Germinal Vesicle Oocytes Research Articles

Follicular Fluid Zinc Level and Oocyte Maturity and Embryo Quality in Women with Polycystic Ovary Syndrome.

Background:Polycystic ovary syndrome (PCOS) is considered to be one of the most common endocrine disorders in women of reproductive age. Zinc, a vital trace element in the body, plays a key role in maintaining health, especially due to its antioxidant role. On the other hand, lack of antioxidants and oxidative stress can adversely affect oocytes quality and consequently fertility rate. The available studies that report the effect of follicular fluid (FF) zinc in terms of the number and quality of the oocytes in infertile women with PCOS, are few and not consistent. We decided to investigate this issue.Materials and Methods:In this cross-sectional study, from the women with PCOS referring to Omolbanin Hospital, Dezful, Iran (February to December 2019), a total of 90 samples (follicular fluid, oocytes, and embryos) were collected from those who had undergone in vitro fertilization (IVF). To measure zinc level in follicular fluid, high performance liquid chromatograpy (HPLC) was utilized. Also, oocytes maturity and embryos quality evaluation was performed using inverted optical microscopy. One-way ANOVA and Fisher’s least significant difference (LSD) were used for data analysis.Results: The amount of FF zinc was not associated with any significant differences in the number of oocytes and metaphase I (MI) and germinal vesicle (GV) oocytes, but a significant decrease was observed in the number of metaphase II (MII) oocytes at zinc values less than 35 µg/dL. The FF zinc levels less than 35 µg/dL were also significantly associated with decreased embryo qualityConclusion:A significant relationship was found between the level of FF zinc and the quality and the number of oocytes taken from the ovaries of infertile patients with PCOS history who were candidates for IVF treatment as well as the number of high quality embryos.

The Effect of Sodium Selenite on Expression of Mitochondrial Transcription Factor A during In Vitro Maturation of Mouse Oocyte.

Background:The aim of the present study was to investigate the effect of Sodium Selenite (SS) supplemented media on oocyte maturation, expression of mitochondrial transcription factor A (TFAM) and embryo quality.Methods:Mouse Germinal Vesicle (GV) oocytes were collected after administration of Pregnant Mare Serum Gonadotropin (PMSG); in experimental group 1, oocytes were cultured and then subjected for in vitro maturation in the absence of SS, and in experimental group 2, they were matured in vitro in the presence of 10 ng/ml of SS up to 16 hr. The control group included MII oocytes obtained from the fallopian tubes after ovarian stimulation with PMSG, followed by human chorionic gonadotropin. Then, the expression of TFAM in MII oocytes in all three groups was investigated using real-time RT-PCR. The fertilization and embryo developmental rates were assessed, and finally the quality of the blastocysts was evaluated using propidium iodide staining.Results:The oocyte maturation rate to MII stage in SS treated group was significantly higher than non-treated oocytes (75.65 vs. 68.17%, p<0.05). Also, the rates of fertilization, embryo development to blastocyst stage as well as the cell number of blastocyst in SS supplemented group were higher than other experimental group (p<0.05). There was a significant decrease in TFAM gene expression in both in vitro groups compared to the group with in vivo obtained oocytes (p<0.05). Moreover, there was a significant increase in TFAM gene expression in oocytes that matured in the presence of SS compared to that of the group without SS (p<0.05).Conclusion:Supplementation of oocyte maturation culture media with SS improved the development rate of oocytes and embryo and also enhanced TFAM expression in MII oocytes which can affect the mitochondrial biogenesis of oocytes.

135 Differential gene expression of bovine long noncoding RNAs in single oocytes aspirated from small and large follicles

In mammals, maternal mRNAs accumulate in the oocyte and play important roles during early embryogenesis in embryonic genome activation. Proper development is dependent upon regulation of these maternal transcripts while simultaneously activating the embryonic genome. Long noncoding RNAs (lncRNAs) regulate various cellular processes, including modulation of gene expression and epigenetic status. Recently, lncRNAs have been functionally characterised as key regulators of embryonic genome activation in humans and mice. Our laboratory identified 1535 lncRNAs in bovine oocytes using RNA-sequencing. The objective of this study was to verify our RNA-seq data and if present, characterise the expression of six highly abundant lncRNAs (2160.1, 5379.1, 18208.1, 23721, 25823.1, and 34876.2) in single oocytes aspirated from varying size follicles at different developmental stages. Single oocytes were aspirated from small (&amp;lt;4mm) and presumably oestrogen active (6–18mm) follicles and were denuded at the germinal vesicle (GV) or MII stage. MII stage was determined by cumulus expansion and the extrusion of the first polar body. Real-time quantitative PCR analysis, using RPL-19 as a housekeeping gene analysed using the standard curve method, revealed detectable expression for each lncRNA in single oocytes from small (SF) and presumably oestrogen active (EA) follicles at both stages. Average expression of each lncRNA from SF was used as a calibrator in normalization. Effect of follicle size and maturity stage and their interaction on the lncRNA expression were examined using a two-factorial ANOVA followed by Tukey’s HSD post hoc test. In five lncRNAs (2160.1, 5379.1, 18208.1, 25823.1, and 34876.2), maturity stage was significant (P&amp;lt;0.05). Moreover, we found significant interaction of stage and size in expression of lncRNA 25823.1 (P&amp;lt;0.05). SF oocytes at the GV stage had the highest relative expression of lncRNA 25823.1. Specific differences were detected between SF MII and SF GV oocytes, indicating higher expression in the earlier developmental stage (P&amp;lt;0.05). The SF oocytes at MII had a 0.63-fold decrease in relative expression with respect to the SF oocytes at GV. In addition, we found a tendency for significant interaction between maturity stage and follicle size in the expression of lncRNA 5379.1 (P&amp;lt;0.1). LncRNA 5379.1 exhibited the same expression pattern as lncRNA 25823.1 with SF oocytes at MII having a 0.75-fold decrease in relative expression relative to SF oocytes at GV. In summary, both lncRNA 25823.1 and 5379.1 had higher expression in oocytes aspirated from small follicles during the earlier developmental stage. The literature indicates that follicle size can be an indicator of oocyte quality and that RNAs accumulated or degraded during maturation are crucial to achieve developmental competence. Therefore, lncRNAs 25823.1 and 5379.1 shows promise to be linked with oocyte quality.

Proteomic Changes of Porcine Oocytes After Vitrification and Subsequent in vitro Maturation: A Tandem Mass Tag-Based Quantitative Analysis.

Cryopreservation of immature germinal vesicle (GV) oocytes is a promising strategy in pigs but still results in reduced oocyte quality due to inevitable cryodamages. Recently, there has been more focus on the molecular changes of oocytes after vitrification, but the alteration in the proteome level remains elusive. The aim of this study therefore was to decipher the proteomic characteristics of porcine GV oocytes following vitrification and in vitro maturation (IVM) by using tandem mass tag (TMT)-based quantitative approach and bioinformatics analysis. A total of 4,499 proteins were identified, out of which 153 presented significant difference. There were 94 up-regulated and 59 down-regulated proteins expressed differentially in the vitrified oocytes. Functional classification and enrichment analyses revealed that many of these proteins were involved in metabolism, signal transduction, response to stimulus, immune response, complement, coagulation cascades, and so on. Moreover, a parallel reaction monitoring technique validated the reliability of TMT data through quantitative analysis for 10 candidate proteins. In conclusion, our results provided a novel perspective of proteomics to comprehend the quality change in the vitrified porcine GV oocytes after IVM.

Enhanced Cryoprotective Effect of Melatonin and Resveratrol by Coencapsulation: Improved In Vitro Development of Vitrified-Warmed Mouse Germinal Vesicle Oocytes.

Oocyte vitrification, as a vital step in reproductive medicine, is strongly associated with lower development caused by cryodamaging factors, such as oxidative stress. In this study, we evaluated the antioxidative synergistic effects of Melatonin (Mel) and Resveratrol (RES) coencapsulated by solid lipid nanocarriers (SLNs) against the pure antioxidant combination (Mel+RES). In this research, the formation of Mel+RES-SLN was confirmed by Fourier-transformed infrared spectroscopy. The average mean diameter, size distribution, polydispersity index, and zeta potential of particles were measured by Zetasizer, and the morphology was evaluated by scanning electron microscopy. In addition, the encapsulation efficiency (EE%) or drug loading capacity (DL%) of the nanocapsule was determined by spectrophotometric methods. Germinal vesicle (GV)-stage oocytes harvested from 6- to 12-week-old female NMRI mice were randomly divided into seven groups for in vitro studies. In these groups, (0, 10-12 M + 0.5 μM, 10-9 M + 2 μM, or 10-6 M + 10 μM) of Mel+RES/Mel+RES-SLN were added into vitrification media. After thawing, oocytes were matured, fertilized, and cultured for 3 days. Extra/intracellular reactive oxygen species (ROS) levels were measured in in vitro maturation medium after 24 hours. Our results revealed a significant improvement in the normal morphology of warmed GV-stage oocytes, GV breakdown (GVBD) rate, Metaphase II (MII)-stage oocyte formation, fertilization rate, early embryo development, and a significant reduction in intra/extracellular ROS level when vitrification media was supplemented with the lowest Mel+RES-SLN concentration. In vitro studies also demonstrated that the highest concentration of Mel+RES-SLN was safe, without a detrimental effect on embryonic development upon treatment. In conclusion, the lowest concentration of Mel+RES-SLN supplementation in GV-stage oocyte vitrification media improved maturation, fertilization, and embryo development rate and decreased extra/intracellular ROS level through an enhanced/controlled intracellular penetration compared to the pure Mel+RES.

Does the Apoptosis Value of Cumulus Cells Play a Role in Rescue Oocyte in Vitro Maturation?

OBJECTIVE: In this study, we aimed to investigate the role of the cumulus cell’s apoptosis parameter in the maturation of immature rescue oocytes. STUDY DESIGN: In this experimental study, donated immature germinal vesicle oocytes were cultured for, in vitro maturation, embryo development in matured germinal vesicle oocytes were compared with apoptotic properties of cumulus cells. RESULTS: In all of the immature oocytes after oocyte in vitro maturation, the maturation rate has been observed as 56.1% and 2PN rate as 63.0%. Afterin vitro maturation of germinal vesicle oocytes, there was no difference in apoptosis rates of the cumulus cells between mature and immature oocytes (p&gt; 0.05). The ratio of 2PN in matured germinal vesicle oocytes showing embryo development was 35.4%. A positive correlation was found between luteinizing hormone values on day 3 and E2 values during HCG days during oocyte maturation and embryo development (p=0.021, p=0.020). In addition, it has been observed that the germinal vesicle oocytes, which have completed their maturation and developed into embryos, have high E2 values during HCG days (p=0.020).CONCLUSION: In our study, it has been demonstrated that in vitro maturation in rescue oocytes from stimulated cycles, embryo development potential could not be explained by the apoptosis parameter.

In vitro maturation with letrozole priming: Can it be a solution for patients with cancerophobia? A pilot study.

Objective:To investigate whether letrozole priming could be used efficiently in patients undergoing in vitro maturation (IVM) as compared with follicle-stimulating hormone (FSH) priming.Materials and Methods:This is a retrospective analysis of 63 patients who underwent IVM due to the high risk of Ovarian Hyperstimulation syndrome (OHSS) (n=39), cancerophobia (n=16), and desire for IVM after failed in vitro fertilization attempts (n=8). Forty-two patients received FSH priming and 21 patients received letrozole priming.Results:The patients who had FSH or letrozole priming were statistically similar with respect to age, body mass index, duration of infertility, basal antral follicle count, serum anti-Müllerian hormone levels, and IVM indications (p>0.05 for all). When compared with the FSH priming group, the number of germinal vesicle oocytes, metaphase II and fertilized oocytes were significantly higher (p=0.003, p=0.001, and p=0.016, respectively), but the number of metaphase I oocytes was significantly lower in the letrozole priming group (p=0.002). The patients who received FSH and letrozole priming had statistically similar rates of implantation (33.3% vs 37.0%, p=0.709), clinical pregnancy (31.5% vs 33.3%, p=0.848), twinning (1.9% vs 3.7%, p=0.611), and live birth (24.1% vs 29.6%, p=0.682).Conclusion:Potential indications for IVM include patients with increased risk for OHSS and contraindication for hyperestrogenism. Aromatase inhibitors can be used to preserve the fertility of patients with estrogen-sensitive cancers. Letrozole priming appears to be an efficient approach in patients who undergo IVM, with likely less cost than FSH priming.

MDR-1 function protects oocyte mitochondria against the transgenerational effects of nitrogen mustard exposure

Oocytes are vulnerable to alkylating agents like nitrogen mustard (NM), which can cause mitochondrial dysfunction associated with increased oxidative stress. Because mitochondria are maternally inherited, NM exposure affects oocyte mitochondrial physiology and compromises future progeny. Multidrug resistance transporters (MDRs) are transmembrane proteins that efflux such cytotoxic substances; MDR-1 is expressed in oocyte plasma and mitochondrial membranes and protects against oxidative stress. Our objective was to investigate how loss of MDR-1 can modulate oocyte response to NM transgenerationally. Wild Type (WT) and Mdr1a mutant female mice were injected intraperitoneally with sterile saline (control) or 0.1 mg/kg NM. 48 h post-injection, females were either sacrificed for F0 studies or mated with control males to yield F1 pups. After weaning, F1 females were sacrificed or mated to yield F2 pups. Germinal vesicle oocytes were assessed for mitochondrial membrane potential and reactive oxygen species (ROS) levels. NM exposed oocytes of both genotypes exhibited significantly higher ROS than controls in F0 and F1. NM F2 oocytes of neither genotype exhibited significantly higher ROS, though variation in Mdr1a mutants led to an upward trend. NM oocytes of both genotypes exhibited significantly disrupted mitochondrial membrane potential in F0. WT regained normalcy by F1 whereas Mdr1a mutants were unable to by F2. Our data suggest that Mdr1a mutants exhibit transgenerational mitochondrial dysfunction following toxic challenge that persists, implying that MDR-1 protects against toxicant-induced mitochondrial stress. Women without functional MDR-1 exposed to environmental toxicants could therefore be at risk for passing on compromised mitochondria to future offspring.

Comparison of Oocyte and Embryo Quality in Women With Polycystic Ovary Syndrome and the Control Group Candidate for In Vitro Fertilization and Intracytoplasmic Sperm Injection

Objectives: Polycystic ovary syndrome (PCOS) is the most common cause of female infertility. The aim of this study was to compare the oocyte and embryo quality between the PCOS women with the control group candidate for in vitro fertilization/ intracytoplasmic sperm injection (IVF/ICSI). Materials and Methods: The present study was designed at the Infertility Research Center of Milad in the prospective cohort format and was carried out on 100 cases of infertile women with confirmed PCOS (case group) and the male factor (control group) as the first IVF cycle candidates. Both groups underwent the ovary stimulation cycle and ICSI under the standard antagonist protocol. The collected data were then processed and analyzed using the SPSS software, version 16. Results: The average age of study cases was 35±3, and oocyte necrosis was the underlying pathological factor in both groups (28% and 26% in the PCOS and control groups, respectively). In addition, most embryones belonged to either grade 1 or 2 or were 8-cell embryos. Furthermore, the highest number of transferred embryos among the patients was related to the 8-cell and grade 1. The occurrences of biochemical pregnancy in the PCOS and control groups were up to 31.91% and 22%, respectively, leading to 72.73% and 60% childbirth in cases of both groups. Finally, there were no significant differences observed with respect to the quality and the quantity of the embryones, the oocyte, the transferred embryo, the germinal vesicle oocytes, and the rate of pregnancy among the two groups (P&gt;0.05). Conclusions: According to the results of the present study, no differences were found concerning the oocyte quality, embryo, and the pregnancy rate between PCOS cases and any other patients requiring ICSI. Therefore, such cases can similarly benefit from ICSI methods as well.

Ageing and ovarian stimulation modulate the relative levels of transcript abundance of oocyte DNA repair genes during the germinal vesicle-metaphase II transition in mice.

Oocyte quality and reproductive outcome are negatively affected by advanced maternal age, ovarian stimulation and method of oocyte maturation during assisted reproduction; however, the mechanisms responsible for these associations are not fully understood. The aim of this study was to compare the effects of ageing, ovarian stimulation and in-vitro maturation on the relative levels of transcript abundance of genes associated with DNA repair during the transition of germinal vesicle (GV) to metaphase II (MII) stages of oocyte development. The relative levels of transcript abundance of 90 DNA repair-associated genes was compared in GV-stage and MII-stage oocytes from unstimulated and hormone-stimulated ovaries from young (5-8-week-old) and old (42-45-week-old) C57BL6 mice. Ovarian stimulation was conducted using pregnant mare serum gonadotropin (PMSG) or anti-inhibin serum (AIS). DNA damage response was quantified by immunolabeling of the phosphorylated histone variant H2AX (γH2AX). The relative transcript abundance in DNA repair genes was significantly lower in MII oocytes compared to GV oocytes in young unstimulated and PMSG stimulated but was higher in AIS-stimulated mice. Interestingly, an increase in the relative level of transcript abundance of DNA repair genes was observed in MII oocytes from older mice in unstimulated, PMSG-stimulated and AIS-stimulated mice. Decreased γH2AX levels were found in both GV oocytes (82.9%) and MII oocytes (37.5%) during ageing in both ovarian stimulation types used (PMSG/AIS; p < 0.05). In conclusion, DNA repair relative levels of transcript abundance are altered by maternal age and the method of ovarian stimulation during the GV-MII transition in oocytes.

Ceratonia siliqua (Carob) extract improved in vitro development of vitrified-warmed mouse germinal vesicle oocytes: assessment of possible mechanism.

Oocyte banking is a vital step for safekeeping and spreading genetic resources of animals. It is also used for fertility preservation of human. Oocyte vitrification is closely related to the lower developmental competence which includes the cryo-injury arisen during vitrification. The aim of the present study was to evaluate the maturation, embryonic development and production of reactive oxygen species (ROS) of mice oocytes following the supplementation vitrification media with different concentrations of Ceratonia siliqua (carob) extracts. In this experimental study, germinal vesicle oocytes collected from 8 to 10week-old female NMRI mice (30-40gr) were randomly divided into six groups of vitrification media supplemented with 0 (control), 5, 10, 20, 30 and 50µg/ml C. siliqua. After thawing, oocytes were put in an in vitro maturation medium (IVM) (α-MEM: Alpha Minimum Essential Medium). 3-4 and 24h (hr) later, the oocyte nuclear maturity was checked. Standard invitro fertilization was performed on the matured oocytes (MII), and embryonic development was followed. Extra- and intra-cellular ROS was measured in IVM medium after 24h of oocyte incubation. The addition of 20 and 30μg/ml C. siliqua extract to vitrification media improved normal morphology of warmed germinal vesicle (GV) oocytes, rate of germinal vesicle break down (GVBD), and metaphase 2 (MII) oocyte formation significantly (p < 0.05). Fertilization rate, (embryonic development to 2 cells stage, 4-8 cells stage, and > 8 cells stage increased in the 30μg/ml C. siliqua group significantly (p < 0.05). Furthermore, supplementation of 30μg/ml C. siliqua in vitrification media significantly decreased extra- and intra-cellular of ROS as well as embryonic fragmentation (p < 0.05). In conclusion, supplementation of GV oocyte vitrification media with carob extract improved maturation, fertilization, and embryonic development rate and decreased extra- and intra-cellular ROS levels.

Proteomic-based identification of oocyte maturation-related proteins in mouse germinal vesicle oocytes.

Oocyte proteins play an important role in oocyte maturation, fertilization and embryonic development. However, the protein composition of mouse germinal vesicle (GV) oocytes is still unclear. Using one-dimensional Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (1D SDS-PAGE) and Reverse-phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS), we constructed a protein profile of mouse GV oocytes. First, our proteomics profile identified 1,405 different proteins from 11,000 mouse GV oocytes lacking zona pellucida. Second, with detailed bioinformatics analysis, a group of proteins that play an essential role in oocyte maturation was screened. In addition, the expression and localization of suppressor of G2 allele of skp1(SUGT1, also called SGT1), heterogeneous nuclear ribonucleoprotein K(Hnrpk), Seruin, Cullin1(Clu1) and nuclear distribution protein C (Nudc) in mouse ovaries and early embryos were also captured and investigated in this study. Moreover, the protein profile was submitted to the Proteomics Identifications Database (PRIDE) and is available via ProteomeXchange with the identifier PXD014314. Our research provides valuable resources for the study of oocyte proteins and oocyte maturation and helps to clarify the mechanisms of oocyte maturation.

Actinomycin D causes oocyte maturation failure by inhibiting chromosome separation and spindle assembly†.

Actinomycin D (ActD) has been considered as one of the most effective and safe chemotherapeutic medications for treating a number of cancers. Although ActD has been used in the treatment of gynecological tumors and pediatric tumors for more than 50years, the toxic effects of ActD on mammalian oocytes remain unknown. In this study, the influence of ActD on mouse and human oocyte maturation and the possible mechanisms were investigated. Notably, ActD inhibited oocyte maturation and arrested oocytes at the metaphase I (MI) stage in a dose-dependent manner. In addition, ActD arrested oocyte maturation when the oocytes were treated at different successive stages, including the germinal vesicle (GV), germinal vesicle breakdown, and MI stages. In ActD-treated oocytes, disordered chromosome condensation and irregular spindle assembly occurred, resulting in incomplete chromosome segregation and oocytes arresting at the MI phase; these results possibly occurred because ActD triggered the formation of reactive oxygen species, resulting in DNA damage and decreased ATP in mouse GV oocytes. Besides, in vivo treatment with ActD also inhibited mouse oocyte maturation. Similar effects were seen in human oocytes. Collectively, our results indicated that ActD exposure disrupted oocyte maturation by increasing DNA damage, which is a finding that might help with optimizing future methods for female fertility preservation before undergoing chemotherapy.

The impact of transcription inhibition during in vitro maturation on the proteome of bovine oocytes†.

Proper oocyte maturation is a prerequisite for successful reproduction and requires the resumption of meiosis to the metaphase II stage (MII). In bovine oocytes, nuclear maturation has been shown to occur in in vitro maturing cumulus-enclosed oocytes (COCs) in the absence of transcription, but their developmental capacity is reduced compared to transcriptionally competent COCs. To assess the impact of transcription during in vitro maturation of bovine COCs on the quantitative oocyte proteome, a holistic nano-LC-MS/MS analysis of germinal vesicle oocytes and MII oocytes matured with or without addition of the transcription inhibitor actinomycin D (ActD) was carried out. Analyzing eight biological replicates for each of the three groups, a total of 2018 proteins was identified. These could be clearly classified into proteins depending or not depending on transcription during oocyte maturation. Proteins whose abundance increased after maturation irrespective of transcription inhibition - and hence independent of transcription - were related to the cell cycle, reflecting the progression of meiosis, and to cellular component organization, which is crucial for cytoplasmic maturation. In contrast, transcription-dependent proteins were associated with cell-cell adhesion and translation. Since a high rate of protein synthesis in oocytes has been shown to correlate with their developmental competence, oocyte maturation in transcriptionally impaired COCs is apparently disturbed. Our experiments reveal that impaired transcription during in vitro maturation of COCs has a substantial effect on specific components of the oocyte proteome, and that transcription is required for specific classes of oocyte proteins predominantly involved in translation.

Dynamic alterations in H4K12 acetylation during meiotic maturation and after parthenogenetic activation of mouse oocytes.

The aim of the study was to investigate the continuous changing pattern of H4K12 acetylation, and the expression levels of histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs) in mouse oocytes during meiosis and after parthenogenetic activation (PA). The immunofluorescence results showed hyperacetylation of lysine-12 on histone H4 (H4K12) in the germinal vesicle (GV) oocytes that then decreased during germinal vesicle breakdown (GVBD), and disappeared in metaphase II (MII). However, it reappeared in the early 1-cell embryos derived after 4 h of PA. The expression levels of some selected HATs and HDACs also validated the changing pattern of H4K12 acetylation during meiosis and PA. In conclusion, H4K12 is deacetylated in GVBD and MII, and re-hyperacetylated after PA.

Does the repeat dose of gonadotropin-releasing hormone agonist trigger in polycystic ovarian syndrome improve in vitro fertilization cycles outcome? A clinical trial study.

Background A repeat dose of Gonadotropin-releasing Hormone (GnRH) agonist could provide long duration of luteinizing hormone (LH) surge and amplitude appropriately.Objective Improvement in oocyte maturity could be obtained by a repeat dose of GnRH agonist.Materials and MethodsIn this randomized double-blinded study, 120 women with polycystic ovarian syndrome and serum estradiol level (E2) 3000 who were candidate for in vitro fertilization with Antagonist protocol were enrolled between July 2018 and July 2019. Participants were randomized in two groups - and final oocyte maturation was triggered with two doses: In group A, a repeat dose of 0.1 mg, 12 hr. after the first dose and in group B, 0.2 mg SC triptorelin (decapeptyl) 35 hr. prior to oocyte retrieval. Serum Estradiol, LH, and progesterone concentration were measured on the trigger day. Serum LH measurement was done three times in both groups. The outcomes were oocyte yield, meiosis (M) I, MII, Maturity rate, germinal vesicle (GV) rate, 2 pronuclear, embryo yield, ovarian hyper stimulation syndrome rates.ResultsMaturity rate (p = 0.89), MI (p = 0.38), MII (p = 0.89), and GV oocytes (p = 0.38) were not statistically different between the two study groups. LH levels measured at 12 hr post-trigger did not relate statistically significant with maturity rate in our participants (p = 0.96). No empty follicular syndrome was reported.ConclusionAlthough, the second dose of GnRH agonist after 12 hr since the first dose could provide duration of LH surge and amplitude and as a result no empty follicular syndrome was seen, the maturity rate, MI, MII, and GV oocytes were not different between the two study groups.

Maternal ageing causes changes in DNA methylation and gene expression profiles in mouse oocytes.

Although it is well known that maternal ageing causes reduced oocyte quality and fertility, little information is known about its effect on germ cell epigenetics. In the present study, we compared the gene expression and DNA methylation profiles in germinal vesicle oocytes from young (8-week-old) and aged (18-month-old) mice using single-cell RNA-sequencing and single-cell whole-genome DNA methylation sequencing. We found significant differences in the data from the two groups. Oocytes from aged mice showed significant changes in the expression of some metabolism-related genes, such as mitochondria-associated genes, that was in line with our expectations. Expression of some genes associated with reproduction also showed significant differences. DNA methylation levels were also changed in oocytes from aged mice. The two groups had significant gaps in hypermethylation and hypomethylation levels on each chromosome. These data provide useful information for further understanding the mechanisms of oocyte ageing.

Comparing four laboratory three-parent techniques to construct human aged non-surrounded nucleolus germinal vesicle oocytes: A case-control study.

Background The three-parent assisted reproductive technique may increase oocyte competence.Objective In this case-control study, the suitability of germinal vesicle transfer (GVT), synchronous ooplasmic transfer (sOT), asynchronous ooplasmic transfer using cryopreserved MII oocyte (caOT), and asynchronous ooplasmic transfer using waste MII oocyte (waOT) for maturation of the human-aged non-surrounded nucleolus germinal vesicle-stage (NSN-GV) oocyte were investigated.Materials and MethodsNSN-GV oocytes were subjected to four methods: group A (GVT), B (sOT), C (caOT) D (waOT), and E (Control). The fusion rates, MI, MII, ICSI observations and cleavage at 2-cell, 4-cell, and 8-cell stages were compared in the groups.ResultsIn GVT, none of the oocytes fused. In sOT, all oocytes fused, 20 achieved the MI, 14 progressed to MII, 8 fertilized, 6 cleaved and 5, 4, and 3 achieved the 2-cells, 4-cells and 8-cells, respectively. In caOT, all oocytes fused and achieved the MI, 8 progressed to MII and fertilized, 6 cleaved and 6, 5, and 5 achieved the 2-cells, 4-cells, and 8-cells respectively. In waOT, all oocytes fused, 5 and 3 progressed to MI and MII, respectively, but only one fertilized, cleaved and reached a 4-cells stage. In group E, 6 and 2 oocytes progressed to MI and MII, respectively, and only one fertilized but arrested at the zygote stage. caOT had the highest survival rate when compared to sOT (p = 0.04), waOT (p = 0.002), and control (p = 0.001).ConclusionThe caOT method was beneficial over sOT, waOT, and GVT in supplementing the developmental capacity of human-aged NSN-GV oocytes.

Transferrin Insufficiency and Iron Overload in Follicular Fluid Contribute to Oocyte Dysmaturity in Infertile Women With Advanced Endometriosis.

Purpose: To screen out specific protein with different concentration in follicular fluid from advanced endometriosis and determine its direct effect on mouse oocytes matured in vitro.Methods: FF samples were obtained from 25 patients (EMS group, n = 15; control group, n = 10) to screen the differential proteins by using iTRAQ Labeling and 2D LC-MS. Transferrin (TRF) in was found significantly decreased in EMS group, which was verified using ELISA in enlarged FF samples (EMS group, n = 31; control group, n = 27). The contents of ferric ion in FFs were detected by ELISA and TRF saturations were calculated in two groups. Germinal vesicle (GV) oocytes of mouse were maturated in vitro interfered with the FFs in five groups, whose concentrations of TRF were modulated, and maturation in vitro rates were compared among groups.Results: The reduced concentration of TRF with three analogs and increased concentration of ferric ion were found in the FF of the EMS group (p < 0.05). The numerical values of TSAT was 54.8% in EMS group, indicating iron overload in the FF. The EMS-FF showed significantly decreased maturation in vitro rate (p < 0.05) of mouse oocytes, which was improved with the supplementation of TRF, compared with the control-FF. The effect was blocked by the TRF antibody (p < 0.05).Conclusions: Being aware of the relatively small sample size, our results possibly suggest that TRF insufficiency and iron overload in FF from advanced EMS contribute to oocytes dysmaturity, which may be a cause of EMS-related infertility.

The effect of conditioned media on mouse oocytes ultrastructure following in vitro maturation

Optimizing the culture condition and vitrification process during in vitro maturation (IVM) of oocyte has been controversial. This study aimed to evaluate the effect conditioned medium (CM) enriched with human mesenchymal stem cells (hUCM) derived from bone marrow and umbilical cord blood on the ultrastructure of immature mouse oocytes during IVM. Three hundred ninety-two germinal vesicle (GV) oocytes were obtained from mouse ovaries. They were divided into fresh IVM (fIVM) and vitrified IVM (vIVM) groups and cultivated in either alpha Minimum Essential Medium (α-MEM) (control) or CMs supplemented with supernatants derived from cultures of bone marrow or umbilical cord mesenchyme stem cells (MSCs). Then the maturation rate was evaluated in the experimental groups. Twelve in vivo fresh mature oocytes were randomly obtained from uterine tubes as the control group. Finally, 43 matured IVM oocytes were randomly selected for ultra-structural evaluations using transmission electron microscopy (TEM). Oocyte maturation rates were significantly higher in fIVM than vIVM groups. The highest maturation rate was observed in the fIVM umbilical cord MSCs while the lowest rate was related to α-MEM vIVM (86.44% vs 72.72%). There was an increase in the number of mitochondria–vesicle (MV) complexes in IVM groups. Also, the distribution of cortical granules showed the highest decline in α-MEM vIVM groups. The highest aggregations of mitochondria–SER (M-SER) were observed in fIVM umbilical cords. Oolemma had an irregular border with small microvilli in vIVM groups. The Zona pellucida was darker in the vIVM than fIVM groups. In conclusion, umbilical cord MSCs showed a superior efficacy than bone marrow cells for supporting oocyte maturation and conserving its ultrastructure.