM glycine to the vitrification media prevented these perturbations. Furthermore, blastocyst development from oocytes that were vitrified with glycine was significantly higher compared to those vitrified without glycine (83.9 % vs. 76.5 % respectively; p?<?0.05) and blastocysts derived from oocytes that were vitrified without glycine had significantly decreased levels of IGF2 and Glut3 compared to control blastocysts however those derived from oocytes vitrified with glycine had comparable levels of these genes compared to fresh controls.

Conclusion

Addition of 1 mM glycine to the vitrification solutions improved the ability of the oocyte to maintain its mitochondrial physiology and subsequent development and therefore could be considered for routine inclusion in cryopreservation solutions.     

The effect of minimal concentration of ethylene glycol (EG) combined with polyvinylpyrrolidone (PVP) on mouse oocyte survival and subsequent embryonic development following vitrification

Purpose

Vitrification techniques employ a relatively high concentration of cryoprotectant in vitrification solutions. Exposure of oocytes to high concentrations of cryoprotectant is known to damage the oocytes via both cytotoxic and osmotic effects. Therefore, the key to successful vitrification of oocytes is to strike a balance between the usage of minimal concentration of cryoprotectant without compromising their cryoprotective actions.

Methods

The minimal concentration of ethylene glycol (EG) on mouse oocyte survival and subsequent embryonic development was evaluated following vitrification-warming and parthenogenetic activation. Polyvinylpyrrolidone (PVP) combined with EG on mouse oocyte survival and subsequent embryonic development as well as morphology of the spindle and chromosome alignment were also evaluated. Vitrification system was adapted with JY Straw and the cooling rate was approximately 442–500 °C/min. In contrast, the warming rate was approximately 2,210–2,652 °C/min.

Results

Survival rate of oocytes increased significantly when 15 % EG was combined with 2 % PVP in vitrification solution (VS). The effect of combination of EG and PVP was not significant when the concentration of EG was 20 % and higher. Although there were no significant differences in embryonic development, the percentage of abnormal spindle and chromosome alignment was significantly higher in the oocytes without 2 % PVP in VS.

Conclusions

Our data provide a proof of principle for oocyte vitrification that may not require a high concentration of cryoprotectant. There are synergic effects of EG combined with PVP for oocyte vitrification, which may provide important information to the field in developing less cytotoxic VS.     

The effects of different concentrations of sodium selenite on the in vitro maturation of preantral follicles in serum-free and serum supplemented media

Purpose

This study was to investigate the effect of sodium selenite (SS) on in vitro maturation of mouse preantral follicles.

Methods

The isolated preantral follicles were cultured in TCM 199 medium supplemented with different concentrations (0, 5, 10, 15 ng/ml) of SS and 3 mg/ml bovine serum albumin (BSA) or 5% Fetal Bovine Serum (FBS). The ovulation was induced by addition of 1.5 IU/ml human chorionic gonadotropin. The size and development of follicles and oocytes were assessed by calibrated eyepiece.

Results

The survival rates of follicles in FBS supplemented groups containing 5 and 10 ng/ml SS (88.23%, 90.83%) were higher than other groups (P?P?P?Conclusion The sodium selenite and FBS improve the in vitro growth and maturation of mouse preantral follicles.     

Effect of long-term caffeine administration to mice on in vitro fertilization and embryo development using oocytes

Purpose

To evaluate the effect of long-term caffeine administration to mice on in vitro fertilization (IVF) of oocytes.

Methods

Mice were injected with different dosages (0, 0.1, and 1.0 mg/mouse/converted day) of caffeine for one month. Subsequently, the fertilization rate and embryo development to blastocyst stage were evaluated in IVF using oocytes from the mice.

Results

The retrieved average oocyte rate was significantly lower (27.4) in mice injected with 1.0 mg caffeine than in the control group (36.5; P < 0.05); the fertilization rate was significantly different between the 0 mg (317/401; 79.1 %) and 1.0 mg group (199/301; 66.1 %) (P < 0.05). At 96 h after insemination, the blastocyst formation rate was significantly decreased in the 1.0 mg group (94/199; 47.2 %) compared with the control (0 mg) group (237/317; 74.8 %) and 0.1 mg group (226/323; 70 %) (P < 0.05). When 1.0 mg caffeine was administered for two weeks, embryo development was significantly impacted.

Conclusions

Our findings suggest that caffeine administration negatively impacts oocytogenesis and embryonic development after IVF.     

Conventional IVF versus ICSI in sibling oocytes from couples with endometriosis and normozoospermic semen

Purpose

This study compares the fertilization rate and embryonic development of oocytes randomly inseminated by conventional IVF or ICSI in patients with endometriosis and normozoospermic semen during IVF cycles.

Methods

Sibling oocytes were randomized to be inseminated either by ICSI or IVF. Rates of fertilization, cleavage, blastulation and embryonic morphology were assessed.

Results

A total of 786 sibling cumulus-oocyte complexes (COC) were randomized between insemination by conventional IVF (387 COC) or ICSI (399 COC). A significantly higher fertilization rate was found in the ICSI group (ICSI versus IVF, 73.3±23 % versus 54.7±31.9 % respectively; P=0.003), yielding a higher mean number of day 2 embryos (5.2±3.4 versus 3.6±2.9 respectively; P=0.002). Triploid fertilization rate (3PN/COC) was significantly higher in the IVF group compared to the ICSI group (3.9±8.7 % versus 0.9±3.1 % respectively; P=0.02). The morphology score and rate of development of day 2 and 3 embryos were not different between the two groups. Comparison of embryo transfer cycles in which either IVF or ICSI only embryos were transferred did not reveal any statistically significant differences in pregnancy or implantation rates.

Conclusion

ICSI appears to be a better treatment option than conventional IVF in endometriosis-associated infertility, since it offers the advantages of higher fertilization rate and mean number of embryos and lower rate of total fertilization failure and triploid fertilization.     

Repeated ovarian stimulation does not affect the expression level of proteins involved in cell cycle control in mouse ovaries and fallopian tubes

Purpose

To understand if repeated cycles (2–4 rounds) of gonadotropin stimulation could affect intracellular localization/content of proteins controlling cell cycle progression in mouse fallopian tubes (FT) and ovaries.

Methods

FT and ovaries of estrous mice (control) and of stimulated mice were analyzed to detect Oct-3/4, Sox-2, p53, β-catenin, pAKT and cyclin D1 localization/content. Spindles and chromosome alignment were analyzed in ovulated oocytes.

Results

After round 4, FT and ovaries of control and stimulated groups showed no differences in Oct-3/4, Sox-2 and β-catenin localization nor in Oct-3/4, Sox-2, p53, β-catenin and pAKT contents. Cyclin D1 level increased significantly in FT of treated mice. Oocytes number decreased meanwhile frequency of abnormal meiotic spindles increased with treatments.

Conclusions

Repetitive stimulations affected oocyte spindle morphology but did not induce changes in a set of proteins involved in cell cycle progression, usually altered in ovarian cancer. The significant increase of cyclin D1 in the FT requires further investigation.     

Effect of embryonic fibroblast cell co-culture on development of mouse embryos following exposure to visible light

Purpose

To determine the effects of visible light on development of mouse embryos and the potential of fibroblast cells to overcome deleterious effects of visible light on mouse preimplantation stage embryos.

Methods

Two-cell mouse embryos were randomly allocated to un-exposed group (control) and exposed group receiving 1600 lx visible light for various time lengths. Both exposed and un-exposed embryos were co-cultured with either Mouse Embryonic Fibroblast (MEF) or Human Embryonic Fibroblast (HEF). Developmental rate of embryos at day 3 (morula), 4 (expanded blastocyst) and 5 (hatching or hatched blastocyst) was evaluated.

Results

Exposure of embryos to visible light for 30 min decreased developmental rate significantly (P?<?0.01). Developmental rate of exposed embryos co-cultured with MEF (58%; p?<?0.05 both at day 4 and 5) and HEF (67%; P?<?0.01 both at day 4 and 5) was higher than control.

Conclusions

Visible light adversely affects embryo development in a time-dependent manner. Feeder cells may enhance embryo development particularly when suboptimal conditions are involved.     

The combination of calcium ionophore A23187 and GM-CSF can safely salvage aged human unfertilized oocytes after ICSI

Purpose

Artificial oocyte activation using calcium ionophores and enhancement of embryonic developmental potential by the granulocyte-macrophage colony-stimulating factor (GM-CSF) have already been reported. In this study, we evaluated the synergistic effect of these two methods on aged human unfertilized oocytes after intracytoplasmic sperm injection (ICSI). Then, we cultured the resulting embryos to the blastocyst stage and screened them for chromosomal abnormalities, to assess the safety of this protocol.

Methods

Aged human oocytes deemed unfertilized after ICSI were activated, either by briefly applying the calcium ionophore A23187 alone (group A) or by briefly applying the ionophore and then supplementing the culture medium with recombinant human GM-CSF (rhGM-CSF) (group B). Next, the development was monitored in a time-lapse incubator system, and ploidy was analyzed by array comparative genomic hybridization (aCGH), after whole embryo biopsy and whole genome amplification. Differences between oocytes and resulting embryos in both groups were evaluated statistically.

Results

Oocytes unfertilized after ICSI can be activated with the calcium ionophore A23187 to show two pronuclei and two polar bodies. Addition of rhGM-CSF in the culture medium of A23187-activated oocytes enhances their cleaving and blastulation potential and results in more euploid blastocysts compared to the culture medium alone.

Conclusions

This study shows that activating post-ICSI aged human unfertilized oocytes with a combination of a calcium ionophore and a cytokine can produce good-morphology euploid blastocysts.

     

Does metformin improve <Emphasis Type="Italic">in vitro</Emphasis> maturation and ultrastructure of oocytes retrieved from estradiol valerate polycystic ovary syndrome-induced rats

Background

Metformin decreases polycystic ovary syndrome (PCOS) symptoms, induces ovulation, and may improve developmental competence of in vitro oocyte maturation. This study was designed to define the effects of metformin on the characteristics of in vitro oocyte maturation in estradiol valerate (EV) PCOS-induced rats.

Methods

Forty-five adult female Sprague–Dawley rats were randomly divided into control; sham and PCOS-induced (treated by a single dose of estradiol valerate, 4 mg/rat, IM) groups. The body weight was measured weekly for 12 weeks. At the end of week 12, the serum levels of testosterone, estrogen, progesterone, LH, and FSH and blood glucose of all the rats were measured. About 380 cumulus oocyte complexes (control, 125; sham, 122; PCOS-induced rats, 133) were incubated in Ham’s F10 in the absence and/or presence of metformin (M 5^?10) for 12, 24, 36, and 48 h. The cumulus cells expansion and nuclear and cytoplasmic maturation of the oocytes was evaluated using 1 % aceto-orcein staining, and transmission electron microscopy (TEM).

Results

No significant differences were observed in the body weight of the rats. The serum level of testosterone was reduced, and progesterone and LH were significantly increased in the PCOS-induced rats (p?<?0.05). However, no significant differences were observed in the serum levels of estrogen and FSH among the groups. Blood glucose level was higher in the PCOS-induced rats than control, (p?<?0.01). The expansion of cumulus cells was observed in the metformin-treated oocytes. The oocytes retrieved from PCOS-induced rats show a stage of meiotic division (GVBD, MI, A-T, and MII) in 57.12 % of metformin-untreated and fairly significantly increased to 64.28 % in metformin-treated oocytes, (p?<?0.05), but no differences were observed in the MII stage within groups. The redistribution of some cytoplasmic organelles throughout the ooplasm, particularly the peripheral cortical granules, was defined in the metformin-treated oocytes.

Conclusions

Single dose of EV can creates a reversible PCO adult rat model. Metformin enhances the COCs to initiate meiotic resumption at the first 6 h of IVM. In our study the metformin inability to show all aspects of in vitro oocyte maturation and may be resulted from deficiency of EV to induce PCOS.