Oocyte vitrification for fertility preservation is an evolving practice requiring a new mindset: societal, technical, clinical, and basic science-driven evolutions.

Infertility is a condition with profound social implications. Indeed, it is not surprising that evolutions in both medicine and society affect the way in vitro fertilization is practiced. The keywords in modern medicine are the four principles, which implicitly involve a constant update of our knowledge and our technologies to fulfill the "prediction" and "personalization" tasks, and a continuous reshaping of our mindset in view of all relevant societal changes to fulfill the "prevention" and "participation" tasks. A worldwide aging population whose life priorities are changing requires that we invest in fertility education, spreading actionable information to allow women and men to make meaningful reproductive choices. Fertility preservation for both medical and nonmedical reasons is still very much overlooked in many countries worldwide, demanding a comprehensive update of our approach, starting from academia and in vitro fertilization laboratories, passing through medical offices, and reaching out to social media. Reproduction medicine should evolve from being a clinical practice to treat a condition to being a holistic approach to guarantee patients' reproductive health and well-being. Oocyte vitrification for fertility preservation is the perfect use case for this transition. This tool is acquiring a new identity to comply with novel indications and social needs, persisting technical challenges, brand-new clinical technologies, and novel revolutions coming from academia. This "views and reviews" piece aims at outlining the advancement of oocyte vitrification from all these tightly connected perspectives.

Dietary supplementation with green tea extract improves the antioxidant status and oocyte developmental competence in Italian Mediterranean buffaloes.

The aim of this work was to assess the antioxidant status and the developmental competence of oocytes recovered by ovum pick-up (OPU) in Italian Mediterranean buffaloes supplemented with green tea extracts (GTE) for 90 days. Buffalo cows (n = 16) were randomly assigned to a control group receiving no supplement and a treatment group, receiving GTE starting 90 days before OPU, carried out for five consecutive sessions. Blood samples were collected before the start of supplementation with GTE (T0) and at day 45 (T1) and day 90 (T2) of supplementation, to measure ferric reducing activity (FRAP), total antioxidant capacity (TAC), superoxide dismutase (SOD) and catalase (CAT). The antioxidant status of follicles was measured as TAC on the follicular fluid collected from the dominant follicle just prior OPU, coinciding with T2, and at the end of five repeated OPU sessions (T3). Another objective was to assess in vitro the protective effects of green tea extracts on hepatic cells exposed to methanol insult. Different concentrations of GTE (0.5 µM and 1 µM) were tested on cultured hepatic cells and viability, morphology and SOD activity were assessed at 24, 48 and 72 h. Supplementation with GTE increased (P < 0.05) the number of total follicles (8.7 ± 0.5 vs 6.9 ± 0.5), the number and the percentage of Grade A + B cumulus-oocyte complexes (COCs) compared with the control (3.7 ± 0.4 vs 2.3 ± 0.3 and 57.5 ± 4.2 vs 40.4 ± 4.9 %, respectively). Oocyte developmental competence was improved in the GTE group as indicated by the higher (P < 0.05) percentages of Grade 1,2 blastocysts (44.8 vs 29.1 %). In the GTE group, plasma TAC was higher both at T1 and T2, while FRAP increased only at T2, with no differences in SOD and CAT. The TAC of follicular fluid was higher (P < 0.05) in the GTE compared to the control both at T2 and at T3 The in vitro experiment showed that co-treatment with methanol and 1 µM GTE increased (p < 0.01) cell viability at 24 h (P < 0.01), 48 h (P < 0.05) and 72 h (P < 0.01) compared with the methanol treatment co-treatment with 1 µM GTE prevented the decrease in SOD activity observed with methanol at 24 and 48 h of culture. In conclusion, the results of in vivo and in vitro experiments suggest that supplementation with GTE increases buffalo oocyte developmental competence, by improving oxidative status and liver function.

Nutritional Strategies to Promote Bovine Oocyte Quality for In Vitro Embryo Production: Do They Really Work?

The ability of bovine oocytes to reach the blastocyst stage (i.e., embryo with around 150 cells in cattle) in vitro can be affected by technical (e.g., culture medium used) and physiological factors in oocyte donors (e.g., age, breed). As such, the nutritional status of oocyte donors plays a significant role in the efficiency of in vitro embryo production (IVEP), and several nutritional strategies have been investigated in cattle subjected to ovum pick-up (OPU). However, there is no clear consensus on the reliability of nutritional schemes to improve IVEP in cattle. Available evidence suggests that a moderate body condition score (i.e., 3 in a 1-5 scale) in cattle is compatible with a metabolic microenvironment in ovarian follicles that will promote embryo formation in vitro. The usefulness of fatty acid and micronutrient supplementation to improve IVEP in cattle is debatable with the current information available. Overall, the supply of maintenance nutritional requirements according to developmental and productive stage seems to be enough to provide bovine oocyte donors with a good chance of producing embryos in vitro. Future nutrition research in cattle using OPU-IVEP models needs to consider animal well-being aspects (i.e., stress caused by handling and sampling), which could affect the results.

Increased Systemic Antioxidant Power Ameliorates the Aging-Related Reduction in Oocyte Competence in Mice.

Ovarian aging is associated with elevated oxidative stress and diminished oocyte developmental competence. We aimed to determine the impact of systemic antioxidant treatment in aged mice. Female outbred CF-1 mice were aged for 9 months prior to an 8-week 45 mg Euterpe oleracea (açaí) daily supplement. The açaí treatment induced a threefold increase in serum antioxidant power (FRAP) compared to both young and aged mice (p < 0.0001). Compared to young mice, aged mice had fewer oocytes and reduced blastocyst development (p < 0.0001); açaí did not affect the oocyte numbers, but improved blastocyst formation (p < 0.05). Additionally, açaí alleviated the aging-related decrease in implantation potential (p < 0.01). The aged mice showed evidence of elevated ovarian ER stress (increased whole-ovary PDIA4 expression, granulosa cell and oocyte GRP78 expression, and oocyte PDIA4 protein), reduced oocyte mitochondrial quality (higher PRKN activation and mitochondrial DNA oxidative damage), and dysregulated uterine glandular epithelium. Antioxidant intervention was sufficient to lessen these effects of ovarian aging, likely in part by the upregulation of NRF2. We conclude that açaí treatment is a promising strategy to improve ER and mitochondrial function in the ovaries, thereby ameliorating the decreased oocyte competence that occurs with ovarian aging.

Improving the cytoplasmic maturation of bovine oocytes matured in vitro with intracellular and/or extracellular antioxidants is not associated with increased rates of embryo development.

The production of reactive oxygen species (ROS) is a normal process that occurs in the cellular mitochondrial respiratory chain. However, an increase in ROS levels during in vitro production of bovine embryos induces oxidative stress, leading to failed embryonic development. Therefore, we investigated whether supplementation of IVM medium with intracellular (cysteine and cysteamine; C + C) and/or extracellular (catalase; CAT) antioxidants improves the culture system, affects the mitochondrial membrane potential, affects the intracellular levels of ROS and glutathione (GSH) in the bovine oocytes at the end of maturation, and thereby affects the subsequent embryonic development. At the end of IVM, the metaphase II rates were unaffected by the treatments (76.7 ± 1.7% to 80.6 ± 5.2%; P > 0.05). The intracellular ROS levels, expressed in arbitrary fluorescence units, found in the oocytes treated with intracellular antioxidants (C + C and C + C + CAT groups; 1.06, averaged) were as low as those observed in immature oocytes (0 hour: 1.00 ± 0.12). Among mature oocytes, higher (P < 0.05) ROS levels were found in the control group (1.91 ± 0.10) when compared to the ROS levels found in oocytes treated with antioxidants. Intracellular GSH levels in all groups were lower (0.17 ± 0.09 to 0.51 ± 0.05; P < 0.05) than those in immature oocytes (1.00 ± 0.08), although GSH levels in the C + C group (0.51 ± 0.05) were greater (P < 0.05) than in the control, CAT, and C + C + CAT groups (0.23; averaged). The mitochondrial membrane potential in all groups was improved (1.6; averaged; P < 0.05) compared to the membrane potential observed in the immature oocytes (1.00 ± 0.05), with the exception of the C + C group (0.94 ± 0.03). There was no effect (P > 0.05) of antioxidant supplementation on embryonic development to the blastocyst stage (36.1%; averaged); however, there was an increased tendency (P = 0.0689) to obtain a higher blastocyst rate for the C + C + CAT group (47.5 ± 5.6%) compared to the control group (29.9 ± 4.8%). In conclusion, despite improvements in specific parameters of cytoplasmic maturation, the addition of intracellular and/or extracellular antioxidants during IVM did not affect embryo development.

N-3 polyunsaturated fatty acid DHA during IVM affected oocyte developmental competence in cattle.

The positive effect of n-3 polyunsaturated fatty acids (FAs) on fertility in ruminants seems to be partly mediated through direct effects on the oocyte developmental potential. We aimed to investigate whether supplementation with physiological levels of docosahexaenoic acid (DHA, C22:6 n-3 polyunsaturated fatty acids) during IVM has an effect on oocyte maturation and in vitro embryo development in cattle. We reported that DHA (0, 1, 10, or 100 µM) had no effect on oocyte viability or maturation rate after 22-hour IVM. Incubation of oocyte-cumulus complexes with 1-µM DHA during IVM significantly increased (P < 0.05) oocyte cleavage rate as compared with control (86.1% vs. 78.8%, respectively) and the greater than 4-cell embryo rate at Day 2 after parthenogenetic activation (39.1% vs. 29.7%, respectively). Supplementation with 1 µM DHA during IVM also induced a significant increase in the blastocyst rate at Day 7 after IVF as compared with control (30.6% vs. 17.6%, respectively) and tended to increase the number of cells in the blastocysts (97.1 ± 4.9 vs. 81.2 ± 5.3, respectively; P = 0.08). On the contrary, 10-µM DHA had no effects, whereas 100-µM DHA significantly decreased the cleavage rate compared with control (69.5% vs.78.8%, respectively) and the greater than 4-cell embryo rate at Day 2 after parthenogenetic activation (19.5% vs. 29.7%). As was shown by real-time polymerase chain reaction, negative effects of 100-µM DHA were associated with significant increase of progesterone synthesis by oocyte-cumulus complexes, a three-fold increase in expression level of FA transporter CD36 and a two-fold decrease of FA synthase FASN genes in cumulus cells (CCs) of corresponding oocytes. Docosahexaenoic acid at 1 and 10 µM had no effect on expression of those and other key lipid metabolism-related genes in CC. In conclusion, administration of a low physiological dose of DHA (1 µM) during IVM may have beneficial effects on oocyte developmental competence in vitro without affecting lipid metabolism gene expression in surrounding CCs, contrarily to 100 µM DHA which diminished oocyte quality associated with perturbation of lipid and steroid metabolism in CC.