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PurposeDetermine if the gene expression profiles of ovarian support cells (OSCs) and cumulus-free oocytes are bidirectionally influenced by co-culture during in vitro maturation (IVM).MethodsFertility patients aged 25 to 45 years old undergoing conventional ovarian stimulation donated denuded immature oocytes for research. Oocytes were randomly allocated to either OSC-IVM culture (intervention) or Media-IVM culture (control) for 24–28 h. The OSC-IVM culture condition was composed of 100,000 OSCs in suspension culture with human chorionic gonadotropin (hCG), recombinant follicle stimulating hormone (rFSH), androstenedione, and doxycycline supplementation. The Media-IVM control lacked OSCs and contained the same supplementation. A limited set of in vivo matured MII oocytes were donated for comparative evaluation. Endpoints consisted of MII formation rate, morphological and spindle quality assessment, and gene expression analysis compared to in vitro and in vivo controls.ResultsOSC-IVM resulted in a statistically significant improvement in MII formation rate compared to the Media-IVM control, with no apparent effect on morphology or spindle assembly. OSC-IVM MII oocytes displayed a closer transcriptomic maturity signature to IVF-MII controls than Media-IVM control MII oocytes. The gene expression profile of OSCs was modulated in the presence of oocytes, displaying culture- and time-dependent differential gene expression during IVM.ConclusionThe OSC-IVM platform is a novel tool for rescue maturation of human oocytes, yielding oocytes with improved nuclear maturation and a closer transcriptomic resemblance to in vivo matured oocytes, indicating a potential enhancement in oocyte cytoplasmic maturation. These improvements on oocyte quality after OSC-IVM are possibly occurring through bidirectional crosstalk of cumulus-free oocytes and ovarian support cells.

PurposeThe main purpose and research question of the study are to compare the efficacy of high-security closed versus open devices for human oocytes’ vitrification.MethodsA prospective randomized study was conducted. A total of 737 patients attending the Infertility and IVF Unit at S.Orsola University Hospital (Italy) between October 2015 and April 2020 were randomly assigned to two groups. A total of 368 patients were assigned to group 1 (High-Security Vitrification™ - HSV) and 369 to group 2 (Cryotop® open system). Oocyte survival, fertilization, cleavage, pregnancy, implantation, and miscarriage rate were compared between the two groups.ResultsNo statistically significant differences were observed on survival rate (70.3% vs. 73.3%), fertilization rate (70.8% vs. 74.9%), cleavage rate (90.6% vs. 90.3%), pregnancy/transfer ratio (32.0% vs. 31.8%), implantation rate (19.7% vs. 19.9%), nor miscarriage rates (22.1% vs. 21.5%) between the two groups. Women’s mean age in group 1 (36.18 ± 3.92) and group 2 (35.88 ± 3.88) was not significantly different (P = .297). A total of 4029 oocytes were vitrified (1980 and 2049 in groups 1 and 2 respectively). A total of 2564 were warmed (1469 and 1095 in groups 1 and 2 respectively). A total of 1386 morphologically eligible oocytes were inseminated by intracytoplasmic sperm injection (792 and 594 respectively, P = .304).ConclusionsThe present study shows that the replacement of the open vitrification system by a closed one has no impact on in vitro and in vivo survival, development, pregnancy and implantation rate. Furthermore, to ensure safety, especially during the current COVID-19 pandemic, the use of the closed device eliminates the potential samples’ contamination during vitrification and storage.

PurposeTo investigate the effectiveness of a biphasic IVM culture strategy at improving IVM outcomes in oocytes from small follicles (< 6 mm) compared with routine Standard IVM in patients with polycystic ovaries.MethodsThis prospective pilot study was performed in 40 women with polycystic ovaries whose oocytes were randomized to two IVM culture methods. Patients received a total stimulation dose of 450 IU rFSH. Cumulus-oocyte complexes (COCs) from follicles < 6 mm and ≥ 6 mm were retrieved and cultured separately in either a prematuration medium with c-type natriuretic peptide followed by IVM (CAPA-IVM), or STD-IVM. Primary outcomes were maturation rate, embryo quality, and the number of vitrified day 3 embryos per patient.ResultsUse of the CAPA-IVM system led to a significant improvement in oocyte maturation (p < 0.05), to a doubling in percentage of good and top-quality day 3 embryos per COC, and to an increased number of vitrified day 3 embryos (p < 0.001), compared to STD IVM. Oocytes from follicles < 6 mm benefited most from CAPA-IVM, showing a significant increase in the amount of good and top-quality embryos compared to STD IVM. CAPA-IVM yielded significantly (p < 0.0001) less GV-arrested oocytes and larger oocyte diameters (p < 0.05) than STD IVM.ConclusionsCAPA-IVM brings significant improvements in maturation and embryological outcomes, most notably to oocytes from small antral follicles (< 6 mm), which can be easily retrieved from patients with a minimal ovarian stimulation. The study demonstrates the robustness and transferability of the CAPA-IVM method across laboratories and populations.

The first author's name is spelled incorrectly. (2014) Mitogenomes of Polar Bodies and Corresponding Oocytes. (2014) Mitogenomes of Polar Bodies and Corresponding Oocytes.

PurposeThe aim of the present study was to improve the in vitro maturation (IVM) procedure using oocytes from surplus ovarian tissue after fertility preservation.MethodsTwenty-five patients aged 17–37 years were included in the study. Maturation was compared between oocytes collected in HEPES-buffered medium or saline, and we determined whether transport on ice prior to oocyte collection affected maturation. Two different IVM media were used that were supplemented with and without recombinant human midkine. Mature oocytes were assessed for aneuploidy using next-generation sequencing (NGS).ResultsOn average, 36 immature oocytes were collected from each patient (range 7–90, N = 895). Oocytes recovered from HEPES-buffered medium matured at a higher rate than oocytes recovered from saline (36% vs 26%, p < 0.01). Ovarian transportation on ice prior to the procedure negatively affected maturation compared with non-transported samples (42% vs 27%, p < 0.01). The addition of midkine improved maturation rate (34% vs 27%, p < 0.05). On average, 11 MII oocytes were obtained per patient (range 1–30). NGS of 53 MII oocytes and their first polar bodies indicated that 64% were euploid.ConclusionsThe study demonstrated unexpectedly high number of immature oocytes collected from surplus ovarian tissue without any stimulation. The overall MII rate was one in three, resulting in a total number of MII oocytes that was similar to the number obtained after ovarian stimulation. If these MII oocytes prove suitable for IVF, they will provide a substantial improvement in fertility preservation for patients and advance IVM as an interesting platform for further improvements in assisted reproduction.

PurposeStandard oocyte in vitro maturation (IVM) usually results in lower pregnancy rates than in vitro fertilization (IVF). IVM preceded by a prematuration step improves the acquisition of oocyte developmental competence and can enhance embryo quality (EQ). This study evaluated the effectiveness of a biphasic culture system incorporating prematuration and IVM steps (CAPA-IVM) versus standard IVM in women with polycystic ovarian morphology (PCOM).MethodsEighty women (age < 38 years, ≥ 25 follicles of 2–9 mm in both ovaries, no major uterine abnormalities) were randomized to undergo CAPA-IVM (n = 40) or standard IVM (n = 40). CAPA-IVM uses two steps: a 24-h prematuration step with C-type natriuretic peptide-supplemented medium, then 30 h of culture in IVM media supplemented with follicle-stimulating hormone and amphiregulin. Standard IVM was performed using routine protocols.ResultsA significantly higher proportion of oocytes reached metaphase II at 30 h after CAPA-IVM versus standard IVM (63.6 vs 49.0; p < 0.001) and the number of good quality embryos per cumulus-oocyte complex tended to be higher (18.9 vs 12.7; p = 0.11). Clinical pregnancy rate per embryo transfer was 63.2% in the CAPA-IVM versus 38.5% in the standard IVM group (p = 0.04). Live birth rate per embryo transfer was not statistically different between the CAPA-IVM and standard IVM groups (50.0 vs 33.3% [p = 0.17]). No malformations were reported and birth weight was similar in the two treatment groups.ConclusionsUse of the CAPA-IVM system significantly improved maturation and clinical pregnancy rates versus standard IVM in patients with PCOM. Furthermore, live births after CAPA-IVM are reported for the first time.

PurposeTo determine how clinical, demographic, and laboratory characteristics influence ovarian tissue oocyte quality.MethodsImmature cumulus-oocyte complexes were isolated from removed ovaries and cultured for 48–52 h in either monophasic standard or biphasic CAPA media for fertility preservation. A total of 355 MII oocytes from 53 patients were described for intracytoplasmic and extracytoplasmic anomalies. Multiple clinical, laboratory, and demographic characteristics were analyzed. Statistically significant differences between independent groups in qualitative variables were identified using Pearson’s χ2 and Fisher’s exact tests. The diagnostic value of quantitative variables was assessed using the ROC curve analysis. Factors associated with the development of dysmorphism, taking patient age into account, were identified using the binary logistic regression analysis.ResultsDysmorphisms were observed in 245 oocytes (69.0%), with a median number of dysmorphisms of 2. Oocyte dysmorphisms were found to be 2.211 times more likely to be detected in patients with ovarian cancer, while the presence of dark-colored cytoplasm was associated with gynecologic surgery in the anamnesis (p = 0.002; OR 16.652; 95% CI, 1.977–140.237; Cramer’s V 0.187). Small polar bodies developed 2.717 times more often (95% CI, 1.195–6.18) in patients older than 35. In the case of ovarian transportation on ice at 4 ℃, the chances of development of cytoplasmic granularity increased 2.569 times (95% CI, 1.301–5.179). The use of biphasic CAPA IVM media contributed to a decrease in the probability of large polar body formation (p = 0.034) compared to the standard monophasic IVM media.ConclusionsBoth patients’ characteristics and laboratory parameters have an impact on the quality of IVM ovarian tissue oocytes.

Oocytes form before birth and remain viable for several decades before fertilization 1 . Although poor oocyte quality accounts for most female fertility problems, little is known about how oocytes maintain cellular fitness, or why their quality eventually declines with age 2 . Reactive oxygen species (ROS) produced as by-products of mitochondrial activity are associated with lower rates of fertilization and embryo survival 3 – 5 . Yet, how healthy oocytes balance essential mitochondrial activity with the production of ROS is unknown. Here we show that oocytes evade ROS by remodelling the mitochondrial electron transport chain through elimination of complex I. Combining live-cell imaging and proteomics in human and Xenopus oocytes, we find that early oocytes exhibit greatly reduced levels of complex I. This is accompanied by a highly active mitochondrial unfolded protein response, which is indicative of an imbalanced electron transport chain. Biochemical and functional assays confirm that complex I is neither assembled nor active in early oocytes. Thus, we report a physiological cell type without complex I in animals. Our findings also clarify why patients with complex-I-related hereditary mitochondrial diseases do not experience subfertility. Complex I suppression represents an evolutionarily conserved strategy that allows longevity while maintaining biological activity in long-lived oocytes. Oocytes prevent the production of reactive oxygen species by remodelling the mitochondrial electron transport chain through elimination of complex I, a strategy that enables their long-term viability.

Advanced maternal age has been reported to impair oocyte quality; however, the underlying mechanisms remain to be explored. In the present study, we identified the lowered NAD+ content and decreased expression of NMNAT2 protein in oocytes from old mice. Specific depletion of NMNAT2 in mouse oocytes disturbs the meiotic apparatus assembly and metabolic activity. Of note, nicotinic acid supplementation during in vitro culture or forced expression of NMNAT2 in aged oocytes was capable of reducing the reactive oxygen species (ROS) production and incidence of spindle/chromosome defects. Moreover, we revealed that activation or overexpression of SIRT1 not only partly prevents the deficient phenotypes of aged oocytes but also ameliorates the meiotic anomalies and oxidative stress in NMNAT2‐depleted oocytes. To sum up, our data indicate a role for NMNAT2 in controlling redox homeostasis during oocyte maturation and uncover that NMNAT2‐ NAD+‐SIRT1 is an important pathway mediating the effects of maternal age on oocyte developmental competence. Loss of NAD+ content and NMNAT2 protein results in the meiotic abnormalities and metabolic dysfunction in oocytes from old mouse. NA supplement and SIRT1 overexpression/activation could partly rescue the defective phenotype of these aged oocytes.

The ultrastructure of human oocytes has been described only qualitatively. To offer a precise organelle spatial distribution and organelle volume during the main maturation stages, we previously conducted stereological studies on prophase-I (GV) and metaphase-I (MI) oocytes, and here we present results on metaphase-II (MII) oocytes. Five donor oocytes from different donors were processed for transmission electron microscopy, and quantification of organelle distribution was performed using point-counting stereology. Statistical tests compared the means of the relative volumes occupied by organelles among oocyte regions. The most abundant organelles were elements of the smooth endoplasmic reticulum (SER), such as SER small vesicles, SER medium vesicles, SER large vesicles and SER isolated tubules, along with mitochondria, followed by SER tubular aggregates, cortical vesicles and lysosomes. Significant differences between oocyte regions were found for lysosomes, cortical vesicles and SER large vesicles. Comparisons of MII oocytes to previous findings in GV and MI oocytes evidenced specific patterns of organelle distribution and relative volumes. This final evaluation thus enables to track organelle spatial reorganization across oocyte stages, which, in addition to gathered knowledge, may be useful to assist in improvements of stimulation protocols, in-vitro maturation media and cryopreservation techniques.