Antioxidants improve IVF outcome and subsequent embryo development in the mouse.

STUDY QUESTION: What is the effect of a combination of three antioxidants (Acetyl-L-Carnitine, N-Acetyl-L-Cysteine and α-Lipoic Acid), present in IVF medium during mouse oocyte and sperm collection, on fertilization and subsequent IVF embryo development? SUMMARY ANSWER: A combination of antioxidants resulted in faster developmental times from the 2-cell stage through to expanded blastocyst stage, accompanied by a significant increase in blastocyst cell number and a reduction of intracellular hydrogen peroxide (H2O2) levels. WHAT IS KNOWN ALREADY: The antioxidant combination Acetyl-L-Carnitine, N-Acetyl-L-Cysteine and α-Lipoic Acid, when present in embryo culture media, has a significant beneficial effect on in vitro fertilized mouse pronucleate oocyte development, especially under oxidative stress. STUDY DESIGN, SIZE, DURATION: IVF was conducted with combined antioxidants supplemented in IVF medium that was used for mouse oocyte collection and fertilization (oocyte IVF medium, 4 h exposure) and sperm collection and preparation (sperm IVF medium, 1 h exposure). PARTICIPANTS/MATERIALS, SETTINGS, METHODS: IVF was conducted under 20% oxygen, in the presence or absence of a combination of antioxidants (10 µM Acetyl-L-Carnitine, 10 µM N-Acetyl-L-Cysteine, 5 µM α-Lipoic Acid) and resultant embryos cultured with and without antioxidants under 20% oxygen. Subsequently, the effects of antioxidants on either oocytes or sperm was evaluated. Embryo development was analysed through time-lapse microscopy followed by differential nuclear staining to determine cell allocation in the blastocyst. Intracellular levels of H2O2 were assessed using an aryl boronate probe after 4 h of incubation with antioxidants. Controls were gametes and embryos that had no antioxidants in the medium. In a separate series of experiments, pronucleate oocytes were collected in handling medium with and without antioxidants for 20 min and subsequent cell numbers analysed. MAIN RESULTS AND THE ROLE OF CHANCE: Antioxidant treatment during both IVF and culture resulted in significantly faster development times to two cell cleavage (P < 0.01), which continued through to the expanded blastocyst stage (P < 0.05). Resultant blastocysts had a significant increase in both trophectoderm (TE) cell numbers, inner cell mass (ICM) and total cell numbers (P < 0.001). The addition of antioxidants to IVF medium or embryo culture media exclusively also resulted in a significant increase in both blastocyst TE and ICM numbers leading to an increase in total cell numbers (P < 0.001). Antioxidant supplementation of either oocyte IVF medium alone, or in both oocyte and sperm IVF medium, lead to significantly faster times to two cell cleavage, which continued through to the expanded blastocyst stage. Blastocyst cell number in both these groups had significantly higher TE cell numbers resulting in an increase in total cell numbers. In contrast, there were no differences in embryo developmental rates and blastocyst cell number when antioxidants were present only in the sperm IVF medium. Levels of H2O2 were significantly reduced in pronucleate oocytes that were cultured in the presence of antioxidants (P < 0.001) compared to control, untreated embryos. Similarly, pronucleate oocytes treated with the combined antioxidants during pronucleate oocyte collection resulted in significantly increased blastocyst ICM numbers compared with controls (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Embryo development was only examined in the mouse. WIDER IMPLICATIONS OF THE FINDINGS: These findings suggest that supplementation of antioxidants to the IVF medium, as well as to embryo culture media, may further assist in maintaining the viability of human embryos in ART, conceivably through the reduction of oxidative stress. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by a research grant from Vitrolife AB (Sweden). The authors have no conflict of interest to declare.

Endometrial signals improve embryo outcome: functional role of vascular endothelial growth factor isoforms on embryo development and implantation in mice.

STUDY QUESTION: Does vascular endothelial growth factor (VEGF) have important roles during early embryo development and implantation? SUMMARY ANSWER: VEGF plays key roles during mouse preimplantation embryo development, with beneficial effects on time to cavitation, blastocyst cell number and outgrowth, as well as implantation rate and fetal limb development. WHAT IS KNOWN ALREADY: Embryo implantation requires synchronized dialog between maternal cells and those of the conceptus. Following ovulation, secretions from endometrial glands increase and accumulate in the uterine lumen. These secretions contain important mediators that support the conceptus during the peri-implantation phase. Previously, we demonstrated a significant reduction of VEGFA in the uterine cavity of women with unexplained infertility. Functional studies demonstrated that VEGF significantly enhanced endometrial epithelial cell adhesive properties and embryo outgrowth. STUDY DESIGN, SIZE, DURATION: Human endometrial lavages (n = 6) were obtained from women of proven fertility. Four-week old Swiss mice were superovulated and mated with Swiss males to obtain embryos for treatment with VEGF in vitro. Preimplantation embryo development was assessed prior to embryo transfer (n = 19-30/treatment group/output). Recipient F1 female mice (8-12 weeks of age) were mated with vasectomized males to induce pseudopregnancy and embryos were transferred. On Day 14.5 of pregnancy, uterine horns were collected for analysis of implantation rates as well as placental and fetal development (n = 14-19/treatment). PARTICIPANTS/MATERIALS, SETTING, METHODS: Lavage fluid was assessed by western immunoblot analysis to determine the VEGF isoforms present. Mouse embryos were treated with either recombinant human (rh)VEGF, or VEGF isoforms 121 and 165. Preimplantation embryo development was quantified using time-lapse microscopy. Blastocysts were (i) stained for cell number, (ii) transferred to wells coated with fibronectin to examine trophoblast outgrowth or (iii) transferred to pseudo pregnant recipients to analyze implantation rates, placental and fetal development. MAIN RESULTS AND THE ROLE OF CHANCE: Western blot analysis revealed the presence of VEGF121 and 165 isoforms in human uterine fluid. Time-lapse microscopy analysis revealed that VEGF (n = 22) and VEGF121 (n = 23) treatment significantly reduced the preimplantation mouse embryo time to cavitation (P < 0.05). VEGF and VEGF165 increased both blastocyst cell number (VEGF n = 27; VEGF165 n = 24: P < 0.001) and outgrowth (n = 15/treatment: 66 h, P < 0.001; 74, 90, 98 and 114 h, P < 0.01) on fibronectin compared with control. Furthermore, rhVEGF improved implantation rates and enhanced fetal limb development (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Due to the nature of this work, embryo development and implantation was only examined in the mouse. WIDER IMPLICATIONS OF THE FINDINGS: The absence or reduction in levels of VEGF during the preimplantation period likely affects key events during embryo development, implantation and placentation. The potential for improvement of clinical IVF outcomes by the addition of VEGF to human embryo culture media needs further investigation. STUDY FUNDING/COMPETING INTERESTS: This study was supported by a University of Melbourne Early Career Researcher Grant #601040, the NHMRC (L.A.S., Program grant #494802; Fellowship #1002028; N.J.H., Fellowship # 628927; J.E.; project grant #1047756) and L.A.S., Monash IVF Research and Education Foundation. N.K.B. was supported by an Australian Postgraduate Award. Work at PHI-MIMR Institute was also supported by the Victorian Government's Operational Infrastructure Support Program. There are no conflicts of interest to declare.

Analysis of fertility-related soluble mediators in human uterine fluid identifies VEGF as a key regulator of embryo implantation.

Embryo implantation requires synchronized dialogue between the receptive endometrium and activated blastocyst via locally produced soluble mediators. During the midsecretory (MS) phase of the menstrual cycle, increased glandular secretion into the uterine lumen contains important mediators that modulate the endometrium and support the conceptus during implantation. This study aimed first to identify the growth factor and cytokine profile of human uterine fluid from fertile women during the midproliferative (MP; nonreceptive) and MS (receptive) phases of the cycle, and from women with unexplained infertility during the MS phase. The second aim was to determine important functions of endometrial secretions for embryo implantation. Analysis of uterine fluid using quantitative Luminex assays revealed the presence of over 30 cytokines and growth factors, of which eight [platelet-derived growth factor-AA, TNF-B, soluble IL-2 receptor-A, Fms-like tyrosine kinase 3 ligand, soluble CD40 ligand, IL-7, interferon-A2, and chemokine (C-X-C motif) ligand 1-3] were previously unknown in human uterine fluid. Comparison of the fertile MP, MS, and infertile MS cohorts revealed vascular endothelial growth factor (VEGF) levels are significantly reduced in uterine fluid during the MS phase in women with unexplained infertility compared with fertile women. Functional studies demonstrated that culturing mouse embryos with either MS-phase uterine fluid from fertile women or recombinant human VEGF significantly enhanced blastocyst outgrowth. Furthermore, treatment of human endometrial epithelial cells with uterine fluid or recombinant human VEGF-A significantly increased endometrial epithelial cell adhesion. Taken together, our data support the concept that endometrial secretions, including VEGF, play important roles during implantation. Identifying the soluble mediators in human uterine fluid and their actions during implantation provides insight into interactions essential for establishing pregnancy, fertility markers, and infertility treatment options.

Blastocysts from patients with polycystic ovaries exhibit altered transcriptome and secretome.

Polycystic ovaries (PCO) is a common phenotype of women presenting for infertility treatment. This study investigated whether blastocysts derived from women with PCO have an altered molecular signature which could be a causative factor contributing to reproductive failure. Morphologically similar blastocysts derived from women with PCO and donor oocyte cycles were analysed for transcription and protein secretion. Unsupervised hierarchical clustering demonstrated that the transcriptome profiles of blastocysts derived from PCO patients and control blastocysts were markedly different with complete branch separation. Statistical analysis revealed 829 genes with significantly different expression: 784 decreased (94.6%) and 45 increased (5.4%) in blastocysts derived from women with PCO compared with controls (P<0.05). Functional annotation of these genes revealed predominant gene ontology biological processes including protein metabolism (30%), transcription (22%), signal transduction (15%), biosynthesis (15%) and cell cycle (14%). Proteomic profiling identified 12 biomarkers that displayed significant decrease in expression in blastocysts derived from women with PCO compared with controls (P<0.05). These data indicate molecular alterations in human blastocysts derived from PCO patients, potentially demonstrating for the first time a link between patient aetiology/phenotype and subsequent embryo development, which in part may explain the observed reduction in reproductive capacity.

Culture and transfer of viable blastocysts: a feasible proposition for human IVF.

In spite of the numerous advances in the field of human assisted reproductive technologies (ART) over the past 20 years, a rate-limiting factor in the overall efficiency of the procedure (the implantation rate) has remained at 10-30%. The development of sequential media has led to the ability to culture routinely the human embryo to the viable blastocyst stage. Transfer of such blastocysts has resulted in a significant increase in implantation rates. Increases in implantation rates following blastocyst transfer have been reported for specific groups of patients culminating in the elimination of high order multiple gestations. Of greater significance, however, is that the introduction of blastocyst transfer to all patients entering infertility clinics is associated with an overall increase in implantation and pregnancy rates. Blastocysts derived from the use of sequential media are readily cryopreserved and produce high implantation rates after transfer. Using a model to account for both total embryo utilization per cycle (transferred plus cryopreserved) and implantation rate, it has been calculated that extended embryo culture and blastocyst transfer is approximately 20% more efficient than the transfer of cleavage stage embryos on day 3. Furthermore, as the score of the blastocysts obtained using sequential media is directly related to implantation and pregnancy rates, it is possible to determine which patients should be offered a single blastocyst transfer, thereby addressing the issue of twins conceived through ART.

Temporal and differential effects of amino acids on bovine embryo development in culture.

The aim of the study was to determine the amino acid requirements of the in vitro-produced bovine embryo as it develops from the zygote to the blastocyst, using a two-step culture system. When added to synthetic oviduct fluid (SOF) for the first 72-h culture, Eagle's nonessential amino acids and glutamine (NeGln) significantly increased development to the 8- to 16-cell stage (Day 4 postinsemination [pi]) and subsequent blastocyst development (Day 7 pi). Glutamine alone during the first 72-h culture did not stimulate development to the 8- to 16-cell stage (p > 0.05); however, the removal of glutamine from NeGln reduced the stimulatory effects of the nonessential amino acids. Replacing glutamine with betaine (an organic osmolyte) in NeGln did not stimulate development to the 8- to 16-cell stage compared to culture in SOF, but it did improve subsequent blastocyst development, indicating an osmolytic function of glutamine during the first 72-h culture. The addition of Eagle's essential amino acids and glutamine to SOF, or to medium already containing nonessential amino acids and glutamine for the first 72-h culture, did not affect cleavage to the 8- to 16-cell stage or subsequent blastocyst development (p > 0.05). Beyond Day 4 pi, culture with 20aa (nonessential and essential amino acids and glutamine) increased blastocyst development, total cell number, and the number of cells in both the trophectoderm and inner cell mass, compared to culture with other groups of amino acids (p < 0.05). Substituting betaine for glutamine in 20aa reduced blastocyst formation, indicating a non-osmolytic function of glutamine during the second 72-h culture. Further, there was a significant negative correlation between the concentration of essential amino acids (quarter, half, or single strength) and embryo development during both the first 72-h and second 72-h culture (p < 0.01), indicating that the concentration of essential amino acids was too high during culture of the bovine embryo. This study identified the temporal and differential effects of amino acids during development of the bovine embryo from the zygote to the blastocyst.

Reactivating tammar wallaby blastocysts oxidize fatty acids and amino acids.

The tammar wallaby, Macropus eugenii, has a ruminant-like digestive system which may make a significant concentration of amino acids and fatty acids available to the blastocyst via uterine fluids. Fluorescent and radioisotope analyses were performed to determine the rate of glutamine and palmitate use by blastocysts recovered on day 0, 3, 4, 5 and 10 after reactivation induced by removal of pouch young (RPY). Between day 0 and 4 glutamine uptake increased from 15.6 +/- 6.6 to 36.1 +/- 2.7 pmol per embryo h-1 (P < 0.01) and ammonium production increased from 8.2 +/- 4.3 to 26.6 +/- 3.0 pmol per embryo h-1 (P < 0.01). Glutamine oxidation did not increase until day 10 after RPY (P < 0.01), but the percentage of glutamine oxidized increased from 4.5 +/- 3.1% during diapause to 31.2 +/- 12.6% (P < 0.01) by day 5 after RPY and increased further to 51.0 +/- 15.8% (P < 0.01) by day 10 after RPY. Palmitate oxidation also increased from 0.3 +/- 0.1 by day 0 blastocysts to 3.8 +/- 1.7 pmol per embryo h-1 (P < 0.01) by day 4 blastocysts. This increase provides a greater potential for ATP production, possibly to supply increased demand due to the coincident resumption of mitoses. The ATP:ADP ratio within blastocysts had reduced by the time of the first measurement at day 3 (0.5 +/- 0.2 pmol per embryo h-1; P < 0.01) compared with day 0 blastocysts (1.4 +/- 0.3 pmol per embryo h-1). It is likely that metabolism of amino acids and fatty acids contributes to the energy supply during reactivation of tammar wallaby blastocysts after embryonic diapause.

Reactivating tammar wallaby blastocysts oxidize glucose.

Metabolic reactivation of the tammar blastocyst appears to be characterized by a change in the pathway of glucose metabolism rather than an absolute increase in substrate uptake. The switch in type of metabolism used was examined to gain information on the timing and physiology of blastocyst reactivation. Fluorescent and radioisotope techniques were used sequentially to determine the activity of pathways of glucose metabolism by individual wallaby blastocysts during diapause and 3, 4, 5, 6, 7, 8, and 10 days after removal of pouch young (RPY). Maternal endometrial and luteal cell metabolism and circulating hormone levels were measured and correlated with blastocyst activity. Observed differences between rates of blastocyst reactivation could be explained by variation in the maternal response between animals. While blastocysts recovered 4 days after RPY oxidized more glucose compared with Day 0 blastocysts (p < 0.05), rates of glycolysis did not change until Day 10. Blastocysts recovered between 4 and 10 days after RPY oxidized a significantly greater percentage of the glucose taken up (p < 0.01). The reduced ATP:ADP ratio within blastocysts recovered 3 days after RPY (p < 0.05) indicates that conditions are suitable for blastocysts to undergo a metabolic switch from glycolytic to oxidative metabolism of glucose on Day 4 after RPY. The increased oxidation results in greater ATP production, which plausibly fuels the increased energy requirements of wallaby blastocysts during the early stages of reactivation.

Concentrations of energy substrates in oviduct fluid in unilaterally ovariectomised pigs.

Nichol et al (1992, Journal of Reproduction and Fertility, 96, 699-707) identified a pre- to post-ovulatory decrease (approx 1 mM) in the amount of glucose in pig oviduct fluid. The present studies investigated whether the decrease was due to metabolism by embryos and/or oviduct tissues, and also whether there was a local influence of the ovary on the oviduct fluid content of energy substrates. Unilaterally ovariectomised pigs were used, in which, through compensation, oviducts that contained twice the normal number of embryos could be compared with oviducts which contained no embryos. Following unilateral ovariectomy and after two oestrous cycles of normal duration, surgery was performed 88 hours after the beginning of standing heat to obtain oviduct fluid samples, just before embryonic entry into the uterus. Luminal fluid samples from the ampulla and ampullary-isthmic junction from oviducts with and without an adjacent ovary were assayed for glucose, pyruvate and lactate concentrations. No significant differences were found between the glucose, pyruvate and lactate concentrations in fluids from the ampulla or ampullary-isthmic junction from oviducts containing embryos compared with absence of embryos (P > 0.05). Therefore, the post-ovulatory decrease was not due to the presence of embryos or to a local effect of the ipsilateral ovary. Consequently, pig oviduct fluid concentrations of glucose, lactate and pyruvate are seemingly regulated by systemic mechanisms.

Modifications made to culture medium by bovine oviduct epithelial cells: changes to carbohydrates stimulate bovine embryo development.

Co-culture remains a common method to support the development of bovine embryos, derived from IVM/IVF procedures. However, the mechanism by which somatic cells confer their benefit to the developing embryo remains undetermined. This study therefore analysed the changes made to the culture medium TCM-199, used in bovine embryo co-culture systems, by somatic cells and determined the effects of specific changes in medium composition on bovine embryo development in culture. Bovine oviduct epithelial (BOE), Buffalo rat liver (BRL) and fibroblast (3T3) cells were compared. The concentrations of glucose, L-lactate, pyruvate, amino acids, NH4+, H+ and the gas tensions of O2 and CO2 were measured in TCM-199 supplemented with 10% fetal calf serum (FCS) prior to and directly following 48 h incubation periods with each cell type. All three somatic cell types modified the carbohydrate composition of the media in a similar manner with the greatest changes made by the BOE cells. Notable alterations were an increase in the levels of L-lactate and pyruvate and a reduction in glucose concentration, which in the case of the BOE cells, fell from 5.55 mM to 2.67 mM. In order to determine the relevance of such changes in carbohydrate concentrations on bovine embryo development, modifications were made to carbohydrate levels in synthetic oviduct fluid (SOF) medium and their effect on blastocyst development in vitro assessed. In SOF medium supplemented with amino acids and BSA (SOFaa), significantly more zygotes developed to the blastocyst stage (64%; P < 0.01) than in SOFaa medium with the concentrations of glucose, D/L-lactate and pyruvate equivalent to those in TCM-199 (11%). Interestingly, when the levels of carbohydrates in SOFaa mimicked those present in TCM-199 following a 48 h incubation with BOE cells, 57% of zygotes reached the blastocyst stage. This improvement was ascribed to the reduction in glucose and increases in D/L-lactate and pyruvate concentrations in the culture system. Results from this study demonstrate that BOE cells create an environment favourable to embryonic development. The analysis of media samples by enzymatic methods meant that only the biologically active L-isomer of lactate was quantified. However, in SOFaa, both the L-isomer and inactive D-isomer are present in equimolar amounts. As such, culture media in which D/L-lactate syrup is used actually contain only 50% biologically active lactate meaning that all D/L-lactate concentrations are reported at twice the effective concentration. Therefore the effect of D/L-lactate concentration on blastocyst development was subsequently determined in this study. Blastocyst development was poor (24-36%) until the total D/L-lactate was present in the culture system at concentrations equal to or greater than 0.82 mM. However, blastocyst cell numbers remained low (60.1 +/- 6.9 - 78.5 +/- 6.6) until a total D/L-lactate concentration of 3.3 mM. This data reinforces that embryo morphological appearance is not sensitive enough to be used as the sole criterion for assessing embryo development.

Carbohydrate uptake by quiescent and reactivated mouse blastocysts.

The mouse, Mus musculus, can maintain blastocysts in embryonic diapause in the uterus while suckling young. This study used microfluorimetry to simultaneously examine glucose and pyruvate uptake by quiescent blastocysts, and at four hourly intervals after administration of the reactivating stimulus, oestradiol-17 beta. Following the non-invasive analysis of energy metabolism, blastocysts were incubated in colcemid (0.2 mg/ml), and mitotic activity determined. Mitoses and cell numbers in reactivated embryos increased significantly within 8 and 12 hours, respectively, after oestradiol-17 beta administration, compared to those of diapause (control) blastocysts (0.5 +/- 0.1 vs. 0.22 +/- 0.03 mitoses/embryo; P < 0.05, and 141.8 +/- 1.5 vs. 133.8 +/- 2.4 cells/ embryo; P < 0.05). Similarly, pyruvate uptake by reactivating blastocysts (9.3 +/- 1.1) was significantly higher than controls (5.8 +/- 0.8 pmol/embryo/hour; P < 0.05), within 4 hours of oestradiol-17 beta, but by 16 hours after oestradiol-17 beta administration, pyruvate uptake by reactivating blastocysts was no longer significantly different from the delayed controls. In contrast, significant differences in glucose uptake between the reactivated and control groups were not evident until 16 hours after oestradiol-17 beta (reactivating, 14.9 +/- 1.5; control, 10.6 +/- 1.7 pmol/embryo/hour; P < 0.05). These results demonstrate that pyruvate rather than glucose could supply the additional energy required during the first 12 hours of reactivation in the mouse, but from 16 hours after injection of the reactivating stimulus oestradiol-17 beta, glucose is the predominant energy source.

Increase in postimplantation development of cultured mouse embryos by amino acids and induction of fetal retardation and exencephaly by ammonium ions.

The effects of amino acids and ammonium on the postimplantation development of cultured preimplantation mouse zygotes were assessed. Development after transfer revealed that the mouse embryo undergoes a switch in nitrogen requirements during the preimplantation period. Although Eagle's nonessential amino acids and glutamine supported the highest implantation and fetal development rates per embryo transferred when zygotes were cultured for 48 h, by 93 h of culture the highest implantation rate was observed when all 20 amino acids were in the culture medium. Furthermore, fetal development per implantation at 69 and 93 h of culture was increased only in the presence of essential amino acids without glutamine. The beneficial effects of amino acids on postimplantation development when embryos were cultured for 4 days required that the medium be renewed after 48 h (at the 6-8-cell stage) to alleviate the build-up of ammonium. Ammonium was shown to induce fetal retardation and exencephaly in a time- and concentration-dependent manner. Renewal of amino-acid-free culture medium reduced fetal mass, providing indirect evidence for the production of an embryo-derived growth factor capable of stimulating postimplantation development. These data demonstrate that inclusion of amino acids in the culture medium for preimplantation embryos significantly increases postimplantation development the preimplantation mouse embryo changes its nitrogen requirement as development proceeds, nonessential amino acids increase the implantation rate while the essential amino acids enhance fetal development, and ammonium in the medium retards fetal development and induces the neural tube defect exencephaly.