A pre-in vitro maturation medium containing cumulus oocyte complex ligand-receptor signaling molecules maintains meiotic arrest, supports the cumulus oocyte complex and improves oocyte developmental competence.

STUDY QUESTION: Can a pre-in vitro maturation (pre-IVM) medium containing signaling molecules rather than chemical/pharmaceutical agents, sustain meiotic arrest and improve developmental competence of in vitro matured oocytes in CF1 outbred mice? SUMMARY ANSWER: A short 2 h period of pre-IVM prevents spontaneous meiotic resumption, improves mitochondria activity in subsequently matured oocytes, and increases developmental competence, pregnancy rate and implantation of resulting embryos. WHAT IS KNOWN ALREADY: Spontaneous resumption of meiosis in vitro is detrimental for oocyte developmental competence. Pre-IVM systems that prevent spontaneous meiotic resumption with chemical/pharmaceutical agents are a promising approach to improving IVM oocyte competence; however, the success of these methods has proven to be inconsistent. STUDY DESIGN, SIZE, DURATION: This study consisted of a series of experiments using cumulus oocyte complexes (COC) derived from outbred mice following ovarian stimulation. The study was designed to examine if a novel, ligand/receptor-based pre-IVM treatment could sustain meiotic arrest in vitro and improve oocyte developmental competence, compared to control IVM. Two pre-IVM durations (2 h and 24 h) were evaluated, and the effect of the mitochondrial stimulator PQQ during 24 h pre-IVM was studied. PARTICIPANTS/MATERIALS, SETTING, METHODS: Murine (outbred CF1) immature COC were cultured in vitro in the presence of C-type natriuretic peptide (CNP) (30 nM), estradiol (100 nM), FSH (1 × 10-4 IU/ml) and bone morphogenic protein 15 (BMP15) (100 ng/ml) for 2 h or 24 h prior to IVM. Meiotic status during pre-IVM and IVM was analyzed using orcein staining, and functionality of gap junction communication was confirmed using the functional gap junction inhibitor carbenoxolone (CBX). Oocytes exposed to pre-IVM treatment were compared to control oocytes collected on the same day from the same females and undergoing standard IVM. Developmental competence and embryo viability was assessed by oocyte mitochondrial activity and ATP concentration, in vitro embryo development following IVF and in vitro culture, blastocyst cell number and allocation, embryo morphokinetics, and embryo transfer. Differences were determined to be significant when P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: Both a short (2 h) and long (24 h) pre-IVM period successfully prevented spontaneous resumption of meiosis. Moreover, gap junctions remained open during the pre-IVM period, as shown by the resumption of meiosis (95.9 ± 2.1%) in the presence of CBX during pre-IVM. A 2 h pre-IVM treatment improved blastocyst development after 96 h of culture per cleaved embryo compared to control (71.9 ± 7.4% versus 53.3 ± 6.2%, respectively), whereas a longer 24 h pre-IVM had no effect on development. A short 2 h period of pre-IVM increased mitochondrial activity in mature oocytes. On the contrary, mitochondrial activity was reduced in mature oocytes following 24 h of arrest and IVM. Treatment of arrested COC with pyrroloquinoline quinone (PQQ) during the 24 h pre-IVM period successfully maintained mitochondrial activity equal to control. However, PQQ was not able to improve blastocyst development compared to pre-IVM 24 h without PQQ. Moreover, ATP concentration in mature oocytes following pre-IVM and/or IVM, did not differ between treatments. A 2 h pre-IVM period prior to IVM improved pregnancy rate following transfer to recipient females. Implantation was also improved after transfer of embryos derived from oocytes arrested for either 2 h or 24 h prior to IVM, compared to control IVM derived embryos (41.9 ± 9%, 37.2 ± 9.5% and 17.2 ± 8.3%, respectively), although fetal development did not differ. LIMITATIONS, REASONS FOR CAUTION: Slower meiotic resumption and enhanced mitochondrial activity likely contribute to improved developmental competence of oocytes exposed to pre-IVM for 2 h, but further experiments are required to identify specific mechanisms. Maintaining oocytes in meiotic arrest for 24 h with this approach could be a potential window to improve oocyte quality. However, an initial attempt to utilize this period of arrest to manipulate quality with PQQ, a mitochondrial stimulator, did not improve oocyte competence. WIDER IMPLICATIONS OF THE FINDINGS: IVM could be an attractive clinical alternative to conventional IVF, with reduced time, cost and reliance on high doses of exogenous hormones to stimulate follicle growth, thus eliminating ovarian hyperstimulation syndrome (OHSS). Currently IVM is not widely used as it results in reduced embryo development and lower pregnancy outcomes compared to embryos produced from in vivo matured oocytes. Our approach to IVM, incorporating a ligand/receptor pre-IVM period, could improve human oocyte quality following IVM leading to routine adoption of this patient friendly technology. In addition, our methodology of pre-IVM containing signaling molecules rather than chemical/pharmaceutical agents may prove to be more consistent at improving oocyte quality than those focusing only on cAMP modulation with pharmacological agents. Finally, a reliable method of maintaining oocytes in meiotic arrest in vitro provides a novel window of opportunity in which the oocyte may be manipulated to address specific physiological deficiencies prior to meiotic resumption. LARGE SCALE DATA: N/A. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Colorado Center for Reproductive Medicine (CCRM, Lone Tree, Colorado USA). We declare no conflict of interest.

Direct and Osmolarity-Dependent Effects of Glycine on Preimplantation Bovine Embryos.

Concentrations of glycine (Gly) in embryo culture media are often lower (~0.1 mM) than those in oviductal or uterine fluids (≥1.2 mM). The objective of this study was to determine direct and osmolarity-dependent effects of physiological concentrations of Gly on blastocyst formation and hatching, cell allocation to the trophectoderm (TE) and inner cell mass (ICM), and metabolic activity of bovine embryos. In experiment 1, zygotes were cultured with 100 or 120 mM NaCl and 0 or 1 mM Gly for the first 72 h of culture. Blastocyst formation and hatching were improved (P<0.05) when embryos were cultured with 100 compared to 120 mM NaCl. Inclusion of 1 mM Gly improved (P<0.05) blastocyst formation compared to 0 mM Gly, but this effect was only significant (P<0.05) for embryos cultured with 120 mM NaCl, suggesting bovine embryos can utilize Gly as an osmolyte. In experiment 2, embryos were cultured with 0.1, 1.1, 2.1, or 4.1 mM Gly (100 mM NaCl) for the final 96 h of culture. Blastocyst development was not affected (P>0.05) by Gly, but hatching (0.1 mM Gly, 18.2%) was improved (P<0.05) when embryos were cultured with 1.1 (31.4%) or 2.1 (29.4%) mM Gly. Blastocyst, TE, and ICM cell numbers were not affected (P>0.05) by Gly in either experiment. Blastocysts produced alanine, glutamine, pyruvate, and urea and consumed aspartate, but this metabolic profile was not affected (P>0.05) by Gly. In conclusion, Gly (1.0 mM) improves the development of both early and late stage embryos, but beneficial effects are more pronounced for early embryos exposed to elevated osmolarity.

Antioxidant supplementation during in vitro culture improves mitochondrial function and development of embryos from aged female mice.

Maternal aging results in reduced oocyte and blastocyst quality, thought to be due, in part, to mitochondrial dysfunction and accumulation of reactive oxygen species. To reduce oxidative stress, the antioxidants α-lipoic acid (ALA; 10µM), α-tocopherol (250µM), hypotaurine (1mM) and N-acetylcysteine (NAC; 1mM), and sirtuin (100ngmL(-1)) were added to embryo culture medium (AntiOX) and compared with a control (CON) without antioxidants to assess blastocyst development after in vitro maturation and fertilisation of oocytes from aged B6D2F1 female mice (13.5 months). Development to the blastocyst stage increased in the AntiOX compared with CON group (87.6% vs 72.7%, respectively; P<0.01), in addition to higher mitochondrial membrane potential and ATP levels in the AntiOX group. Expression of genes associated with oxidative stress (PI3K, FOXO3A and GLRX2) was upregulated in the CON compared with AntiOX group. In addition to AntiOX, a medium containing only NAC and ALA (rAntiOX) was used to culture embryos from young CF1 females (6-8 weeks). More blastocysts developed in the rAntiOX compared with CON group (64.1% vs 43.3%, respectively; P<0.01), although AntiOX (48.0% blastocysts) did not result in improved development in young mice. Antioxidants improved mitochondrial activity, gene expression and development in embryos of older female mice, whereas a reduced level of antioxidants during culture was beneficial to embryos from young mice.