OBJECTIVE: To demonstrate clinical techniques for in vitro maturation (IVM) treatment, including stimulation recommendations, small follicle pick-up procedures, and compact cumulus-oocyte complex (COC) search practice.
METHODS: This video utilizes live-action footage from surgery and embryology practice for a representative IVM treatment cycle, with step-by-step instructions and recommendations for practice procedures.
METHODS: In vitro fertilization (IVF) clinic.
METHODS: Patients undergoing IVM treatment. The patient(s) included in this video gave consent for publication of the video and posting of the video online, including on social media, the journal website, scientific literature websites, and other applicable sites.
METHODS: Identification of treatment cohorts, IVM definitions, and recommendations for stimulation treatments. A visual demonstration of COC extraction techniques from small antral follicles includes tubing, needle types, considerations when using double lumen or sheath needles, needle pressure, ultrasound, needle flushing, and aspiration technique. Visual demonstration of oocyte search and IVM preparation, including filtering follicular aspirate, prevention of COC cooling, identification of compact COCs, and general parameters of different IVM approaches.
METHODS: Clinical techniques for small follicle ovum pick up and compact COC identification for IVM treatment.
RESULTS: Successful IVM treatment of patients can be achieved using minimal ovarian stimulation, effective small follicle retrieval, and efficient compact COC identification with flexibility in approach depending on clinical constraints and preference.
CONCLUSIONS: In vitro maturation treatment is an efficacious and safe treatment for high ovarian reserve and hyper-responding patients undergoing IVF treatment, in which the retrieval of multiple immature COCs and their ex vivo maturation can be achieved with little to no in vivo stimulation. Practice procedures vary between treatment centers and IVM techniques. This video provides practice recommendations paired with a visual demonstration of techniques to assist in standardizing the approach and expanding the practice to more centers.

Zinc plays a crucial role in the growth and reproductive functions of animals. Despite the positive effects of zinc that have been reported in oocytes of cows, pigs, yaks, and other animals, the influence of zinc on sheep is little known. To investigate the effect of zinc on the in vitro maturation of sheep oocytes and subsequent parthenogenesis-activated embryonic development, we added different concentrations of zinc sulfate to the in vitro maturation (IVM) culture medium. The IVM culture medium with zinc improved the maturation of sheep oocytes and the subsequent blastocyst rate after parthenogenesis activation. Notably, it also enhanced the level of glutathione and mitochondrial activity while reducing levels of reactive oxygen species. Thus, zinc addition to the IVM medium improved the quality of oocytes with a positive effect on the subsequent development of oocytes and embryos.

Fibroblast growth factor 10 (FGF10) is an important regulator of the mammalian cumulus-oocyte complex that plays a crucial role in oocyte maturation. In this study, we investigated the effects of FGF10 supplementation on the in vitro maturation (IVM) of buffalo oocytes and its related mechanisms. During IVM, the maturation medium was supplemented with a range of concentrations of FGF10 (0, 0.5, 5, and 50 ng/mL) and the resulting effects were corroborated using aceto-orcein staining, TUNEL apoptosis assay, detection of Cdc2/Cdk1 kinase in oocytes, and real-time quantitative PCR. In matured oocytes, the 5 ng/mL-FGF10 treatment resulted in a significantly increased nuclear maturation rate, which increased the activity of maturation-promoting factor (MPF) and enhanced buffalo oocyte maturation. Furthermore, it treatment significantly inhibited the apoptosis of cumulus cells, while simultaneously promoting its proliferation and expansion. This treatment also increased the absorption of glucose in cumulus cells. Thus, our results indicate that adding an appropriate concentration of FGF10 to a maturation medium during IVM can be beneficial to the maturation of buffalo oocytes and improve the potential of embryo development.

Multiple effects of stress on health have been reported; however, reproductive alterations in oocytes and cumulus cells have not been fully described. In females, chronic stress has been shown to produce alterations in the estrous cycle, to decrease oocyte in vivo maturation, and to increase the percentage of abnormal oocytes. The aim of this study was to evaluate whether the oocytes from chronically stressed female rats could recover and mature in vitro by providing them with all the necessary culture conditions, as well as to evaluate the functionality of the GAP junctions, and the viability and DNA integrity of the cumulus cells, which are crucial for the complete maturation and development of the oocyte. For this, rats were stressed daily by cold water immersion (15 °C) during 15 min for 30 consecutive days. Corticosterone serum levels in rats increased as an indicator of stress. Chronic stress decreased the percentage of in vitro matured oocytes because the cumulus cells presented irreparable damage to their DNA that led to their death, being unable to establish bidirectional communication with the oocyte for its meiotic resumption through the GAP junctions, which were also damaged. These findings could partially explain an association between stress and infertility.

Oocyte in vitro maturation (IVM) has been proposed as an alternative to conventional ovarian stimulation (COS) in subfertile women with polycystic ovary syndrome. To evaluate the effectiveness and safety of IVM compared with COS in women with predicted hyperresponse to gonadotropins, we searched the published literature for relevant studies comparing any IVM protocol with any COS protocol followed by in vitro fertilization or intracytoplasmic sperm injection. A systematic review was undertaken on 3 eligible prospective studies. Live birth rate was not significantly lower after IVM vs. COS (odds ratio [95% confidence interval] of 0.56 [0.32-1.01] overall, 0.83 [0.63-1.10] for human chorionic gonadotropin (hCG)-triggered IVM [hCG-IVM] and 0.45 [0.18-1.13] for non-hCG-triggered IVM [non-hCG-IVM]), irrespective of the stage of transferred embryos. Data from nonrandomized studies generally showed either significantly low or statistically comparable rates of live birth with IVM vs. COS. Most studies have not identified any significant difference between IVM and COS with respect to the rates of obstetric or perinatal complications, apart from a potentially higher rate of hypertensive disorders during pregnancy. The development of offspring from IVM and COS with in vitro fertilization or intracytoplasmic sperm injection appears to be similar. Additional research is needed to identify which patient populations will benefit most from IVM, to define the appropriate clinical protocol, and to develop the optimal culture system.

This study investigated the effects of age and FSH treatment on the ovarian response, follicular fluid (FF) biochemical composition, nuclear maturation, and molecular profile of cumulus-oocytes complexes (COCs) recovered from prepubertal gilts. Thirty-five prepubertal gilts were separated according to age [140 (n = 20) or 160 (n = 15) days], and within each age, the gilts were allotted to receive either 100 mg of FSH [treated; G140+FSH (n = 10) and G160+FSH (n = 7)] or saline solution [control; G140+control (n = 10) and G160+control (n = 8)]. Thus, four experimental groups were included in this study. In the FSH-treated gilts, the percentage of medium follicles increased (P < 0.0001) in the same proportion with which the percentage of small follicles decreased (P < 0.0001). In addition, the glucose concentration in the FF obtained from medium follicles increased (P < 0.05), while that of triglycerides decreased (P < 0.05) in the FSH-treated gilts. The FSH stimulation also improved (P < 0.05) the number of grade I COCs obtained from medium follicles and the meiotic maturation and BCB + rates. FSH treatment only upregulated (P < 0.05) HMGCR expression in immature COCs from prepubertal gilts. The metaphase II and BCB + rates, FF glucose and plasma IGF-1 levels were greater (P < 0.05) in prepubertal gilts at 160 than at 140 days of age. Age had no effect (P > 0.05) on the transcript abundance of the target genes in immature COCs. Hence, oocytes obtained from 140-day-old prepubertal gilts appeared less meiotically competent than those of 160-day-old prepubertal gilts. Our study suggests a possible strategy of using FSH treatment to improve oocyte quantity, quality, and nuclear maturation in 140 and 160-day-old prepubertal gilts.

To study whether fertility preservation strategies using ovarian stimulation or without using it impact long-term disease-free survival of patients with breast cancer.
Retrospective bicentric cohort study.
Two university hospitals.
In this study, 740 women with breast cancer, aged 18-43 years, who received primary fertility preservation between 2013 and 2019 after a diagnosis of localized breast cancer were included.
Overall, 328 patients underwent at least 1 ovarian stimulation cycle (STIM group) and 412 had a technique without hormonal administration (no STIM group).
Disease-free survival and overall survival up to May 2021 were compared between the 2 groups by log-rank test. Cox proportional-hazard regression model was used for multivariable analyses.
Out of the 740 women who underwent fertility preservation, follow-up data were available for 269 women in the STIM group (82%) and 330 (80%) in the no STIM group. Kaplan-Meier estimates of disease-free survival at 4 years were 87.9% (82.8%-92.2%) and 83.1% (78.4%-87.3%) in the STIM and no STIM groups, respectively. After adjustment on prognostic parameters, no significant difference in breast cancer recurrence rate was observed between the STIM and no STIM groups (hazard ratios, 0.83 [0.64-1.08]). Kaplan-Meier estimate of overall survival at 4 years was 97.6% (95.3%-99.2%) and 93.6% (90.9%-95.9%) in the STIM and no STIM groups, respectively. Overall survival was higher in the STIM group than no STIM group (log-rank test). After adjustment on prognostic parameters, the risk of death remained significantly lower in the STIM group (Hazard Ratio, 0.55 [0.35-0.85]).
In our cohort, STIM for fertility preservation in breast cancer did not significantly impact disease-free survival but was associated with higher overall survival. The disease-free survival and overall survival of young patients with breast cancer were not impacted by fertility preservation techniques irrespective of the timing of chemotherapy (neoadjuvant or adjuvant) and the use of ovarian stimulation. Nevertheless, because death and recurrence were rare events, these results should be taken with caution.

Autophagy plays an important role in mammalian oocyte maturation and early embryonic development and rapamycin is well known for inducing autophagy. Although previous studies have reported the effects of rapamycin on oocytes in vitro maturation (IVM) in different species, few studies have been reported on the role of rapamycin in yak oocytes IVM and embryonic development. Therefore, the objective of this study was to examine the effect of rapamycin treatment on yak oocytes IVM and early embryonic development. Specifically, immature yak oocytes during IVM or parthenogenetic (PA) embryos were treated with different rapamycin concentrations to select an optimal dose. Then evaluated its effect on maturation rates, cleavage, and blastocyst formation rates, mitochondrial membrane potential, ROS levels. Related genes and proteins expression in matured oocytes and blastocysts were also evaluated. The results show that 10 nM rapamycin treatment during IVM significantly improved oocyte maturation rates of oocytes and blastocyst formation rates. Treatment with 10 nM rapamycin reduced ROS level but increased mitochondrial membrane potential. Correspondingly, mRNA and protein expressions of LC3, Beclin-1, and Bcl-2 up-regulated while Bax down-regulated in matured yak COCs. When parthenogenetic embryos were treated with different rapamycin concentrations, 10 nM rapamycin treatment showed higher 8-cell and blastocyst formation rates. Also, CDX2, POU5F1, SOX2, and Nanog levels in blastocysts were upregulated. In summary, our findings demonstrate that rapamycin treatment improves oocytes maturation probably by increasing mitochondrial membrane potential, reducing ROS levels, and regulating the apoptosis in mature yak oocytes. Rapamycin treatment also improves embryonic developmental competence in the yak.

The in vitro maturation (IVM) of equine oocytes is still not efficient and does not yield consistent results. The specific requirements of equine oocytes during this process are still largely unknown, which hinders the development of assisted reproductive techniques (ART) in this species. Because the ovarian follicle microenvironment supports oocytes in their acquisition of developmental competence, follicular fluid seems to be a substantial source of bioactive factors that could support the IVM process. Extracellular vesicles (EVs) are cell-secreted molecules in body fluids that are able to deliver molecular signals and transfer genetic information (mRNA, miRNA) between donor and recipient cells. Hence, our hypothesis is that follicular fluid EVs (ffEVs) from small (<20 mm) ovarian follicles can improve the in vitro maturation rate of mare oocytes. To test our hypothesis, equine ovarian follicular fluid was aspirated and ffEVs were isolated by ultracentrifugation, then characterized using nanoparticle tracking analysis and flow cytometry. Additionally, ffEVs were labeled using the ExoGlow-protein EV labeling kit (System Biosciences, Palo Alto, CA). Cumulus-oocyte complexes (COCs) were matured using a one-step method (Method I, continuous culture for 24-38 h) or a two-step method (Method II, initial denudation after 24 h), in the presence (200 μg protein/ml) or absence of ffEVs. The results show the internalization of ffEVs by equine cumulus cells and, for the first time, also by oocytes. The ffEV treatment during two-step culture had a positive effect on the maturation rate of compacted COCs compared to the control group (45.7% and 20.5%, respectively; p < 0.05). No effect of supplementation was observed on the maturation rate during one-step culture. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of small follicles can improve the IVM rate of mare oocytes, suggesting that ffEVs play an important role during this process and may enhance the development of equine ART.

OBJECTIVE: To evaluate the meaning of meiotic maturation kinetics and duration of pronucleus presence (DPP) for parthenogenetic activation outcome.
METHODS: Retrospective study.
METHODS: University hospital.
METHODS: Eight patients with endometrioid adenocarcinoma and 65 patients who underwent in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI).
METHODS: After collection of oocytes from nonstimulated ovaries of patients with endometrioid adenocarcinoma, in vitro maturation (IVM) and parthenogenetic activation performed with time-lapse imaging; after ICSI, embryos similarly incubated with time-lapse imaging.
METHODS: Timing of the release of the first polar body (fPB), DPP, and developmental stage with IVM and parthenogenetic activation; after ICSI, assessment of DPP and preimplantation developmental stage.
RESULTS: With IVM, 55.2% of oocytes matured; 53.1% of fPBs were released within 24 hours, and 46.9% of fPBs were released after 24 hours. Regarding developmental stage, oocytes that released fPB later during IVM tended to develop more than oocytes that released the fPB within 24 hours. For embryos from parthenogenetic activation the DPP was statistically significantly shorter than the DPP of embryos from ICSI. With ICSI, the DPP was statistically significantly shorter in embryos that developed to ≥8 cells than embryos whose final development included ≤7 cells. The development rate in parthenogenetic activation was statistically significantly lower than that in ICSI.
CONCLUSIONS: Embryo development is negatively affected by DPP that is too short or too long. When the DPP was short with parthenogenetic activation, embryo development did not proceed, indicating that DPP is an important determinant of parthenogenetic activation outcomes as with the timing of fPB release.