Human Germinal Vesicle Oocytes Research Articles

Chromatin Morphology in Human Germinal Vesicle Oocytes and Their Competence to Mature in Stimulated Cycles.

The search for simple morphological predictors of oocyte quality is an important task for assisted reproduction technologies (ARTs). One such predictor may be the morphology of the oocyte nucleus, called the germinal vesicle (GV), including the level of chromatin aggregation around the atypical nucleolus (ANu)-a peculiar nuclear organelle, formerly referred to as the nucleolus-like body. A prospective cohort study allowed distinguishing three classes of GV oocytes among 135 oocytes retrieved from 64 patients: with a non-surrounded ANu and rare chromatin blocks in the nucleoplasm (Class A), with a complete peri-ANu heterochromatic rim assembling all chromatin (Class C), and intermediate variants (Class B). Comparison of the chromatin state and the ability of oocytes to complete meiosis allowed us to conclude that Class B and C oocytes are more capable of resuming meiosis in vitro and completing the first meiotic division, while Class A oocytes can resume maturation but often stop their development either at metaphase I (MI arrest) or before the onset of GV breakdown (GVBD arrest). In addition, oocytes with a low chromatin condensation demonstrated a high level of aneuploidy during the resumption of meiosis. Considering that the degree of chromatin condensation/compaction can be determined in vivo under a light microscope, this characteristic of the GV can be considered a promising criterion for selecting the best-quality GV oocytes in IVM rescue programs.

Chromatin Configuration in Diplotene Mouse and Human Oocytes during the Period of Transcriptional Activity Extinction.

In the oocyte nucleus, called the germinal vesicle (GV) at the prolonged diplotene stage of the meiotic prophase, chromatin undergoes a global rearrangement, which is often accompanied by the cessation of its transcriptional activity. In many mammals, including mice and humans, chromatin condenses around a special nuclear organelle called the atypical nucleolus or formerly nucleolus-like body. Chromatin configuration is an important indicator of the quality of GV oocytes and largely predicts their ability to resume meiosis and successful embryonic development. In mice, GV oocytes are traditionally divided into the NSN (non-surrounded nucleolus) and SN (surrounded nucleolus) based on the specific chromatin configuration. The NSN-SN transition is a key event in mouse oogenesis and the main prerequisite for the normal development of the embryo. As for humans, there is no single nomenclature for the chromatin configuration at the GV stage. This often leads to discrepancies and misunderstandings, the overcoming of which should expand the scope of the application of mouse oocytes as a model for developing new methods for assessing and improving the quality of human oocytes. As a first approximation and with a certain proviso, the mouse NSN/SN classification can be used for the primary characterization of human GV oocytes. The task of this review is to analyze and discuss the existing classifications of chromatin configuration in mouse and human GV oocytes with an emphasis on transcriptional activity extinction at the end of oocyte growth.

Effects of microvibration stimulation on developmental potential of discarded germinal vesicle oocytes of human.

This research aims to study the effects of continuous microvibration stimulation on the parthenogenetic development of human germinal vesicle oocytes. Ninety-five discarded germinal vesicle oocytes from intracytoplasmic sperm injection treatment (ICSI) cycles performed at Amcare Women's & Children's Hospital between January and December 2021 were used for conventional static culture as well as 10Hz microvibration culture. We investigated the differences between the two groups in terms of oocyte maturation rate, parthenogenetic activation rate, and parthenogenetic blastocyst formation rate. The static culture and 10Hz microvibration culture of 95 oocytes showed that the parthenogenetic blastocyst formation rate in the microvibration culture group was significantly higher than those in the traditional static culture group. A continuous microvibration stimulation can significantly improve the parthenogenetic developmental potential of human immature oocytes.

Evaluation of Origio IVM medium quality in in vitro maturation of human germinal vesicle oocytes gained during intra cytoplasmic sperm injection cycles

Different medium has been introducing in last two decays for IVM of human oocytes germinal vesicles but they are very expensive and gained by order with different qualification, and different maturation rates. Objective: the aim of study to evaluate the efficacy of Origio IVM medium used in in vitro maturation of germinal vesicles gained during ICSI cycles. Patients and methods: 81 oocytes in germinal vesicle stage involved in in vitro maturation (IVM) program with Origio medium, they represent 35 cases (couples) that underwent IVF-ICSI operation, gained from them, (18) of them excluded because they are morphologically abnormal, the rest (63) GV introduced in program, they incubated for 24 hours and reassessed again to evaluate their maturation, the study done in the High institute for infertility diagnosis and assisted reproductive technologies, Al- Nahrain University from the period December 2018 to February 2020 .

Evaluation of fertilization medium quality in in vitro maturation of human germinal vesicle oocytes gained during intra cytoplasmic sperm injection cycles

Background: in vitro maturation of human germinal oocytes is a promising technique in assisted reproductive technologies, different medium has been introducing in last two decays for this purpose but they are very expensive and gained by order with different qualification Objective: the aim of study to evaluate the efficacy of fertilization medium used in in vitro maturation of germinal vesicles gained during ICSI cycles. Patients and methods: 42 oocytes in germinal vesicle stage involved in in vitro maturation (IVM) program with fertilization medium, they represent 22 cases (couples) that underwent IVF-ICSI operation, gained from them, (9) of them excluded because they are morphologically abnormal, the rest (33) GV introduced in program, they incubated for 24 hours and reassessed again to evaluate their maturation, the study done in the High institute for infertility diagnosis and assisted reproductive technologies, Al- Nahrain University from the period December 2018 to February 2020 . Results: out of (33) Gv incubation for 24 hours (6) of them remain in GV stage, (8) of them pass to M1 maturation stage, and another (8) passes to M2 stage of maturation, the rest 11 they were atretic.

O-289 Effects of Taxol on the developmental potency of human and mouse GV oocytes

Abstract Study question Is it possible to improve the quality of oocytes with delayed maturation or aging? Summary answer Yes, modification of oocyte microtubules at the germinal vesicle (GV) stage inhibits abnormal chromosome separation in in-vitro maturation and enhances early cleavage. What is known already Oocyte aging is characterized by an increase of aneuploidy and a decrease of the developmental potency with maternal ages, which will results in low frequencies of the blastocyte formation and implantation. In addition to the 1st meiotic division, it has become clear that the 2nd meiotic division also significantly contributes to aneuploidy production which is followed by pre-implantation embryo loss. However, no method for overcoming the oocyte aging has been established. Study design, size, duration Human GV oocytes with delayed maturation obtained from consented female patients were used. Mouse GV oocytes collected from 9-15 month old ICR mice were also used. After exposure to Taxol for 1hr, the oocytes were matured in vitro and examined cytogenetically and cytologically at the GV stage, the MII stage and pronuclear stage. Participants/materials, setting, methods RNA transcripts of the GV stage oocytes were compared before and after Taxol exposure with microarray in both species. Chromosome aberrations at the MII stage and blastocyst formation rate were examined in human IVM oocytes. Mouse IVM oocytes were evaluated on 2nd polar body (PB) extrusion and O2 consumption (CRAS3.0, Crino Co Inc.) at the pronuclear stage after parthenogenetic activation with electro-stimulation. Main results and the role of chance After Taxol treatment, premature chromosome separation was significantly reduced from 96% to 7% and the blastocyst formation rate increased from 3% to 16% in human. In mice, normal PB extrusion rate increased from 29% to 92%, and oxygen consumption was higher in some pronuclear oocytes. In human GV oocytes, taxol treatment altered gene expression only in a few factors related to chromosome attachment and segregation. On the other hand, in mouse GV oocytes, gene expression was significantly altered by oocyte aging and Taxol treatment. Limitations, reasons for caution The present results were obtained in human GV oocytes with delayed maturation which were collected after PMS and HCG administrations. It is not clear whether Taxol is effective on the GV oocytes collected before HCG administration. Wider implications of the findings Our results indicate that the combination of biochemical modification and IVM in the GV stage may be able to restore the aging of human oocytes. Trial registration number not applicable

Exploring the Developmental Potential of Human Germinal Vesicle Oocytes Aiming at Fertility Preservation: Can We Increase the Yields of Competent Oocytes through IVM Combined with Vitrification?

The rescue in vitro maturation (rIVM) of germinal vesicle oocytes (GVs) has been proposed to improve the total number of mature oocytes in women undergoing fertility preservation. Currently, there is no consensus about the clinical utility of this practice, and heterogeneity in the protocols used may influence the final outcomes. This study investigated the developmental potential of mature metaphase II (MII) human oocytes obtained from GVs after rIVM and the impact of applying vitrification at different timepoints either before or after rIVM. After randomization, oocytes were assigned to undergo rIVM and thereafter vitrification or intracytoplasmic sperm injection (ICSI), or to undergo direct vitrification-warming and thereafter rIVM and ICSI. The likelihood of obtaining MII oocytes was just slightly higher in the fresh rIVM group compared to the vitrification-warming-rIVM group. When comparing fresh rIVM that underwent subsequently ICSI, the fertilization and developmental rates up to the blastocyst stage were seen to be reduced in both groups that underwent vitrification either before or after rIVM. Although some blastocysts were obtained in the fresh rIVM-ICSI group, the efficacy of these methods was low overall, suggesting that the further development of protocols for IVM conducted early after denudation is needed to improve the final results of rIVM aiming at fertility preservation.

Age-related gene expression profiles of immature human oocytes.

What is the difference between the gene expression profiles of single human germinal vesicle (GV) oocytes from women of different ages? There were no statistically significant differences in gene expression profiles of human GV oocytes from women of different ages (range: 25-43). It is well established that reproductive capacity declines as women age, which is attributed to oocyte quality since this decline is counterbalanced in older women receiving young donor oocytes. Altered gene expression of human oocytes at different stages of development in relation to female age is one of the suggested mechanisms that could explain the decrease in oocyte quality. Between 2012 and 2014, 40 human GV oocytes of 40 women were obtained during follicular aspiration as part of routine ICSI treatment. Gene expression profiles of 38 GV oocytes were determined in four different age groups: 25-30, 31-35, 36-38 and 39-43 years of age. GV oocytes were donated for research and frozen between 3.5 and 7.5 h after follicular aspiration. Subsequently, GV oocytes were thawed and prepared for gene expression profile analysis using Agilent microarrays containing ~42 000 Human Gene Expression probe-sets. Gene expression profiles were visualized by hierarchical clustering and the top 500 most differing genes were determined by multidimensional scaling (MDS). Transcripts were analysed in a class comparison between the four age groups and for indicators of biological age: antral follicle count (AFC) and the total dosage of FSH needed for ovarian stimulation. Individual transcripts were analysed using linear regression. A false discovery rate <0.05 was considered statistically significant. Visualization of gene expression profiles of GV oocytes with hierarchal clustering and MDS demonstrated no clear grouping of samples based on female age, AFC or FSH dosage. The gene expression profile of GV oocytes classified in four age groups revealed no significantly differentially expressed genes between the four different age groups. There were also no significantly differentially expressed genes in the linear regression analysis for individual transcripts against age. Not applicable. Immature (GV) oocytes obtained from ovarian stimulation cycles were used. Findings may therefore differ for oocytes at other developmental stages and for in-vivo matured oocytes under physiological conditions. Due to our relatively large, but still limited study sample (40 GV oocytes), we cannot exclude that there might be smaller age-related gene-expression differences, i.e. due to a lack of power. We did not find an effect of female age on gene expression profiles of individual human GV oocytes. Other studies have suggested that gene-expression profiles are affected in mature oocytes, which might imply that female age affects oocyte maturation. Alternatively, other mechanisms in human oocytes might cause the age-related fertility decline. This study received no external funding and there are no competing interests.

The effects of different types of media on in vitro maturation outcomes of human germinal vesicle oocytes retrieved in intracytoplasmic sperm injection cycles.

ObjectiveOptimizing in vitro maturation (IVM) media to achieve better outcomes has been a matter of interest in recent years. The aim of this prospective clinical trial was to investigate the effects of different media on the IVM outcomes of immature oocytes at the germinal vesicle (GV) stage.MethodsA total of 400 immature oocytes at the GV stage with normal morphology were retrieved from 320 infertile women aged 31±4.63 years during stimulated intracytoplasmic sperm injection (ICSI) cycles. They were divided into groups of homemade IVM medium (I, n=100), cleavage medium (II, n=100), blastocyst medium (III, n=100), and Sage IVM medium (IV, n=100) and cultured for 24 to 48 hours at 37℃. ICSI was performed, and the rates of fertilization and embryo formation were compared across the four groups.ResultsIn the 400 retrieved GV oocytes, the total maturation rates showed significant differences in groups I to IV (55%, 53%, 78%, and 68%, respectively, p<0.001). However, there were no significant differences in the fertilization, embryo formation, or arrest rates of metaphase II oocytes across these groups. In all groups, GV maturation was mostly completed after 24 hours, with fewer oocytes requiring 48 hours to mature (p<0.01). Moreover, the rate of high-quality embryos was higher in group IV than in the other groups (p=0.01).ConclusionThe quality of the IVM medium was found to affect clinical IVM outcomes. Additionally, blastocyst medium may be a good choice in IVM/ICSI cycles as an alternative IVM medium.

Vitrification of Human Germinal Vesicle Oocytes: before or after In Vitro Maturation?

BackgroundThe use of immature oocytes derived from stimulated cycles could be of great importance, particularly for urgent fertility preservation cases. The current study aimed to determine whether in vitro maturation (IVM) was more successful before or after vitrification of these oocytes.Materials and MethodsThis prospective study was performed in a private in vitro fertilization (IVF) center. We collected 318 germinal vesicle (GV) oocytes from 104 stimulated oocyte donation cycles. Oocytes were divided into two groups according to whether vitrification was applied at the GV stage (group 1) or in vitro matured to the metaphase II (MII) stage and then vitrified (group 2). In the control group (group 3), oocytes were in vitro matured without vitrification. In all three groups, we assessed survival rate after warming, maturation rate, and MII-spindle/chromosome configurations. The chi-square test was used to compare rates between the three groups. Statistical significance was defined at P<0.05 and we used Bonferroni criterion to assess statistical significance regarding the various pairs of groups. The Statistical Package for the Social Sciences version 17.0 was used to perform statistical analysis.ResultsThere was no significant difference in the survival rate after vitrification and warming of GV (93.5%) and MII oocytes (90.8%). A significantly higher maturation rate occurred when IVM was performed before vitrification (82.9%) compared to after vitrification (51%). There was no significant difference in the incidence of normal spindle/ chromosome configurations among warmed oocytes matured in vitro before (50.0%) or after (41.2%) vitrification. However, a higher incidence of normal spindle/chromosome configurations existed in the in vitro matured oocytes which were not subjected to vitrification (fresh oocytes, 77.9%).ConclusionIn stimulated cycles, vitrification of in vitro matured MII oocytes rather than GV oocytes seems to be more efficient. This approach needs to be verified in nonstimulated fertility preservation cases.

In human germinal vesicle oocytes mitochondrial stress disrupts meiotic spindles without affecting mean telomere length

The architecture and structure of the meiotic spindle influence embryo development(1) and risk of aneuploidy in women(2). The factors that disrupt the meiotic spindle remain incompletely understood. Dysfunctional mitochondria produce reactive oxygen species (ROS), which may directly perturb spindles(3). ROS also can disrupt spindles by inducing telomere attrition, since telomeres are essential for spindle formation and are especially susceptible to ROS(4). We studied the impact of ROS, produced by uncoupling mitochondria, on the area and retardance (measure of molecular order) of meiotic spindles and on mean telomere length of individual human oocytes.

Human GV oocytes generated by mitotically active germ cells obtained from follicular aspirates.

Human female germline stem cells (FGSCs) have opened new opportunities for understanding human oogenesis, delaying menopause, treating infertility, and providing a new strategy for preserving fertility. However, the shortage of adult human ovaries tissues available impedes their future investigations and clinical applications. Here, we have established FGSC lines from scarce ovarian cortical tissues that exist in follicular aspirates (faFGSCs), which are produced and discarded in in vitro fertilization centers worldwide. The faFGSCs have characteristics of germline stem cells involved in the gene expression profile, growth characteristics, and a normal karyotype consistent with that of FGSCs obtained from ovarian cortexes surgically removed from patients (srFGSCs). Furthermore, faFGSCs have developmental potentials including spontaneous differentiation into oocytes under feeder-free conditions, communicating with granulosa cells by gap junctions and paracrine factors, entering meiosis after RA induction, as well as forming follicles after injection into human ovarian cortical tissues xenografted into adult immunodeficient female mice. Lastly, we developed a strategy guiding FGSCs differentiated into germinal vesicle (GV) stage oocytes in vitro and revealed their developmental mechanisms. Our study not only provides a new approach to obtain human FGSCs for medical treatment, but also opens several avenues to investigate human oogenesis in vitro.

Cytoplasm replacement following germinal vesicle transfer restores meiotic maturation and spindle assembly in meiotically arrested oocytes

Both the cytoplasmic and nuclear compartments are essential for the acquisition of meiotic competence. This study assessed the role of the cytoplasm in meiosis resumption in meiotically arrested oocytes at the germinal vesicle (GV) stage. Mouse oocytes at GV stage were meiotically arrested with 3-isobutyl-1-methylxanthine (IBMX). GV transfer was performed between IBMX-treated and non-treated (control) mouse oocytes, and between control mouse and human GV oocytes. Extrusion of first polar body (PB) was examined as an indication of nuclear maturation. Meiotic spindle assembly and chromosome alignment were examined by immunostaining. Results indicated that oocytes arrested with IBMX for 24 and 48 h exhibited reduced ability for meiotic maturation and for extruding the first PB when compared with controls (P < 0.01). IBMX-treated oocytes reconstituted with cytoplasm, but not GV, of control oocytes restored the assembly of meiotic spindle and meiotic maturation. Mouse oocytes reconstituted with GV of human oocytes underwent meiosis similar to that observed in mice, but not humans. Additionally, human oocytes reconstituted by mouse GV underwent meiosis similar to that observed in humans, but not mice. These findings suggest that cytoplasm replacement by GV transfer could represent a potential therapeutic option for women who do not produce mature oocytes during IVF.

A randomized controlled trial of gonadotropin-releasing hormone agonist versus gonadotropin-releasing hormone antagonist in Iranian infertile couples: oocyte gene expression.

BackgroundThe main objective of the present work was to compare the effects of the gonadotropin-releasing hormone agonist (GnRH-a) and GnRH antagonist (GnRH-ant) on the gene expression profiles of oocytes obtained from Iranian infertile couples undergoing in vitro fertilization (IVF).MethodsFifty infertile couples who underwent IVF between June 2012 and November 2013 at the Infertility Center of Tehran Women General Hospital, Tehran University of Medical Sciences, were included in this study. We included women that had undergone IVF treatment because of male factor, tubal factor, or unexplained infertility. The women randomly underwent controlled ovarian stimulation (COS) with either the GnRH-a (n = 26) or the GnRH-ant (n = 24). We obtained 50 germinal vesicle (GV) oocytes donated by women in each group. After the sampling, pool of 50 GV oocytes for each group was separately analyzed by quantitative polymerase chain reaction (qPCR).ResultThe expression levels of Adenosine triphosphatase 6 (ATPase 6), Bone morphogenetic protein 15 (BMP15), and Neuronal apoptosis inhibitory protein (NAIP) genes were significantly upregulated in the GnRH-ant group compared to the GnRH-a group, with the fold change of 3.990 (SD ± 1.325), 6.274 (SD ± 1.542), and 2.156 (SD ± 1.443), respectively, (P < 0.001). Growth differentiation factor 9 (GDF9) mRNA did not have any expression in the GnRH-a group; however, GDF9 mRNA was expressed in the GnRH-ant group. Finally, it was found that the genes involved in the DNA repairing and cell cycle checkpoint did not have any expression in either group.ConclusionThe present study showed, for the first time, the expression levels of genes involved in the cytoplasmic maturity (BMP15, GDF9), adenosine triphosphate production (ATPase 6), and antiapoptotic process (NAIP), in human GV oocytes were significantly higher in the GnRH-anta group than in the GnRH-a group in COS. Higher expression level of these genes when GnRH-ant protocol is applied, this protocol seems to be a more appropriate choice for women with poly cystic ovarian syndrome, because it can probably improve the expression of the aforementioned genes.Trial registrationCurrent Controlled Trials: IRCT 2014031112307 N3.

Morphological and molecular markers are correlated with maturation-competence of human oocytes

Does the position of the germinal vesicle (GV) in human oocytes correlate with molecular and morphological parameters as well as with maturation-competence? The position of GV in human oocytes correlates with density of microtubule (MT) filaments, concentration of Fyn, nucleolus localization and the ability of the oocytes to complete maturation following GV breakdown (GVBD). Our knowledge is confined to oocytes of young mice where maturation-competence is correlated with a central GV and regulated by MTs and the presence of a chromatin ring. Fyn kinase is localized at the spindle and cortex of mouse oocytes and plays a role in both maturation and MT stabilization. Spatial localization of the GV and nucleolus (central or peripheral), the presence of a chromatin ring, the localization of Fyn, MT density and oocyte maturation were assessed in 153 human oocytes, 335 oocytes from young mice (2-month-old) and 146 oocytes from old mice (12-month-old). GV human oocytes were donated by consenting female patients (n = 57), 21-45-year-old undergoing IVF/ICSI. As a control, GV mouse oocytes were collected from female mice after injection of pregnant mares' serum gonadotrophin. Human and mouse GV oocytes allocated for immunocytochemistry were fixed on day of retrieval, stained with specific antibodies and imaged using a confocal laser-scanning microscope. Human and mouse oocytes allocated for maturation were incubated for 48 and 24 h, respectively. GVBD and extrusion of the first polar body (PBI) were assessed using differential interference contrast optics. GV location was peripheral and independent of age in 69.9% of the human oocytes, but GV location did vary with age in mice oocytes; it was central in 89.9% of the oocytes retrieved from young-mice and peripheral in 52.1% of the oocytes retrieved from old mice (P < 0.05). A central GV, whether in human or mouse oocytes, was highly correlated with a central nucleolus, absence of Fyn at the GV and a dense MT network (P < 0.05), whereas a peripheral GV correlated with peripheral nucleolus, presence of Fyn at the GV and a flimsy MT network. After 48 h in culture, no degeneration was observed in human central-GV oocytes, however, 12/95 (12.6%) of the peripheral-GV oocytes degenerated (P < 0.05). No correlation was observed between GV position and presence of a chromatin ring. The percentage of human oocytes that extruded the PBI after completing GVBD was significantly higher (73.7%) in central than in peripheral-GV oocytes (45.8%; P < 0.05). In mice oocytes, central location of the GV correlated with maturation competence in young (P < 0.05) but not old mice. The fact that the human GV oocytes used in this study were exposed to gonadotrophic stimulation but failed to mature in vivo might be a sign of their low quality and this should be considered when drawing conclusions from the data. Furthermore, our observation that only peripheral-GV human oocytes were degraded may indicate that they are of a lower quality than central-GV human oocytes. We suggest that the central location of GV within the oocytes, which is associated with an absence of Fyn at the GV and the presence of thick filamentous MTs in the ooplasm, may serve as a predictor of successful maturation and provide new insights for the use of IVM.

Effects of vitrification on mitochondrial distribution and spindle configuration during in vitro maturation of human germinal vesicle-stage oocytes

Cryopreservation of human immature oocytes may be useful tool for fertility preservation in the future. However, vitrification of germinal vesicle (GV)-stage oocytes has poor result as for later development after fertilization. This may be due to abnormality of cytoplasmic maturation of the freeze-thawed oocytes. To elucidate this mechanism, we have investigated the distribution of mitochondria and spindle and chromosome configuration of in vitro matured oocytes with or without vitrification. GV oocytes were obtained from consenting patients undergoing ICSI treatment at our clinic. GV oocytes were randomly assigned to 1 of 3 groups: (1) GV group, GV oocytes; (2) IVM group, GV oocytes matured in vitro to metaphase II (MII); and (3) Vit group, oocytes vitrified at GV stage and matured in vitro. Confocal microscopy was applied to compared the mitochondrial distribution and configuration of the meiotic spindle and chromosomes in those oocytes. The maturation (MII) rate was 71.9% in the IVM group and 73.3% in the Vit group. There were no significant differences between the IVM and Vit groups. Compared to the IVM group (17.5%), the Vit group (45.9%) showed significant increase in diffuse mitochondria distribution. There was also a significantly lower proportion of oocytes with normal spindle configuration in the Vit group (18.9%) compared with the IVM group (47.5%). There were no significant differences in chromosome configuration between the IVM and Vit groups. This study revealed that vitrification of human GV oocytes has a detrimental effect on the spindle configuration of oocytes that have been thawed and matured in vitro which may explain decreased ability of further development.

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.

Spontaneous in vitro maturation and artificial activation of human germinal vesicle oocytes recovered from stimulated cycles

To define germinal vesicles (GV) by morphometric and morphologic examination and by chromatin compaction and to assess their spontaneous nuclear and cytoplasmic competence. 131 GV were cultured for 42.7 ± 2.4 h. Nuclear maturation was evaluated at four time points. Sixty-seven in vitro and twenty-five in vivo metaphase II (MII) were activated. Parthenotes with 2 PB and one pronucleus (NA) were studied for ploidy. A total of 74.8% GV matured to MII: 55% at 21.4 ± 2.4 h and 47.3% in the following 24 h. Artificial activation induced NA in 79.2% of in vivo-MII and in 22.4% of in vitro-MII. All NA were haploid. GV spontaneously mature at the nuclear level. Their NA are haploid, but their cytoplasmic competence is compromised. Variables were not found to be predictors of oocyte competence, probably due to our population being homogeneous with respect to most of the variables studied.

In vitro maturation of human germinal vesicle-stage oocytes: role of epidermal growth factor-like growth factors in the culture medium

In vitro maturation (IVM) of oocytes is a promising technique to reduce the costs and avert the side effects of gonadotrophin stimulation for IVF. The pregnancy rates from oocytes matured in vitro are still lower than those of in vivo stimulation cycles, indicating that optimization of IVM remains a challenge. Recently, it was demonstrated that LH exerts its action on ovulation, at least in part, through stimulation of the production of the epidermal growth factor family members amphiregulin (Areg) and epiregulin (Ereg) in pre-ovulatory follicles, and they, in turn, serve as paracrine mediators of LH. We aimed to investigate the effect of supplementation of the medium with Areg and Ereg on the maturation rate of immature oocytes. A total of 105 sibling human germinal vesicle (GV) oocytes obtained after gonadotrophin stimulation were cultured in a complex defined medium either with or without supplemented recombinant human Areg (75 ng/ml) and Ereg (75 ng/ml) for 24 h. Significantly more oocytes reached the metaphase II stage at 24 h in media supplemented with Areg and Ereg (75.5 versus 36.5%, P < 0.001). In vitro matured oocytes retrieved from the two subgroups had no statistically significant difference in fertilization and cleavage rates or morphology scores. Overall, a significantly higher number of Day 2 (52.8 versus 26.9% P < 0.01) and Day 3 (45.2 versus 23%, P < 0.05) embryos originated from GV oocytes cultured in the Areg- and Ereg-enriched medium. Supplementation of the maturation medium with Areg and Ereg improves the maturation of human GV oocytes in vitro.

In vitro maturation (IVM) of murine and human germinal vesicle (GV)–stage oocytes by coculture with immortalized human fallopian tube epithelial cells

To improve the maturation rate of murine and human germinal vesicle (GV) oocytes using human tubal epithelial cells (hTECs). Murine and human GV oocytes were randomized to human tubal fluid (HTF) media alone or cocultured with mouse embryonic fibroblasts (MEFs) or primary hTECs or immortalized hTECS (ihTECs) for various times. Rates of maturation to meiosis II (MII) were compared between groups. TECs were isolated from discarded salpingectomy specimens. One batch was immortalized with TERT and SV40 large T-antigen. GV oocytes (n = 710) were isolated from 8-week-old-mice at 40 hours after pregnant mare's serum gonadotropin stimulation. Discarded human GV oocytes (n = 62) were obtained from intracytoplasmic sperm injection cycle IVF center patients. Oocytes were cultured in HTF media alone or with MEFs, hTECs, or ihTECs. Maturation rates were assessed by standard morphological criteria and compared. The maturation rate of murine GV oocytes to MII at 12 and 24 hours increased significantly in coculture with hTECS and ihTECS compared with MEF and HTF media alone. In addition, the development rate after IVF was significantly higher with hTECS and ihTECS than in MEF and HTF media alone. Maturation of human GV oocytes to MII at 24 and 48 hours was significantly higher in hTECS and ihTECS compared with HTF media alone. Coculture with either primary or immortalized TECs might improve oocyte quality and significantly raise in vitro maturation rates for GV oocytes.