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.

     

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.     

Regulation of microRNA biosynthesis and expression in 2102Ep embryonal carcinoma stem cells is mirrored in ovarian serous adenocarcinoma patients

Background

Tumours with high proportions of differentiated cells are considered to be of a lower grade to those containing high proportions of undifferentiated cells. This property may be linked to the differentiation properties of stem cell-like populations within malignancies. We aim to identify molecular mechanism associated with the generation of tumours with differing grades from malignant stem cell populations with different differentiation potentials. In this study we assessed microRNA (miRNA) regulation in two populations of malignant Embryonal Carcinoma (EC) stem cell, which differentiate (NTera2) or remain undifferentiated (2102Ep) during tumourigenesis, and compared this to miRNA regulation in ovarian serous carcinoma (OSC) patient samples.

Methods

miRNA expression was assessed in NTera2 and 2102Ep cells in the undifferentiated and differentiated states and compared to that of OSC samples using miRNA qPCR.

Results

Our analysis reveals a substantial overlap between miRNA regulation in 2102Ep cells and OSC samples in terms of miRNA biosynthesis and expression of mature miRNAs, particularly those of the miR-17/92 family and clustering to chromosomes 14 and 19. In the undifferentiated state 2102Ep cells expressed mature miRNAs at up to 15,000 fold increased levels despite decreased expression of miRNA biosynthesis genes Drosha and Dicer. 2102Ep cells avoid differentiation, which we show is associated with consistent levels of expression of miRNA biosynthesis genes and mature miRNAs while expression of miRNAs clustering to chromosomes 14 and 19 is deemphasised. OSC patient samples displayed decreased expression of miRNA biosynthesis genes, decreased expression of mature miRNAs and prominent clustering to chromosome 14 but not 19. This indicates that miRNA biosynthesis and levels of miRNA expression, particularly from chromosome 14, are tightly regulated both in progenitor cells and in tumour samples.

Conclusion

miRNA biosynthesis and expression of mature miRNAs, particularly the miR-17/92 family and those clustering to chromosomes 14 and 19, are highly regulated in both progenitor cells and tumour samples. Strikingly, 2102Ep cells are not simply malfunctioning but respond to differentiation specifically, a mechanism that is highly relevant to OSC samples. Our identification and future manipulation of these miRNAs may facilitate generation of lower grade malignancies from these high-grade cells.     

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 impact of vitrification on murine germinal vesicle oocyte In vitro maturation and aurora kinase A protein expression

Purpose

Investigate the effect of vitrification on in vitro maturation (IVM) and expression of Aurora kinases A, B, and C in germinal vesicle (GV)-stage oocytes.

Methods

GV-stage oocytes from B6D2F1 female mice 7–11 weeks of age were vitrified after collection, thawed, and matured in vitro for 0, 4, 8, and 12 h (hrs). The rate of germinal vesicle breakdown (GVBD), spindle apparatus assembly, and Aurora kinase mRNA and protein expression during IVM was measured.

Results

Oocyte vitrification was associated with significant delays in both GVBD and normal spindle apparatus assembly at 4 and 8 h of IVM (p < 0.05). There was no difference in mRNA levels between control and vitrified oocytes for any of the Aurora kinases. Aurora A protein levels were reduced in vitrified compared to control oocytes at 0 h (p = 0.008), and there was no difference at 4 and 8 h (p = 0.08 and 0.69, respectively) of IVM.

Conclusions

Vitrified oocytes have delayed GVBD and normal spindle assembly during in vitro maturation. Reduced levels of Aurora A protein immediately post-thaw may be associated with the impaired oocyte maturation manifested by the delayed progression through meiosis I and II, and the atypical timing of the formation of meiotic spindles in vitrified GV-stage oocytes.     

Effect of single-oocyte culture system on in vitro maturation and developmental competence in mice

Purpose

To investigate whether single-culture systems influence the quality of in vitro-matured oocytes, we examined the maturation and developmental competence of oocytes obtained by grouped in vitro maturation (IVM) or single IVM.

Methods

In vitro-matured oocytes were obtained using the culture drop (CD) method for the grouped IVM experiments, and the CD and hanging drop (HD) method for the single IVM experiments. To evaluate oocyte developmental competence, we performed in vitro fertilization and culture, and counted the number of blastocysts. To evaluate the oocyte cytoplasmic maturation, we measured the maturation promoting factor (MPF) expression levels.

Results

Oocytes cultured singly had lower maturity and developmental competence than the grouped IVM oocytes. However, enhanced oocyte fertility and blastocyst quality was achieved by the HD single IVM method. Additionally, the MPF activity level increased in all culture methods, compared to the control; however, it lagged behind nuclear maturation.

Conclusions

These results suggest that the HD method is efficient for single IVM.     

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.     

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.     

Expression and intracytoplasmic distribution of staufen and calreticulin in maturing human oocytes

Purpose

In this study we hypothesized that the mRNA vector Staufen mediates RNA relocalization during meiotic maturation, and by virtue of its interactions with endoplasmic reticulum, provides a possible mechanism by which protein synthesis is regulated.

Methods

We assessed the expression of staufen (STAU) and calreticulin (CALR), the latter adopted as a marker of the endoplasmic reticulum, in human oocytes at different stages of maturation: GV, metaphase MI and MII. Oocytes were subjected to polymerase chain reaction in order to investigate the expression of STAU and CALR. The corresponding protein products were identified by immunofluorescence and confocal laser scanning microscopy.

Results

STAU and CALR were constantly expressed and selectively localized during oocyte maturation. At the GV stage the both proteins displayed a dispersed distribution localization throughout the cytoplasm. Progressing to the MII stage, STAU tended to compartmentalize towards the cortical area of the oocyte clustering in granules of larger sizes. At the MII stage, CALR assumed a pattern reminiscent and possibly coincident with the position of the meiotic spindle.

Conclusions

The changing pattern of STAU distribution during meiotic maturation of human oocytes implicates a novel mechanism for the regulation of protein synthesis based on mRNA localization. Moreover, the unique disposition of CALR at the MII spindle uncovers a physical interaction with endoplasmic reticulum that may mediate cytoskeletal remodelling during oocyte maturation.     

Retrieval and in vitro maturation of human oocytes from ovaries removed during surgery for endometrial carcinoma: a novel strategy for human oocyte research

Purpose

To collect human oocytes from ovaries removed as part of surgical treatment for endometrial carcinoma, and to induce in vitro maturation of such oocytes to obtain material for research on human ovarian aging.

Methods

Design: Prospective clinical study. Setting: University Hospital. Patients: Eight patients aged 35–44 years with a preoperative diagnosis of Stage I endometrial cancer agreed to participate in this project. Interventions: Surgically removed ovaries were punctured; oocytes were collected from follicular fluid and matured in vitro. Immunofluorescent detection of microtubules and DNA labeling were performed after in vitro maturation. Main Outcome Measures: Number of oocytes collected and their in vitro maturation stage.

Results

In total, 87 oocytes were collected, 11 of which had completed metaphase II. Of the oocytes collected, 75 % were from three patients in their 30s, while the remaining 25 % were from five patients in their 40s. Several stages of oocytes were collected and the detection of microtubule arrangement and chromatin in various stages using fluorescence was possible.

Conclusion

Material for research on human ovarian aging can be obtained from ovaries removed during surgery for endometrial cancer.     

Cysteamine supplementation during in vitro maturation (IVM) of rabbit oocyte improves the developmental capacity after intracytoplasmic sperm injection

Purpose

Current approaches to in vitro maturation (IVM) may result in low efficiency and inadequate quality of the oocytes due to insufficient cytoplasmic maturation. Although positive effects of the cysteamine supplementation in IVM medium for oocyte nuclear maturation or male pronuclear formation have been confirmed, it is still controversial whether the cysteamine addition affects embryo development after IVM. We aimed here to confirm the effect of cysteamine addition into IVM medium for subsequent embryo development in vitro.

Methods

We administered the cysteamine to the IVM culture of rabbit immature oocytes at various concentrations and observed the developmental rate, speed to reach blastocyst stage and cell numbers at the blastocyst stage.

Results

Cysteamine supplementation improved developmental rate to blastocyst stage of the IVM oocytes. On the other hand, addition of glutathione (GSH) inhibitor buthionine sulfoximine inhibited GSH accumulation in the oocytes and subsequent embryo development to the blastocyst stage.

Conclusions

Controlling the GSH quantity of IVM oocytes may be an important factor for success of embryo development, and it is quite probable that a cysteamine supplementation can contribute to an increase of GSH content in oocyte.     

Prematuration of bovine oocytes with butyrolactone I reversibly arrests meiosis without increasing meiotic abnormalities after in vitro maturation

Objectives

Asynchrony between nuclear and cytoplasmic maturation, and possibly damage to the oocyte meiotic spindle, limits the application of in vitro maturation (IVM) in assisted reproduction. Several studies have suggested that Prematuration with meiosis blockers may improve oocyte quality after IVM, favoring early embryogenesis. Thus, we investigated the effect of Prematuration with the nuclear maturation inhibitor butyrolactone I (BLI) on the meiotic spindle and chromosomal configuration of bovine oocytes.

Study design

Immature oocytes obtained from cows slaughtered in a slaughterhouse (n = 840) were divided into the following groups: (1) control (n = 325), submitted only to IVM in TCM199 for 24 h; (2) BLI 18 h (n = 208) submitted to meiotic blockage with 100 μM BLI for 24 h (Prematuration) and then induction of IVM in TCM199 for 18 h; and (3) BLI 24 h (n = 307), pre-matured with 100 μM BLI for 24 h followed by 24 h of IVM in TCM199. The oocytes were then fixed, stained by immunofluorescence for morphological visualization of both microtubules and chromatin, and evaluated.

Results

Meiotic arrest occurred in 90.2% of the oocytes cultured with BLI. Maturation rates were similar for all groups (80.3%, 73.6% and 82.7% for the control, BLI 18 h and BLI 24 h groups, respectively). We observed 81.3% normal oocytes in metaphase II in the control group, and 80.0% and 81.2% in the BLI 18 h and BLI 24 h groups, respectively. The incidence of meiotic anomalies did not differ between groups (18.7%, 20.0% and 18.8% for the control, BLI 18 h and BLI 24 h, respectively).

Conclusion

Prematuration with butyrolactone I reversibly arrests meiosis without damaging the meiotic spindle or the chromosome distribution of bovine oocytes after in vitro maturation.     

Fertility preservation for trans men: frozen-thawed in vitro matured oocytes collected at the time of ovarian tissue processing exhibit normal meiotic spindles

Purposes

At the moment of sex reassignment surgery (SRS), the ovarian tissue is sometimes cryopreserved as fertility preservation option for female-to-male trans men, also called trans men. During this preparation, cumulus-oocyte-complexes (COCs) can be found and in vitro matured. It is not known if these oocytes are developmentally competent. In order to use these oocytes for fertility preservation and subsequent fertilization, a normal spindle structure before and after vitrification is necessary.

Methods

A total of 680 COCs were collected from trans men (n = 16) at the time of SRS and after testosterone treatment. The COCs were subjected to in vitro maturation and those that reached the metaphase II stage (MII) were collected and split into two groups; group 1 was immediately fixed for spindle staining and group 2 was first vitrified and warmed followed by spindle staining. Statistical analysis was performed by Fisher’s exact test.

Results

After 48 h in vitro maturation, 38.1% of COCs were at MII stage. Those oocytes were split in two groups: (1) 126 MII oocytes in the noncryopreservation group and (2) 133 MII oocytes underwent cryopreservation through vitrification. The oocyte survival rate, after 2 h warming, was 67.7%. Both the noncryopreserved and the vitrified group showed comparable results concerning normal spindle structure and chromosomes alignment, 85.7% vs. 92.2% (P = 0.27).

Conclusions

Spindle structure analysis and chromosomal alignment after vitrification seem normal in in vitro matured COCs collected during the tissue processing of ovaries in trans men at the time of SRS. The MII oocytes do not seem to be morphologically affected by prolonged testosterone treatment.

     

Human blastocysts exhibit unique microrna profiles in relation to maternal age and chromosome constitution

Purpose

To determine microRNA (miRNA) expression in human blastocysts relative to advanced maternal age and chromosome constitution.

Methods

Cryopreserved human blastocysts were warmed and underwent a trophectoderm biopsy for comprehensive chromosomal screening. Select blastocysts were then lysed, reverse transcribed, and pre-amplified prior to running real-time PCR. Statistical analysis was performed using an internal constant housekeeping miRNA. Significant microRNA’s of interest were then analyzed for their predicted genes and biological pathways. Additional cryopreserved blastocysts were warmed and stained for the SIRT1 protein for validation.

Results

Human blastocysts exhibit unique miRNA expression profiles in relation to maternal age and chromosome constitution. miR-93 was exclusively expressed in blastocysts from women in their forties and further up-regulated with an abnormal chromosome complement. Up-regulated miR-93 resulted in an inverse down-regulation of targets like SIRT1, resulting in reduced oxidative defense.

Conclusions

MiRNAs play an important role in aging as well as chromosome constitution and have downstream effects that regulate proteins which can compromise embryonic development.     

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.     

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.     

Dynamic changes in mitochondrial distribution in human oocytes during meiotic maturation

Purpose

The change of mitochondrial distribution in human oocytes during meiotic maturation was assessed using 223 human oocytes donated from patients undergoing fertility treatment between June 2013 and February 2016.

Methods

Live cell images of fluorescence-labelled mitochondria in human oocytes were analysed to investigate dynamic changes in mitochondrial distribution during meiotic maturation using a confocal microscope combined with an incubator in the presence or absence of colchicine and cytochalasin B, inhibitors for tubulin and actin filament, respectively. Subcellular distribution of mitochondria in human oocytes was also assessed at various stages using a transmission electron microscope (TEM).

Results

Live cell imaging analysis revealed that the mitochondria-occupied cytoplasmic area decreased from 83 to 77 % of the total cytoplasmic area around 6 h before germinal vesicle breakdown (GVBD) and that mitochondria accumulated preferentially close to the perinuclear region. Then, the mitochondria-distributed area rapidly increased to 85 % of total cytoplasm at the time of GVBD. On the other hand, there was no significant change in mitochondrial distribution before and after polar body extrusion. Such changes in mitochondrial localization were affected differently by colchicine and cytochalasin B. Most of mitochondria in the cytoplasm formed cluster-like aggregates before GVBD while they distributed homogeneously after GVBD.

Conclusions

Most mitochondria localized predominantly in the non-cortical region of the cytoplasm of GV stage-oocytes, while the mitochondria-occupied area decreased transiently before GVBD and increased rapidly to occupy the entire area of the cytoplasm at GVBD by some cytoskeleton-dependent mechanism.

     

Cytogenetic analysis of human oocytes parthenogenetically activated by puromycin

Purpose

Treatment of aged human oocytes by puromycin allows a high rate of parthenogenetic activation and development until the first cleavage division. This technique was used for the study of the chromosome complement of oocytes which remained unfertilized after in vitro fertilization. Three hundred four unfertilized oocytes were treated with 10 Μg/ml puromycin for 6–8 hr and further cultured for 12–15 hr.

Results

Activation occurred in 90.5% of the oocytes. Heterozygous diploids with two pronuclei predominated (61%), which is in contrast to the mouse, where the majority of oocytes activated by puromycin are uniform haploids (89%).

Conclusions

Therefore we conclude that puromycin treatment induces retention of the second polar body in human oocytes, unlike in mouse oocytes treated in the same way. Chromosome analysis performed on 182 oocytes suggested a nondisjunction (ND) rate for the second meiotic division of 12.7%. This is a low figure considering the fact that puromycin itself has been reported to induce nondisjunction. For the first meiotic division a ND rate of only 5.6% was found. This rate is lower than the one found in metaphase II arrested oocytes and we believe that this difference is due to the technical differences between the study of meiotic and that of mitotic chromosomes.     

The impact of vitrification on immature oocyte cell cycle and cytoskeletal integrity in a rat model

Purpose

To test the effects of varying vitrification protocols on the cell cycle status and chromosomal integrity in cumulus-enclosed GV stage rat oocytes.

Methods

Vitrified and thawed rat oocytes were labeled with fluorescent markers for chromatin, cell cycle activation, and f-actin and analyzed by conventional and laser scanning confocal microscopy.

Results

In all vitrification groups, significant alterations in cumulus cell connectivity, cell cycle status, and cytoplasmic actin integrity were observed following warming compared to fresh control oocytes. Based on the protein phosphorylation marker MPM-2, it is clear that warmed oocytes rapidly enter M-phase but are unable to maintain chromosome integrity as a result of multiple chromatin fusions. A prominent reduction in f-actin is evident in both the ooplasm and at the cortex of vitrified oocytes. Finally, an irreversible but irregular retraction of TZPs occurs on the majority of oocytes subjected to any of the vitrification protocols.

Conclusions

These findings draw attention to undesirable consequences of immature oocyte vitrification that compromise cell cycle status and chromatin and cytoskeleton integrity that may not be evident until after fertilization.     

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.