P–439 Carbohydrate metabolism profile during oocyte final maturation reveals culture induced aberrations in in vitro grown and matured mouse antral follicles

Study question Are there significant differences in carbohydrate metabolism trends between in vivo and in vitro grown mouse antral follicles during oocyte final maturation? Summary answer Glucose metabolism characterization during GV to MII transition revealed altered metabolic patterns mainly in cumulus cells of in vitro grown and matured mouse antral follicles. What is known already For some cancer patients fertility restoration is dependent on using efficient in vitro follicle culture systems. As human donor ovarian tissue available for research is limited, establishing such culture systems relies on data generated from animal models. The culture system previously developed in our laboratory supports in vitro growth of mouse preantral follicles with good oocyte maturation rates but lower developmental competence compared to in vivo grown oocytes. Tracking and comparing the metabolic changes after meiotic maturation in in vitro and in vivo follicles could serve as a screening tool for improving culture conditions and identifying metabolic quality markers. Study design, size, duration Mouse secondary follicle culture was performed. In vitro grown oocytes, their corresponding cumulus (CC) and granulosa cells (GC) were collected from antral follicles, at germinal vesicle stage (GV) on day 9, and at metaphase 2 (MII) on day 10, after hCG/EGF stimulation. In vivo age-matched controls were obtained after intraperitoneal injections with eCG for GV, or with eCG and hCG for MII. In vivo GC after ovulation were not included. Participants/materials, setting, methods Glucose metabolism trends were compared during final maturation between in vitro grown antral follicles and their in vivo controls. Follicles that failed to resume meiosis in vitro were also included. Enzymatic spectrophotometric assays were used to measure glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, and the antioxidant capacity in individual cell types. Pools of 5 oocytes and corresponding somatic cells were collected, from 3 independent experiments. Unpaired t-test was performed with significance when p < 0.05. Main results and the role of chance Important differences were detected between in vivo and in vitro conditions. GV to MII transition in in vivo follicles leads to a metabolic boost in CC as indicated by: i. significant increase in glycolysis, PPP and TCA cycle activity; ii. higher total antioxidant capacity (TAC) (p < 0.05) and small molecule antioxidant capacity (SMAC) (p < 0.01). After ovulation, the only significant change in oocytes was an increase in nicotinamide adenine dinucleotide phosphate (NADP+) level (p < 0.01), possibly due to increased reduced-NADP recycling. Meiotic maturation triggered no significant differences in any of the metabolic pathways for in vitro grown oocytes. Contrary to their in vivo controls, in vitro CC showed significant upregulations limited to aconitase, lactate dehydrogenase (LDH) and glutathione-s-transferase (GST) activity (p < 0.05). In vitro GC showed increased G6PDH activity (p < 0.05), suggesting PPP upregulation. Significant differences were detected between in vivo GV follicles and the in vitro failed-to-mature ones. Oocytes from impaired follicles have higher NADP+ levels (p < 0.0001) than their in vivo immature counterparts. CC showed higher phosphofructokinase (PFK), LDH, catalase activity and increased NADP + (p < 0.01), TAC and SMAC (p < 0.05) compared to in vivo GV CCs. GCs from failed-to-mature follicles have significantly higher LDH and superoxide dismutase (SOD) activity than in vivo GV GC (p < 0.05). Limitations, reasons for caution The altered metabolic patterns described here in in vitro follicles during oocyte GV to MII transition are probably the cumulative effects of both growth and maturation in vitro. Wider implications of the findings: We explored extensively and directly, for the first time, several enzymes and metabolites involved in follicle glucose and redox metabolism in different cell types separately. Understanding of the follicle metabolic requirements is essential for the optimization of follicle culture systems and could lead to development of oocyte quality markers. Trial registration number Not applicable

Reversing complete mechanical transzonal projections disruption during mouse in vitro follicle culture with unaltered oocyte competence

In vitro oocyte growth is widely studied as an alternative fertility preservation approach. Several animal models are used to generate extensive information on this complex process regulated by the constant and dynamic interaction between the oocyte and its somatic compartment throughout follicle growth and maturation. A 2-dimensional (2D) attachment mouse secondary follicle culture system was used to assess the oocyte’s capacity to overcome disconnection from its somatic companions at different developmental stages for final competence acquisition. To test this, complete mechanical denudation of oocytes from preantral and early antral follicles was performed. Established endpoints were the oocyte’s potential to reconnect with somatic cells and the impact of connectivity disruption on mature oocyte quality. This study proves that oocytes from preantral and early antral cultured mouse follicles can overcome complete denudation, restoring likely functional transzonal projections (TZPs) with no significant differences in meiotic and developmental competence compared to those from intact cultured follicles. These novel findings constitute good premises for developing successful strategies to rescue human oocyte competence in the context of in vitro culture approaches such as non-hCG triggered in vitro maturation (IVM).

In vitro ovarian follicle growth: a comprehensive analysis of key protocol variables†

Folliculogenesis is a complex process that requires integration of autocrine, paracrine, and endocrine factors together with tightly regulated interactions between granulosa cells and oocytes for the growth and survival of healthy follicles. Culture of ovarian follicles is a powerful approach for investigating folliculogenesis and oogenesis in a tightly controlled environment. This method has not only enabled unprecedented insight into the fundamental biology of follicle development but also has far-reaching translational applications, including in fertility preservation for women whose ovarian follicles may be damaged by disease or its treatment or in wildlife conservation. Two- and three-dimensional follicle culture systems have been developed and are rapidly evolving. It is clear from a review of the literature on isolated follicle culture methods published over the past two decades (1980–2018) that protocols vary with respect to species examined, follicle isolation methods, culture techniques, culture media and nutrient and hormone supplementation, and experimental endpoints. Here we review the heterogeneity among these major variables of follicle culture protocols.

Optimization of Novel Follicle Culture System to Generate Developmentally Competent Oocytes

This study aimed to develop a novel culture system for porcine ovarian follicles that yields developmentally competent oocytes. We mechanically isolated ovarian follicles of various sizes 325–500 mm and treated them with ovine follicle stimulating hormone OFSH at different concentrations 0–400 mIU. Follicle diameter, antrum formation and cumulus oocyte complex COC recovery rate were significantly higher p andlt; 0.05 under the 0 and 50 mIU OFSH treatments compared with the remaining concentrations 100, 200 and 400 mIU. Additionally, follicles cultured for 3 and 4 d differed significantly p andlt; 0.05 in follicle diameter, antrum formation rate and COC recovery from those cultured for 5 and 6 d. Follicle characteristics did not differ across diameter: those at 250–300, 301–400 and 401–500 mm in vitro had antrum formation rates of 90%, 92% and 90%, along with COC recovery of 78%, 82% and 85%, respectively. Furthermore, nuclear maturation percentages for oocytes that experienced germinal vesicle breakdown (GVBD) were 10%, 13% and 14%, depending on the size of the originating follicle (250–300, 301–400 and 401–500 mm). Nuclear maturation for metaphase II (MII) oocytes derived from follicles of those three sizes were 1%, 2% and 1%, respectively. After 3 d of culture, the 250–300 mm group differed significantly from other size groups in follicle diameter and COC recovery. This study provides insight into establishing effective protocols of ovarian follicle culture, thus improving efforts to preserve large-mammal fertility.

Evaluating the Effects of Different Concentrations of Human Follicular Fluid on Growth, Development, and PCNA Gene Expression of Mouse Ovarian Follicles

Follicle culture in vitro provides a method for investigating stages of folliculogenesis that can lead to preserving fertility through cryopreservation techniques. This study aims to assess the effects of various concentrations of human follicular fluid (hFF) on growth, development, and expression of the proliferating cell nuclear antigen (PCNA) gene in mouse ovarian follicles in vitro. Preantral follicles were isolated from 14-day NMRI mouse ovaries. The follicles were cultured in basic media enriched with FBS, FSH, and insulin-transferrin-selenium, and supplemented with different concentrations of hFF (10, 20, and 30%) for 12 days. During the culture period, survival rate and follicular maturation, follicular diameter, levels of estrogen and progesterone secretion, and PCNA gene expression rate were evaluated. Survival rate, maturation, and antrum formation were significantly higher in the 10% hFF group than in the 20 and 30% hFF groups. On day 4, follicle diameter in the 10% hFF group was also higher than in the 20 and the 30% hFF group. In comparison with other groups, significantly higher estrogen and progesterone production levels were measured in the 10% hFF group. PCNA gene expression was also higher with 10 than 20 and 30% hFF concentrations. The present study suggests that addition of 10% hFF to mice ovarian preantral follicle culture media enhances follicle growth and oocyte maturation.

Ovarian Follicles Rescued 3 Days after Cyclophosphamide Treatment in Adolescent Mice: An Experimental Study Aiming at Maximizing Methods for Fertility Preservation through In Vitro Follicle Culture

There is currently a lack of knowledge about the feasibility of performing procedures for fertility preservation after chemotherapy treatment has been initiated. In this experimental controlled study using adolescent mice, we aimed to investigate if the chance of rescuing and growing in vitro secondary follicles (SeF) would be affected three days after a single injection of cyclophosphamide (CPA). The main outcomes included were: (1) The number of SeF with good morphologic quality obtained per ovary 3 days after CPA injection, (2) SeF development in culture, (3) small follicle density (SFD) on histology, and (4) apoptosis markers, including terminal deoxynucleotidyl transferase dUTP nick end-labelling (TUNEL), mRNA expression, and distribution of p 53 upregulated modulator of apoptosis (Puma) and phosphatase and tensin homolog (Pten). We found a 60% reduction of SeF obtained per ovary in all CPA-treated groups vs. controls. However, in vitro survival rates at culture day 12 and antrum formation were similar among all groups. On histology, SFD was only significantly reduced in the high CPA dose group. Apoptotic cells were mainly found in large growing follicles of CPA groups. Our study indicates the feasibility of SeF isolation and in vitro follicle culture 3 days following CPA treatment and a still preserved SFD, particularly following a low-dose CPA treatment.