Maturation Of Human Oocytes Research Articles

Ultrastructure of human mature oocytes after vitrification

Since the introduction of human assisted reproduction, oocyte cryopreservation has been regarded as an attractive option to capitalize the reproductive potential of surplus oocytes and preserve female fertility. However, for two decades the endeavor to store oocytes has been limited by the not yet optimized methodologies, with the consequence of poor clinical outcome or of uncertain reproducibility. Vitrification has been developed as the promising technology of cryopreservation even if slow freezing remains a suitable choice. Nevertheless, the insufficiency of clinical and correlated multidisciplinary data is still stirring controversy on the impact of this technique on oocyte integrity. Morphological studies may actually provide a great insight in this debate. Phase contrast microscopy and other light microscopy techniques, including cytochemistry, provided substantial morpho-functional data on cryopreserved oocyte, but are unable to unraveling fine structural changes. The ultrastructural damage is one of the most adverse events associated with cryopreservation, as an effect of cryo-protectant toxicity, ice crystal formation and osmotic stress. Surprisingly, transmission electron microsco py has attracted only limited attention in the field of cryopreservation. In this review, the subcellular structure of human mature oocytes following vitrification is discussed at the light of most relevant ultrastructural studies.

Dynamic changes of histone H3 trimethylated at positions K4 and K27 in human oocytes and preimplantation embryos

To investigate the distribution patterns of H3K4me3 and H3K27me3 in human oocytes and preimplantation embryos. Experimental study. University reproductive medical center. Patients undergoing IVF cycles. Oocytes and embryos were collected from patients undergoing IVF cycles. The distribution patterns of H3K4me3 and H3K27me3 in oocytes and embryos were analyzed by indirect immunofluorescent staining and scanning confocal microscopy. H3K4me3 and H3K27me3 signals were detectable at each stage of oocyte and embryonic development. However, only one of the pronuclei showed signal for H3K27me3 in each of the zygotes, whereas H3K4me3 staining was always uniform in all zygotes. The level of H3K4me3 decreased steadily from germinal vesicle to metaphase II stage, obviously increased from zygote stage to four-cell stage, and reached the lowest at eight-cell stage. A sharp increase was then observed at blastocyst stage. The level of H3K27me3 slightly changed from germinal vesicle stage to zygote stage, then decreased steadily and reached the lowest at eight-cell stage, followed by a significant increase at blastocyst stage. The levels of H3K4me3 and H3K27me3 show dynamic changes during human oocyte maturation and preimplantation embryonic development. Asymmetric distribution of H3K27me3 exists in human zygote pronuclei, whereas H3K4me3 is always uniform in all of the pronuclei.

The Safe Use of a PTEN Inhibitor for the Activation of Dormant Mouse Primordial Follicles and Generation of Fertilizable Eggs

BackgroundPrimordial ovarian follicles, which are often present in the ovaries of premature ovarian failure (POF) patients or are cryopreserved from the ovaries of young cancer patients who are undergoing gonadotoxic anticancer therapies, cannot be used to generate mature oocytes for in vitro fertilization (IVF). There has been very little success in triggering growth of primordial follicles to obtain fertilizable oocytes due to the poor understanding of the biology of primordial follicle activation.Methodology/Principal FindingsWe have recently reported that PTEN (phosphatase and tensin homolog deleted on chromosome ten) prevents primordial follicle activation in mice, and deletion of Pten from the oocytes of primordial follicles leads to follicular activation. Consequently, the PTEN inhibitor has been successfully used in vitro to activate primordial follicles in both mouse and human ovaries. These results suggest that PTEN inhibitors could be used in ovarian culture medium to trigger the activation of primordial follicle. To study the safety and efficacy of the use of such inhibitors, we activated primordial follicles from neonatal mouse ovaries by transient treatment with a PTEN inhibitor bpV(HOpic). These ovaries were then transplanted under the kidney capsules of recipient mice to generate mature oocytes. The mature oocytes were fertilized in vitro and progeny mice were obtained after embryo transfer.Results and ConclusionsLong-term monitoring up to the second generation of progeny mice showed that the mice were reproductively active and were free from any overt signs or symptoms of chronic illnesses. Our results indicate that the use of PTEN inhibitors could be a safe and effective way of generating mature human oocytes for use in novel IVF techniques.

Integration of immunodeficiency virus in oocytes via intracytoplasmic injection: possible but extremely unlikely

To determine if human oocytes can be infected with HIV-1 via intracytoplasmic injection and to determine the infection threshold. Twenty-eight donated immature and unfertilized human oocytes from HIV-negative women were injected with 4 × 10(4) HIV-1 virions and 13 oocytes were used as uninjected controls. To determine the infection threshold, 543 cat oocytes were injected with 4 × 10(4), 4 × 10(2), or 40 copies of feline immunodeficiency virus (FIV) and 376 oocytes were used as controls. Academic hospital. PATIENT(S)/ANIMAL(S): Donated immature human oocytes and mature cat oocytes. Injection with HIV-1 or FIV. Viral integration as measured by fluorescent in situ hybridization with HIV-1-specific probes or by nested FIV polymerase chain reaction. We detected viral integration in three of 28 (11%) human oocytes injected with 4 × 10(4) copies of HIV-1. When injected with high dose FIV (4 × 10(4) copies) 16%-49% of cat oocytes showed viral integration. This decreased to 2%-7% and 0.6%-1.8% when an intermediate (4 × 10(2) copies) or low (40 copies) dose was injected, respectively. Human and cat oocytes can be infected with HIV-1 and FIV respectively, when injected with high amounts of virus. The probability of viral integration is extremely low when small amounts of virus particles are injected. Taking into account the small volume injected during intracytoplasmic injection, the chances of viral integration are 0.00002%.

Effects of combined epidermal growth factor, brain-derived neurotrophic factor and insulin-like growth factor-1 on human oocyte maturation and early fertilized and cloned embryo development

Human cloned blastocysts generated from oocytes following in vitro maturation (IVM) are a potential resource for embryonic stem cells (ESC) with homologous immune systems. The purpose of this study was to evaluate the effects of multiple growth factors [epidermal growth factor (EGF), brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1)] on human oocyte maturation, early embryo development, blastocyst formation and ESC line generation. Patients (n= 344) undergoing IVF owing to male factor infertility were enrolled in this study. Metaphase II oocytes were separated into four grades based on their morphology. Spindle assembly from IVM oocytes with or without growth factor treatment was assessed by immunostaining. Piezo-assisted micromanipulation technology was used to produce fertilized (ICSI) and cloned [(somatic cell nuclear transfer (SCNT)] embryos. Embryos received four different growth factor treatments; embryo development rates from pronuclear to blastocyst stage and embryo grading (for quality) at the 8-cell stage were analyzed. The presence of receptors on human cumulus cells and IVM oocytes was assessed by immunofluorescence. The blastocysts generated from fertilized and cloned embryos were used for ESC derivation. The combination of EGF, BDNF and IGF-1 can effectively increase oocyte maturation rate in vitro, and significantly improve the oocyte quality in terms of morphology and normal spindle levels (P< 0.05). Also, the developmental competence of fertilized oocytes to 8-cell and blastocyst stages was improved by the addition of growth factors (P< 0.05). However, there were no significant differences among the four groups in 8-cell grading. Blastocyst formation in cloned embryos cultured with the three growth factors was higher than the control group (23.1 versus 4.3%, P< 0.05). Receptors for the three growth factors were present in cumulus cells and IVM oocytes, and four human ESC lines were derived from fertilized blastocysts but none from cloned blastocysts. This study demonstrated that EGF, BDNF and IGF-1 can improve oocyte maturation rate and quality in vitro, and consequently increase early embryo development and blastocyst formation, which is very beneficial in improving the reprogramming efficiency of SCNT. The present study has identified a valuable culture system for IVM and cloned human embryos, potentially using these embryos to derive human therapeutic ESC.

In-vitro maturation of human oocytes: before or after vitrification?

This study aims to determine if in-vitro maturation (IVM) of human immature oocytes should be performed before or after vitrification. A total of 184 immature oocytes were randomly divided into two different groups: 100 were vitrified at metaphase II (MII) stage 24 h-48 h after IVM (group 1) and 84 were immediately vitrified at germinal vesicle (GV) or metaphase I (MI) stages and in vitro matured after warming (group 2). Survival rate after warming was similar in both groups (86.9% versus 84.5%). However, oocyte maturation rate per collected oocyte was significantly higher for oocytes matured before vitrification (group 1, 46%) than for oocytes vitrified before IVM (group 2, 23.8%) (p < 0.01). Consequently, the number of MII oocytes inseminated per oocyte collected was significantly higher for group 1 (40%) than for group 2 (23.8%) (p < 0.05). IVM procedure is more efficient when it is performed before oocyte vitrification.

Expression and localization of opioid receptors during the maturation of human oocytes

The endogenous opioid system has been characterized in some female reproductive organs, but little is known about the expression of these receptors in human oocytes. This study investigated the presence and differential distribution of the opioid receptors during the maturation of human oocytes. A total of 821 human oocytes from an intracytoplasmic sperm injection (ICSI) programme were studied including 213 at germinal-vesicle (GV) stage and 164 at metaphase-I (MI) stage and 444 failed fertilization metaphase-II (MII) oocytes. Additionally 31 MII oocytes corresponding to cases where ICSI was not attempted and 50 failed fertilization MII oocytes from the IVF programme were included. Western blot analysis revealed the presence of the delta (OPRD1), kappa (OPRK1) and mu (OPRM1) opioid receptors in human oocytes. The OPRK1 and OPRM1 immunostaining patterns changed during the maturation of the oocyte, while the OPRD1 pattern was the same throughout. In particular, OPRD1 were detected in peripheral tissue from the GV to the MII stage. OPRK1 were found peripherally at the GV stage, more internally at MI and homogeneously at MII. Finally, OPRM1 were located peripherally at the GV stage and homogeneously in MI and MII oocytes. Opioids may have a role in oocyte maturation, acting via receptors.The opioid system has been well characterized in the central nervous system, but it is now known that opioids also act in reproductive organs. However, little is known about the presence and function of this system in human oocytes and its role in their maturation. In this study, we investigated the presence and differential distribution of three (delta, kappa and mu) opioid receptors (proteins which bind the opioids) during the maturation of human oocytes. A total of 821 human oocytes (from 253 patients) not suitable for intracytoplasmic sperm injection (ICSI) or which did not develop into an embryo after ICSI were studied. Thus, we have verified the presence of the delta, kappa and mu opioid receptors in human oocytes. The kappa and mu localization changed during the maturation of the oocyte, while the Delta localization was the same throughout. In particular, the delta receptor was detected in the periphery of the oocyte. On the other hand, the kappa receptor was found peripherally at the beginning, more internally during maturation and homogeneously at the end of maturation. Finally, the Mu receptor was located peripherally at the beginning of maturation and homogeneously in the rest of the maturation stages. This finding suggests a possible role for opioids, acting via receptors, in the maturation of the oocyte.

Effects of Cumulus Cells on Vitreous Cryopreservation of Human Mature Oocytes and Clinical Pregnancy Outcomes

The purpose of this study was to explore the effects of cumulus cells on vitreous cryopreservation of human mature oocytes and clinical pregnancy outcomes. The study was divided into group A (cumulus cells were removed from the oocytes before freezing) containing 24 participants and 193 oocytes and group B (cumulus cells were retained with the oocytes before freezing) containing 26 participants and 240 oocytes. Based on no significant differences in age, duration of infertility, infertile causes, and number of retrieved oocytes between both groups when oocytes were retrieved from infertile women, we found that the survival rate of post thaw oocytes (88% vs 58%), cleavage rate (80% vs 56%), and high-quality embryo rate (75% vs 59%) were significantly higher in group B than in group A. Under the conditions that there were no significant differences between the 2 groups in the general status of the participants undergoing embryo transfer, the embryo implantation rate (37% vs 15%) and the clinical pregnancy rate (50% vs 17%) were significantly higher in group B than in group A, all with Ps < .05. We conclude that the retention of cumulus cells can improve the developmental competence of vitrified-thawed human mature oocytes and clinical pregnancy outcomes.

Human oocyte maturation in vitro

Oocytes from medium-sized antral follicles have already completed their growth phase and, if released from the follicular environment and cultured in vitro, are able to resume the meiotic process and mature. However, in vitro maturation (IVM) does not entirely support all the nuclear and cytoplasmic changes that occur physiologically as an effect of the ovulatory stimulus. Regardless, oocyte IVM is widely applied for the breeding of agriculturally important species. In assisted reproduction technology, IVM has been proposed as an alternative treatment to circumvent the drawbacks of standard ovarian stimulation regimens. Initially introduced to eliminate the risks of ovarian hyperstimulation syndrome afflicting women presenting with polycystic ovaries, subsequently IVM has been suggested to represent an additional approach suitable also for normovulatory patients. So far, in children born from IVM cycles, no doubts of an increased incidence of congenital abnormalities have been raised. Many more births would be achieved if novel IVM systems, currently dominated by empiricism, could be conceived according to more physiological criteria. Recent findings shedding new light on the control of meiotic progression, the support of cumulus cells to the oocyte cellular reorganization occurring during maturation, and the modulation of the stimulus that promotes oocyte maturation downstream the mid-cycle gonadotropin signal are likely to provide crucial hints for the development of more efficient IVM systems.

Clinical application of in vitro maturation of human immature oocytes for infertile women with polycystic ovary syndrome

To investigate clinical effect and safety of in vitro maturation (IVM) of human immature oocytes in infertile women with polycystic ovary syndrome by comparing with conventional in vitro fertilization(IVF)and intracytoplasmic sperm injection(ICSI). From Jan. 2003 to Dec. 2009, 157 infertile women with PCOS underwent 162 cycles IVM in Center for Reproductive Medicine, the First Affiliated Hospital of Anhui Medical University. In the mean time, 109 patients with PCOS underwent 114 IVF/ICSI cycles as control group 1 and 106 patients with other factors underwent 106 IVF/ICSI cycles as control group 2. Treatment and outcome of pregnancy and infant were compared among those 3 groups. No statistically significant difference were found in terms of the positive rate of hCG in urine [35.7% (56/157), 42.2% (46/109), 44.3% (47/106)], the rate of clinical pregnancy [29.3% (46/157), 37.6% (41/109), 41.5% (44/106)], the rate of entopic pregnancy [1.9% (3/157), 1.8% (2/109), 0.9% (1/106)], the rate of miscarriage [18.6% (8/43), 12.8% (5/39), 20.9% (9/43)] and the rate of live-birth [22.3% (35/157), 31.2% (34/109), 32.1% (34/106)] among three groups (IVM group, control group 1, control group 2, P > 0.05). The rate of preterm labor, low weight newborn, mean birth weight, ratio of male to female did not show significantly difference among 3 groups (P > 0.05). The average control ovarian stimulation was 6 days, the median dose of gonadotropin (Gn) was 675 IU, and the total hospital cost was (8392 ± 1328) RMB in IVM group, which were statistically lower than those in the other two control groups (P < 0.01). The rate of multiple pregnancy was 4.7% (2/43) and ovarian hyperstimulation syndrome (OHSS) 0 in IVM group, which were significantly lower than those in the other control group (P < 0.01). In vitro maturation is an effective treatment in infertile women with PCOS, it could obtain the similar pregnancy outcome and reduce total cost, the dosage of gonadotropin-releasing hormone and rate of OHSS compared with conventional IVF/ICSI.

Three dimensional culture of fresh and vitrified mouse pre-antral follicles in a hyaluronan-based hydrogel: a preliminary investigation of a novel biomaterial for in vitro follicle maturation

BackgroundFolliculogenesis within the ovary requires interaction between somatic cell components and the oocyte. Maintenance of 3-dimensional (3-D) architecture and granulosa-oocyte interaction may be critical for successful in vitro maturation of follicles. Testing of novel biomaterials for the 3-D culture of follicles may ultimately lead to a culture model that can support the longer in vitro culture intervals needed for in vitro maturation of human oocytes from ovarian tissue biopsies.MethodsA novel tyramine-based hyaluronan (HA) hydrogel was tested for its biocompatibility with ovarian follicles. The HA was prepared at concentrations from 2 to 5 mg/ml. HA hydrogel was also formulated and tested with matrix proteins (ECM). Enzymatically isolated pre-antral follicles from the ovaries of 10–12 day SJL pups were divided amongst control (CT) and HA treatments. The growth of both fresh and vitrified follicles was assessed after encapsulation in the hydrogel. The basal culture medium was MEM alpha supplemented with FSH, LH, ITS and 5% FBS. Maturation was triggered by addition of hCG and EGF after in vitro culture (IVC). Outcome parameters monitored were follicle morphology, survival after IVC, antrum formation, GVBD and MII formation. Differences between treatments were analyzed.ResultsHA and ECM-HA encapsulated follicles looked healthy and maintained their 3-D architecture during IVC. In control cultures, the follicles flattened and granulosa:oocyte connections appeared fragile. Estradiol secretion per follicle was significantly higher by Day 12 in ECM-HA compared to HA or CT (4119, 703 and 1080 pg/ml, respectively). HA and ECM-HA cultured follicles had similar survival rates (62% and 54%, respectively), percent GV breakdown (96–97%), MII formation (47–48%) and oocyte diameters at the end of IVC. Control cultures differed significantly in percent GVBD (85%) and MII formation (67%) . Vitrified-warmed follicles encapsulated in HA had an oocyte maturation rate to MII of 54% as compared to 57% in non-embedded follicles.ConclusionsInitial testing of this new and unique HA-based hydrogel was quite promising. The ease of follicle encapsulation in HA, its optical transparency and ability to be molded combined with its support of follicle growth, estradiol secretion and resumption of meiosis make this HA-hydrogel particularly attractive as model for 3-D ovarian follicle culture.

Cryopreservation of human failed-matured oocytes followed by in vitro maturation: vitrification is superior to the slow freezing method

BackgroundOocyte cryopreservation is an important method used in a number of human fertility circumstances. Here, we compared the survival, in vitro maturation, fertilization, and early embryonic development rates of frozen-thawed human immature oocytes using two different cryopreservation methods.MethodsA total of 454 failed-matured oocytes [germinal vesicle (GV) and metaphase I (MI) stages] were collected from 135 patients (mean age 33.84 +/- 5.0 y) who underwent intracytoplasmic sperm injection (ICSI) cycles between February 2009 and December 2009 and randomly divided into a slow freezing group [1.5 mol/L-1, 2-propanediol (PROH) + 0.2 mol/l sucrose] and vitrification group [20% PROH + 20% ethylene glycol (EG) + 0.5 mol/l sucrose].ResultsThe vitrification protocol yielded a better survival rate than the slow freezing protocol at each maturation stage assessed. Regardless of the maturation stage (GV + MI), the slow freezing protocol had a significantly lower survival rate than the vitrification protocol (p < 0.001). In addition, a significant difference was found in the survival rates between GV and MI oocytes regardless of the protocol used (90.1 vs. 64.7%, respectively; p < 0.01). We also found that the maturation rates of GV and MI oocytes from the slow freezing and vitrification groups were 16.7 vs. 24.4% and 50.8 vs. 55.4%, respectively. Regardless of the protocol used, the GV oocytes had significantly lower viability than MI oocytes after 36 h of in vitro maturation (21.2 vs. 54.0%, respectively; p < 0.01). In addition, the GV and MI oocytes from the slow freezing group had a markedly lower maturation rate than those from the vitrification group (33.6 vs. 43.1%, respectively), but no statistical difference was found between the two groups (P > 0.05). For the GV-matured oocytes, no fertilized eggs were obtained in the slow-freezing group, while a 19.0% (4/21) fertilization rate was observed in the vitrification group. For the MI-matured oocytes, fertilization rates for the slow freezing and vitrified groups were 36% and 61.1%, respectively, but no significant difference was found between the two groups (PIn the Methods section in the MS, all procedures were compliant with ethical guidelines, i.e. approved by the Ethical Committee of our university and Informed Consent signed by each patient. > 0.05). In the GV vitrification group, no embryo formed; however, in the MI slow freezing group, 12 oocytes were fertilized, but only two achieved cleavage and were subsequently blocked at the 2-cell stage. In the MI vitrification group, a total of 22 embryos were obtained, five of which developed to the blastocyst stage.ConclusionsVitrification is superior to the slow freezing method in terms of the survival and developmental rates for the cryopreservation of human failed-matured oocytes. In addition, GV oocytes appeared to be more resistant than MI oocytes to the low temperature and cryoprotectant used during cryopreservation.

Implantation rates of embryos generated from slow cooled human oocytes from young women are comparable to those of fresh and frozen embryos from the same age group.

Previous reports of slow cooling of human mature oocytes have shown a reduced clinical efficiency relative to fresh oocytes. This study reports that equivalent fertilization and implantation rates to those obtained using fresh oocytes and cryopreserved embryos can be achieved with human mature oocytes dehydrated in 1.5 M propanediol and 0.2 M sucrose at 37°C and cryopreserved using slow cooling rates.

High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization

In vitro-matured bovine oocytes do not tolerate vitrification as well as mature murine or human oocytes. Delayed first cleavage in vitrified and in vitro-fertilized bovine oocytes may be responsible for the decreased yield of blastocysts in vitro. Because formation of sperm-aster and the subsequent assembly of microtubule network play an important role for migration and fusion of both pronuclei, aster formation in vitrified-warmed oocytes was analyzed by confocal laser-scanning microscopy. At 10 h post-insemination (hpi), proportions of oocytes fertilized normally were comparable between the vitrified and fresh control groups (67 and 70%, respectively). Proportions of oocytes that exhibited microtubule assembly were similar between the two groups (95% each), but the proportion of oocytes with multiple asters was higher in the vitrified group when compared with the fresh control group (68 vs 29%, P < 0.05). Both migration and development of two pronuclei were adversely affected by multiple aster formation. In the next experiment, multiple asters observed in 5.5 vs 8 hpi pronuclear zygotes were located near the male pronucleus, suggesting that those multiple asters were not the cytoplasmic asters of maternal origin. In conclusion, multiple aster formation frequently observed in vitrified-warmed bovine oocytes may be related to loss of ooplasmic function responsible for normal microtubule assembly from the sperm-aster.

Insufficient histone-3 lysine-9 deacetylation in human oocytes matured in vitro is associated with aberrant meiosis

To characterize histone acetylation during meiosis in human oocytes matured invitro or invivo. Experimental study. University reproductive medical center. Patients undergoing routine intracytoplasmic sperm injection (ICSI) cycles. Immature and mature oocytes were collected from patients undergoing intracytoplasmic sperm injection. Immunohistochemical assessment of the levels of acetylated lysine-9 of histone-3 (H3K9) and lysine-12 of histone-4 (H4K12) combined with spindle and chromosome configurations in invitro- and invivo-matured human oocytes. Transcript levels of histone deacetylases (HDACs) 1 and 2 were measured by single-cell quantitative polymerase chain reaction. Acetylation of H3K9 and H4K12 decreased during human oocyte maturation. Residual H3K9 acetylation was found in 37.7% of oocytes matured invitro, compared with 11.8% in oocytes matured invivo. Abnormal metaphase spindle was more frequent in oocytes with residual histone acetylation than without (51.6% vs. 25.4%, respectively). Treatment with the HDAC inhibitor trichostatin A increased the incidence of an abnormal metaphase but had no adverse effect on maturation efficiency. Furthermore, expression of HDAC1 transcripts was significantly lower in oocytes matured invitro versus invivo. Reduced HDAC1 expression and insufficient histone deacetylation are associated with metaphase defects in human oocytes matured invitro.

Improved Developmental Competence and Spindle Assembly of In Vitro Maturation of Human Oocytes by Prolonged Culture Duration.

In-vitro maturation of human oocytes is a promising technology for the treatment of human infertility, however it is still unclear that how to obtain the similar development competence of matured oocytes during the period of oocyte maturation compared with fresh MII oocytes. The purpose of this study was to investigate the relation between prolong culture time after maturation and subsequent embryo development results, and develop an in vitro oocyte maturation (IVM) method for a wide range of patients. Immature oocytes (including MI and GV oocytes) that underwent meiotic maturation during brief in vitro culture and they were subjected to parthenogenesis using artificial activation method through different prolonged culture duration. Morphology of oocytes and 8-cell embryos, fertilization, cleavage and blastocyst development was evaluated in each group. A total of 486 immature oocytes were collected in 122 IVM-ICSI cycles. Oocyte was separated into four groups with different prolonged culture duration. The overall activation rate is significant lower in both 0- and 4-hour groups compared with other two groups, and there is no difference in cleavage rate in all groups. In the stage of zygotic genomic activation, there is no difference among 0-, 2- and 4-hour groups, but significant higher than 8-hour group. In both 2- and 4-hour group, the efficiency of blastocyst formation is significant higher than that of other two groups. The Grading results at 8-cell stage showed that embryos with high quality was exceeded to 50 percent in 0-, 2- and 4-hour groups, whereas this proportion is less than 30 percent in 8-hour group. Immunostaning results demonstrated that the spindle is not separated completely between nuclear and polar body in 0-hour group, and the spindle lost partial or total disorganization or complete lack of microtubules, and the chromosomes began to condense in 8-hour group. This present study concluded that correctly prolonged culture duration will benefit for the development competence of oocytes matured in vitro, and facilitated the application of immature oocytes in clinical. (poster)

Effect of Incubation Temperature and Warming Solutions on the Viability and Maturation of Vitrified-thawed Human Immature Oocytes

Objective To investigate the viability and maturation of frozen-thawed human immature oocytes exposed to different temperature of vitrification and warming solutions. Methods The immature oocytes in germinal vesicle (GV) and matephase I (Mi) stages were collected from our ICSI patients and exposed to different temperature of vitrification and warming solutions before frozen in the freezing/thawing procedures. The different temperature groups were as follows: Group A, equilibration solution at 37 °C, vitrification solution at room temperature, warming solution at 37 °C; Group B, both vitrification and warming solution at room temperature; Group C, both vitrification and warming solutions at 37 °C; Group D, the frozen-thawed oocytes and the fresh oocytes were cultured for in vitro maturation. The survival rate and maturation rate were compared among groups. The oocytes were examined using immunofluorescent stainingand confocal microscopy to check the spindle configuration and chromosome arrangement. Results The survival rates and Mn rates of GV stage oocytes in groups A, B, C were 100%(15/15), 81.3%(13/16), 68.8%(11/16) and 33.3%(2/6), 83.3%(10/12),72.7% (8/11), respectively. The survival rate of group C was significantly lower than that of the control (P Conclusion The appropriate operation temperature of vitrification and warming solutions can improve the outcomes of the vitrified-thawed human immature oocytes.

Maturitas ex vivo: the case for oocyte in vitro maturation in human ARTs

Maturitas ex vivo: the case for oocyte in vitro maturation in human ARTs

Evaluation of zona pellucida birefringence intensity during in vitro maturation of oocytes from stimulated cycles

BackgroundThis study evaluated whether there is a relationship between the zona pellucida birefringence (ZP-BF) intensity and the nuclear (NM) and cytoplasmic (CM) in vitro maturation of human oocytes from stimulated cycles.ResultsThe ZP-BF was evaluated under an inverted microscope with a polarizing optical system and was scored as high/positive (when the ZP image presented a uniform and intense birefringence) or low/negative (when the image presented moderate and heterogeneous birefringence). CM was analyzed by evaluating the distribution of cortical granules (CGs) throughout the ooplasm by immunofluorescence staining. CM was classified as: complete, when CG was localized in the periphery; incomplete, when oocytes presented a cluster of CGs in the center; or in transition, when oocytes had both in clusters throughout cytoplasm and distributed in a layer in the cytoplasm periphery Nuclear maturation: From a total of 83 germinal vesicle (GV) stage oocytes, 58 of oocytes (69.9%) reached NM at the metaphase II stage. From these 58 oocytes matured in vitro, the high/positively scoring ZP-BF was presented in 82.7% of oocytes at the GV stage, in 75.8% of oocytes when at the metaphase I, and in 82.7% when oocytes reached MII. No relationship was observed between NM and ZP-BF positive/negative scores (P = 0.55). These variables had a low Pearson's correlation coefficient (r = 0.081). Cytoplasmic maturation: A total of 85 in vitro-matured MII oocytes were fixed for CM evaluation. Forty-nine oocytes of them (57.6%) showed the complete CM, 30 (61.2%) presented a high/positively scoring ZP-BF and 19 (38.8%) had a low/negatively scoring ZP-BF. From 36 oocytes (42.3%) with incomplete CM, 18 (50%) presented a high/positively scoring ZPBF and 18 (50%) had a low/negatively scoring ZP-BF. No relationship was observed between CM and ZP-BF positive/negative scores (P = 0.42). These variables had a low Pearson's correlation coefficient (r = 0.11).ConclusionsThe current study demonstrated an absence of relationship between ZP-BF high/positive or low/negative score and nuclear and cytoplasmic in vitro maturation of oocytes from stimulation cycles.

Defective deacetylation of histone 4 K12 in human oocytes is associated with advanced maternal age and chromosome misalignment

Chromosome segregation errors during human oocyte meiosis are associated with low fertility in humans and the incidence of these errors increases with advancing maternal age. Studies of mitosis and meiosis suggest that defective remodeling of chromatin plays a causative role in aneuploidy. We analyzed the histone deacetylation pattern during the final stages of human oocyte maturation to investigate whether defective epigenetic regulation of chromatin remodeling in human oocytes is related to maternal age and leads to segregation errors. Human surplus oocytes of different meiotic maturation stages [germinal vesicle (GV), metaphase (M)I and MII] were collected from standard IVF/ICSI treatments. Oocytes were analyzed for acetylation of different lysines of histone 4 (H4K5, H4K8, H4K12 and H4K16) and for α-tubulin. Human GV oocytes had an intense staining of the chromatin for all four histone 4 lysine acetylations. MI and MII stage oocytes showed either normal deacetylation or various amounts of defective histone deacetylation. Residual H4K12 acetylation was more frequently found in oocytes obtained from older women, with a significant correlation between defective deacetylation and maternal age (r = 0.185, P = 0.007). Eighty-eight percent of the oocytes with residual acetylation had misaligned chromosomes, whereas only 33% of the oocytes that showed correct deacetylated chromatin had misaligned chromosomes (P < 0.001). We conclude that defective deacetylation during human female meiosis increases with maternal age and is correlated with misaligned chromosomes. As chromosome misalignment predisposes to segregation errors, our data imply that defective regulation of histone deacetylation could be an important factor in age-related aneuploidy.