IVF Medium Research Articles

Characterization of human pre-implantation embryonic exosomes

To isolate and characterize exosomes secreted from human embryos. Prospective experimental study Spent IVF culture media was collected prospectively and pooled from 50 embryos at two different stages of early embryonic development on day 3 and at blastocyst. The 4 groups assessed were: control media without protein supplement (plasmanate), control media with plasmanate, day 3 embryo media, and blastocyst media. Exosome size and number were measured using nanosight tracking analysis (NTA). Characterization of embryonic exosome cargo was performed through mass spectrometry proteomic analysis. Exosomal protein cargo unique to embryos was submitted to bioinformatic analysis for molecular pathways and biological processes. NTA identified exosomes in control media with plasmanate, pooled day 3 embryo media, and pooled blastocyst media, which had significantly higher concentrations compared to control media without plasmanate. The particle size distribution for all 3 groups showed isolated peaks in the known exosome size range of 50-150 nm. Moreover, day 5 embryo media had significantly higher protein amount per mL media and per exosome compared to day 3 media and control media with plasmanate. Mass spectrometry proteomic analysis identified 273 distinct proteins in the control with plasmanate, day 3, and day 5 embryonic exosomes, of which 193 were shared by all groups. We identified 30 unique proteins with increased expression in day 3 and/or day 5 exosomes. These proteins were enriched in components of the WNT and p53 pathways, which have been shown in mouse and human models to play vital roles in embryonic development and implantation. Importantly, 11 of these genes have been shown previously via knockout models to play roles in blastocyst development, implantation and early embryogenesis. Furthermore, 10 of these unique embryonic proteins are involved in biological processes of immune cell activation and vesicle mediated exocytosis, which further supports their potential immune function and packaging of these proteins into exosomes. By analyzing spent IVF media, we have shown that pre-implantation embryos secrete exosomes with unique cargo that may serve potential roles in implantation, early embryogenesis and immune regulation. Future directions include molecular functioning studies with isolated embryonic exosomes as well as correlating exosome proteomic profile to embryonic outcomes such as blastocyst morphology and aneuploidy.

Non-invasive metabolomic profiling of culture media of ICSI- and IVF-derived early developmental cattle embryos via Raman spectroscopy

The aim of the present study was to compare differences in composition between in vitro cultured early developmental embryos resulting from either in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Non-invasive metabolomic profiling of culture media was conducted with laser tweezer Raman spectroscopy (LTRS), providing molecular information that was used to aid the diagnosis or treatment of embryos that were adversely affected by ICSI treatment, ultimately improving the ICSI embryonic developmental potential. Cattle embryos were generated via ICSI and IVF with development to the 2-, 4-, 8-, 16-,32-cell, and blastocyst stages with individual in vitro culturing occurring for 4 h. The culture media for embryos in different developmental stages were separately analyzed using LTRS. The resulting composition of culture media used for culturing IVF- and ICSI-derived embryos was mainly altered in contents of carbohydrates, lipids, DNA, and proteins. Bands at 1004 cm−1 (phenylalanine) and 1529 cm−1 (-C = C-carotenoid) had specific patterns related to the metabolicactivity of embryos; using LTRS, and these may be considered as biomarkers for embryonic development. Furthermore, the vibrations of lipids at different stages increased more with assessment of ICSI culture media than in IVF media. Discriminant function analysis can be utilized for the classification of culture media used for culture of ICSI- and IVF-derived embryos. In conclusion, LTRS can be used for development of an independent assay to assess embryo status during both ICSI and IVF procedures, which provides novel insights into differences in structure and components of single cells.

Brief co-incubation of gametes benefits the outcomes of newborns.

The objective of this study was to determine whether ammonium accumulates in IVF media during fertility process and whether the brief co-incubation of gametes (bIVF) benefited the outcomes of newborns. Ammonium levels in IVF media during gamete co-incubation were measured and the effects of bIVF on neonatal outcomes were evaluated retrospectively in this study. A total of 609 live newborns cycles were included in this study. The results showed that ammonium levels in the conventional IVF (cIVF) media was significantly increased than that in bIVF and control media (27.32±5.60 vs 20.71±3.89, P = 0.03; 27.32±5.60 vs 19.46±1.31, P = 0.01, respectively). In the cIVF group, the mean gestational age was significantly lower (37.36 ± 2.29 vs. 37.74 ± 1.94weeks, P = 0.031) and the incidence of preterm birth (< 37weeks) was higher than that in the bIVF group (25.80 vs. 17.63%, P = 0.015). Singleton cycles and twin cycles were then analyzed respectively. The gestational age and birth weight of the singleton cycles were similar between the two groups. However, of the twin cycles, the gestational age was significantly decreased and the rate of preterm birth was increased significantly in the cIVF group (35.76 ± 2.31 vs. 36.48 ± 1.73, P = 0.013; 53.33 vs. 31.52%, P = 0.002, respectively). There is an ammonium accumulation in IVF media during co-incubation of gametes. And bIVF reduces the risk of preterm birth (< 37weeks), especially with regard to preterm birth of the twin cycles, and seems to be a safe alternative method for improving the neonatal outcomes compared with cIVF.

In Vitro Maturation and Fertilization of Donkey Oocytes

Many wild and domestic donkey breeds are now at risk of extinction, therefore there is an urgent need for genetic conservation of these breeds. The development of in vitro conditions that support oocyte maturation, fertilization, and embryo development may improve in vitro production of donkey embryos. This work was designed to identify the optimal condition for in vitro production of donkey embryos. Two experiments were conducted. In experiment 1, grade 1 and 2 cumulus-oocyte complexes were subjected to in vitro maturation in the following media: tissue culture medium 199 (TCM199), Christopher Rozenkrans 1aa medium (CR1aa), Christopher Rozenkrans 2aa medium (CR2aa), potassium simplex optimized medium (KSOMaa), centrifuged follicular fluid (CFF), or non-CFF from preovulatory follicles. In experiment 2, in vitro–matured donkey oocytes were subjected to in vitro fertilization (IVF) in Fert-TALP medium supplemented with 0%, 25%, 50%, 75%, or 100% CFF. After fertilization, the cleavage rates and embryo development were assessed across groups. The results of experiment 1 revealed that non-CFF and KSOMaa significantly increase cumulus cell expansion. The percentage of oocytes reaching the metaphase II stage nuclear maturation was significantly higher for donkey oocytes matured in non-CFF. Also, the percentage of degenerated oocytes was lower for donkey oocytes matured in vitro in non-CFF compared with those in TCM199, CR1aa, CR2aa, KSOMaa, or CFF. In experiment 2, IVF of in vitro–matured donkey oocytes using 100% CFF as IVF medium produced a significantly higher fertilization rate with respect to the number of fertilized oocytes extruding the second polar body. In conclusion, non-CFF from preovulatory follicles gave the highest cumulus cell expansion and nuclear maturation rates for in vitro–matured donkey oocytes. Hundred percent CFF was the best medium for IVF of matured donkey oocytes; however, cleavage rate and embryo development are still very low and need further investigations.

Creatine enhances the duration of sperm capacitation: a novel factor for improving in vitro fertilization with small numbers of sperm.

Why are many sperm required for successful fertilization of oocytes in vitro, even though fertilization occurs in vivo when only a few sperm reach the oocyte? Creatine produced in the ovary promotes efficient fertilization in vivo; however, in vitro, creatine is not contained in the in vitro fertilization (IVF) medium. The IVF medium enables capacitation of sperm. However, the IVF medium does not fully mimic the in vivo environment during fertilization. Consequently, fertilization in vitro is more inefficient than in the oviduct. Follicular and oviductal fluids were collected and then analyzed for creatine and glucose levels. To determine the physiological functions of creatine, the creatine antagonist 3-guanidinopropionic acid (GPA) was injected into hormonally primed mice. Using conventional IVF protocols, sperm were pre-incubated in IVF medium with creatine and then co-cultured with 10 ovulated cumulus-oocyte complexes (1-1000 per oocyte) in 50 μl medium droplets. Glucose and creatine levels were measured using commercial enzymatic assay kits. The effect of creatine in vivo was assessed by mating experiments using mice treated with or without GPA just before ovulation. To assess the functions of sperm incubated in IVF medium containing creatine, we analyzed (1) the motility of sperm using computer-assisted sperm assay, (2) the capacitation level of sperm by western blot analyses, and (3) the condition of sperm acrosomes by peanut agglutinin lectin-FITC staining. Oviductal creatine levels were significantly increased following ovulation. Injecting mice with GPA just before ovulation significantly reduced the number of fertilized oocytes. The addition of creatine to IVF medium enhanced sperm capacitation by increasing ATP levels. Successful fertilization was achieved with as few as five sperm/oocyte in the creatine group, and the number of fertilized oocytes was significantly higher than in the control without creatine (P < 0.01). In the present study, a pharmacological approach, creatine antagonist (GPA) treatment, but not a knockout mouse model, was used to understand the role of creatine in vivo. The role of creatine in fertilization processes can only be shown in a mouse model. A modified IVF technique using creatine-containing medium was developed and shown to markedly improve fertilization with small numbers of sperm. This approach has the potential to be highly beneficial for human assisted reproductive technologies, especially for patients with a limited number of good quality sperm. This work was supported in part by JSPS KAKENHI Grant numbers JP24688028, JP16H05017 (to M.S.), and JP15J05331 (to T.U.), the Japan Agency for Medical Research and Development (AMED) (16gk0110015h0001 to M.S.), and National Institutes of Health (NIH-HD-076980 to J.S.R). The authors have nothing to disclose.

The role of Kisspeptin in bovine in vitro embryo production

The aim of this study was to investigate the effect of Kisspeptin (Kp) on the medium used in different stages of in vitro production of bovine embryo (IVEP), evaluating cleavage (CR) and blastocyst (BR) rates. The study was divided into three experiments that analyzed, respectively, the action of Kp on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) of bovine embryos. In experiment 1, the oocytes were matured in IVM medium and distributed into the following treatments: maturation (IVM Control, n = 102), maturation with addition of 10-7 M Kp (Kp 10-7 IVM, n = 90), and hormone-free maturation luteinizing hormone (LH) and follicle-stimulating hormone (FSH) with the addition of 10-7M Kp (No hormones + Kp 10-7, n = 84), following maturation to normal stages of IVEP. In experiment 2, the oocytes were fertilized in IVF medium, in the following treatments: TALP-FERT without Kp (Control IVF, n = 103) and TALP-FERT with the addition of 10-7M Kp (Kp 10-7 IVF, n = 119), usually following the other steps. Finally, in the third experiment, the oocytes passed through all phases and were divided into IVC in two treatments: SOF medium without Kp (Control IVC, n = 109) and SOF medium with the addition of 10-7M Kp (Kp 10-7, N = 106). The data were analyzed by PROC GLIMMIX of the SAS program. In experiment 1, the means of CR and BR were similar (P &gt; 0.05) between treatments (IVM Control76.47% and 37.25%, Kp 10-7 MIV80% and 33.33%, and No hormones + Kp 10-770.24% and 30.95%, respectively). In experiment 2, the means of CR were similar for the IVF Control and Kp 10-7 IVF groups (P &gt; 0.05), 76.70% and 86.55% respectively. But, the mean of the BR of the group Kp 10-7 IVF was 38.66%, which was higher (P &lt; 0.05) than that of the FIV Control group, which was 31.07%. In the third experiment, the means of CR and BR (P &gt; 0.05) were similar between the IVC Control and Kp 10-7 IVC groups (CR 83.50% and 78.30%, and BR 26.60% and 23.60%, respectively). Although at this concentration of 10-7M during IVC no change in embryo production is seen, Kp presents the same performance as both gonadotrophins in oocyte maturation and modulates the fertilization process, providing more blastocysts. With these findings, it can be seen that Kp presents a regulatory action on bovine reproduction, and can be an excellent tool to maximize IVEP indexes.

Common medium versus advanced IVF medium for cryopreserved oocytes in heterologous cycles

Granulocyte-macrophage colony-stimulation factor plays different crucial roles during embryo implantation and subsequent development. Here we aimed to evaluate the effects of embryo cell culture medium, with the inclusion of granulocyte-macrophage colony-stimulation factor (GM-CSF), on embryo development and pregnancy rate. To this end, we took advantage of our retrospective observational study to correlate the outcomes from two different culture media. We included in this study 25 unselected patient from our IVF Center that underwent heterologous IVF cycle with crypreserved oocytes. We analyze the fertilization rate, pregnancy rate, and embryo quality at different day of transfer obtained from two different media composition. Our results show that the rate of fertilization and the pregnancy rate were increased using medium added with this particular type of cytokines (GM-CSF).

120 Theophylline: the Key for Bulls with Low In Vitro Blastocyst Rates?

Although many efforts have been made to improve the sperm normal penetration ability for in vitro embryo production (IVP), sperm capacitation in bovine remains a bottleneck for in vitro fertilization (IVF). The variable responses of sperm to the different supplements in IVF medium makes the process suboptimal for every bull. Theophylline, a methylxanthine, has recently been reported to enhance and maintain in vitro blastocyst production due to prolonged motility and longevity of bovine sperm (Kang et al. 2015 J. Reprod. Dev. 61, 99-105). Here, we evaluated the effects of theophylline supplementation during IVF on developmental rates of bulls with known low and average blastocyst production under standard conditions. A total of 1498 cumulus–oocyte complexes were obtained by slicing from bovine ovaries. Cumulus-oocyte complexes were submitted to in vitro maturation (IVM) for 24 h. In vitro fertilization was performed for 19 h with (2.5 mM) or without theophylline using frozen–thawed sperm from 5 different bulls at a final sperm concentration of to 2 × 106 cells mL−1. Two of the bulls had a previous report of low blastocyst rates (A, B) and 3 showed an average blastocyst production (C, D, E). After IVF, presumptive zygotes were cultured in vitro for 8 days until the blastocyst stage. Cleavage and blastocyst rates were evaluated 3 and 8 days after IVF, respectively. R software (https://www.r-project.org/) was used to evaluate cleavage and blastocyst rates using a two-sample t-test. The supplementation of theophylline significantly increased blastocyst rates for bulls A, B, and E (Table 1; P &lt; 0.05). The blastocyst rates did not differ for bull C and D with or without the use of theophylline (Table 1; P &gt; 0.05). These preliminary results show that in vitro blastocyst production for low performance bulls can be improved by theophylline supplementation during IVF. The underlying mechanism probably involves phosphodiesterase (PDE) inhibition to increase intra-sperm/oocyte cAMP levels improving oocyte-sperm interaction. Table 1.Total oocytes and mean (± SEM) cleavage and blastocyst rates of bull sperm treated or not with theophylline The authors thank Susanne Meese and Ulrich Witschi from Swissgenetics for the cooperation and donation of frozen bull sperm.

Antioxidants improve IVF outcome and subsequent embryo development in the mouse

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? 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. 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. 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). 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. 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). Embryo development was only examined in the mouse. 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. This work was funded by a research grant from Vitrolife AB (Sweden). The authors have no conflict of interest to declare.

Mg2+]o/[Ca2+]o determines Ca2+ response at fertilization: tuning of adult phenotype?

Alteration of the postnatal phenotype has sparked great concern about the developmental impact of culture media used at fertilization. However, the mechanisms and compounds involved are yet to be determined. Here, we used the Ca2+ responses from mouse eggs fertilized by ICSI as a dynamic and quantitative marker to understand the role of compounds in egg functioning and establish possible correlations with adult phenotypes. We computed 134 Ca2+ responses from the first to the last oscillation in media with specific formulations. Analyses demonstrate that eggs generated two times as many Ca2+ oscillations in KSOM as in M16 media (18.8 ± 7.0 vs 9.2 ± 2.5). Moreover, the time increment of the delay between two consecutive oscillations, named TIbO, is the most sensitive coefficient characterizing the mechanism that paces Ca2+ oscillations once the egg has been fertilized. Neither doubling external free Ca2+ nor dispermic fertilization increased significantly the total number of Ca2+ oscillations. In contrast, removing Mg2+ from the M16 boosted Ca2+ oscillations to 54.0 ± 35.2. Hence, [Mg2+]o/[Ca2+]o appears to determine the number, duration and frequency of the Ca2+ oscillations. These changes were correlated with long-term effects. The rate of female's growth was impacted with the 'KSOM' females having only half the fat deposit of 'M16' females. Moreover, adult animals issued from M16 had significantly smaller brain weight vs 'KSOM' and 'control' animals. TIbO is a new Ca2+ coefficient that gauges the very early functional impact of culture media. It offers the possibility of establishing correlations with postnatal consequences according to IVF medium formulation.Free French abstract: A French translation of this abstract is freely available at http://www.reproduction-online.org/content/154/5/675/suppl/DC2.

English

The aim of the current study is to demonstrate the effect of honey on the sperms characteristics (sperm concentration, sperm motility, grade of activity and sperm normal morphology) as well as pregnancy rate in mice. Sperms were obtained from caudal epididymis of male mice and prepared by adding10% of honey to the IVF medium using direct sperms activation technique for 30 minute incubation period before artificial insemination. The study revealed a significant (P > 0.05) increase in active sperm motility (grade A and grade B) 49% and pregnancy rate 53.3% in female mice artificially inseminated with sperms. On the other hand, there were no significant differences in sperm concentrations and normal sperm morphology. In conclusion, the honey was beneficial in improving male fertility of mice by enhancing sperm motility and pregnancy rate of female mice.

The copper transporter (SLC31A1/CTR1) is expressed in bovine spermatozoa and oocytes: Copper in IVF medium improves sperm quality

Adequate dietary intake of copper (Cu) is required for normal reproductive performance in cattle. The objective of this study was to investigate the pregnancy rates from cattle with deficient, marginal and adequate Cu plasma concentration at the beginning of artificial insemination protocol. Moreover, we determined Cu concentrations present in bovine oviductal fluid (OF), and the effects of Cu on fertilizing ability of bovine spermatozoa. Also, the presence of Cu transporter, SLC31A1 (also known as CTR1), in spermatozoa and in vitro matured oocyte were investigated. We found no differences in pregnancy rates among animals with adequate, marginal, and deficient Cu concentrations measured in plasma at the beginning of fixed-time artificial insemination (FTAI) protocol. Copper concentrations in OF were 38.3 ± 2.17 μg/dL (mean ± SEM) regardless of cupremia levels. The addition of 40 μg/dL Cu to IVF medium enhanced total and progressive motility, sperm viability, functional sperm membrane integrity (HOST), sperm–zona binding, and pronuclear formation. On the other hand, the presence of Cu in IVF medium did not modify acrosome integrity and cleavage rates after IVF, but impaired blastocyst rates. Cu transporter SLC31A1 was detected in bovine spermatozoa in the apical segment of acrosome, and in the oocyte matured in vitro. In conclusion, the results obtained in the present study determined that cupremia levels at the beginning of FTAI protocol did not influence the pregnancy rates at 60 d after insemination. The presence of CTR1 in bovine mature oocyte and spermatozoa, as well as the beneficial effect of Cu on sperm quality would suggest an important role of this mineral during the fertilization process.

Pig sperm preincubation and gamete coincubation with glutamate enhance sperm-oocyte binding and in vitro fertilization

As the taste receptor for monosodium glutamate (umami) is expressed in both murine and human spermatozoa and the presence of α-gustducin and α-transducin, G proteins involved in the umami taste signaling, has been described in boar germ cells, the aim of this study was to evaluate if monosodium glutamate (MSG) would exert any effect on sperm-oocyte binding, in vitro fertilization (IVF) and sperm parameters during in vitro induced capacitation.For sperm-zona pellucida binding assay, boar spermatozoa were preincubated for 1 h and then coincubated for 1 h with denuded in vitro matured oocytes in presence of different concentrations of MSG (0, 0.1, 1, 10 mM). MSG 1 and 10 mM significantly (P < 0.05) increased the mean number of sperm bound to ZP compared with control (12.3 ± 9.0, 17.8 ± 11.3, 17.6 ± 10.8, MSG 0, 1 and 10 mM respectively).For in vitro fertilization trials, both sperm preicubation (1 h) and gamete coincubation (1 h) were performed in presence of different concentrations of MSG (0, 0.1, 1, 10 mM). After 19 h of culture in fresh IVF medium, oocytes were fixed. MSG 1 mM significantly (P < 0.05) increased the penetration rate compared with control (53.7 ± 20.4 vs. 36.8 ± 16.2).The addition of MSG during in vitro induced capacitation of boar spermatozoa did not cause any significant difference, compared with control, on the percentage of viable cells, spermatozoa with intact acrosome and the percentage of spermatozoa displaying tyrosine-phosphorylation of sperm tail proteins.In order to evaluate whether the effect elicited by MSG could be due to glutamate uptake in boar spermatozoa, fertilization trials were performed in presence of either 1 mM MSG or 1 mM MSG + 100 μM DL-threo-beta-hydroxyaspartic acid (THA), a non selective inhibitor of glutamate uptake. A significant increase (P < 0.05) in the penetration rate in both MSG and MSG + THA groups compared to control was recorded (39.8 ± 15.7, 53.7 ± 22.1, 52.2 ± 23.7, Control, MSG and MSG + THA respectively) while no difference in penetration rate between MSG and MSG + THA treatment was observed suggesting that sperm glutamate transporters are not involved in the pathway mediating this effect.Our study demonstrates for the first time that glutamate exerts a positive effect on sperm-oocyte binding and fertilization. Further studies are needed to clarify the mechanism by which glutamate exert his effect.

Improving porcine in vitro fertilization output by simulating the oviductal environment

Differences between the in vitro and in vivo environment in which fertilization occurs seem to play a key role in the low efficiency of porcine in vitro fertilization (IVF). This work proposes an IVF system based on the in vivo oviductal periovulatory environment. The combined use of an IVF medium at the pH found in the oviduct in the periovulatory stage (pHe 8.0), a mixture of oviductal components (cumulus-oocyte complex secretions, follicular fluid and oviductal periovulatory fluid, OFCM) and a device that interposes a physical barrier between gametes (an inverted screw cap of a Falcon tube, S) was compared with the classical system at pHe 7.4, in a 4-well multidish (W) lacking oviduct biological components. The results showed that the new IVF system reduced polyspermy and increased the final efficiency by more than 48%. This higher efficiency seems to be a direct consequence of a reduced sperm motility and lower capacitating status and it could be related to the action of OFCM components over gametes and to the increase in the sperm intracellular pH (pHi) caused by the higher pHe used. In conclusion, a medium at pH 8.0 supplemented with OFCM reduces polyspermy and improves porcine IVF output.

The mechanics clarifying counterclockwise rotation in most IVF eggs in mice

In mammalian fertilization, a small spermatozoon interacts with an egg that is a few thousand times larger in volume. In spite of the big difference in size and mass, when spermatozoa are bound to eggs, they begin rotating the eggs in in vitro observation. This was dubbed the ‘fertilization dance’. Interestingly, some papers reported that the rotation was counterclockwise, although the reason for this skewed rotation was not clarified. We focused on a chirality of helical beating of spermatozoa and found that eggs rotate counterclockwise in simulations under a certain geometrical condition where the eggs were situated. This theory of egg rotation was validated by demonstrating egg rotation in a clockwise direction by floating eggs to the upper surface of the IVF medium. The enigma of skewed rotation of IVF eggs was clarified.

Logistics of large scale commercial IVF embryo production.

The use of IVF in agriculture is growing worldwide. This can be explained by the development of better IVF media and techniques, development of sexed semen and the recent introduction of bovine genomics on farms. Being able to perform IVF on a large scale, with multiple on-farm experts to perform ovum pick-up and IVF laboratories capable of handling large volumes in a consistent and sustainable way, remains a huge challenge. To be successful, there has to be a partnership between veterinarians on farms, embryologists in the laboratory and animal owners. Farmers must understand the limits of what IVF can or cannot do under different conditions; veterinarians must manage expectations of farmers once strategies have been developed regarding potential donors; and embryologists must maintain fluent communication with both groups to make sure that objectives are met within predetermined budgets. The logistics of such operations can be very overwhelming, but the return can be considerable if done right. The present mini review describes how such operations can become a reality, with an emphasis on the different aspects that must be considered by all parties.

Recombinant fetuin-B protein maintains high fertilization rate in cumulus cell-free mouse oocytes.

Does fetuin-B inhibit premature zona pellucida (ZP) hardening in mouse oocytes in vitro and thus increase IVF rate? Supplementation of oocyte in vitro maturation (IVM) media with recombinant mouse fetuin-B (rmFetuB) increased fertilization rate without affecting mouse embryo development into blastocysts. Mice deficient in fetuin-B are infertile owing to premature ZP hardening. Premature ZP hardening also occurs during oocyte IVM leading to decreased fertilization rate. We fertilized batches of 20-30 mouse metaphase II (Mll) stage oocytes from C57BL/6 mice with fresh sperm, and studied early embryo development until blastocyst hatching. Oocytes were maintained with or without rmFetuB during IVM and IVF. Exogenous rmFetuB was added to media prior to oocyte isolation. ZP hardening was quantified by chymotrypsin digestion timing and by counting attached sperm. In the absence of cumulus cells, rmFetuB dose-dependently inhibited ZP hardening and increased IVF rate (P =0.039). Fetuin-B at ≥0.03 mg/ml also inhibited physiological, fertilization-triggered ZP hardening (indicated by increased sperm binding, P =0.0002), without increasing embryo death. Exogenous rmFetuB increased IVF rate for up to 5 hours of IVM (P =0.02 at 1 hour, P =0.01 at 5 hours of IVM). Mll stage oocytes in this study were isolated from the ampullae of fetuin-B expressing mice. Thus, oocytes were protected against premature ZP hardening by endogenous fetuin-B. In humans and livestock, oocytes are usually isolated by follicle puncture before ovulation. In this situation, the deprivation of endogenous fetuin-B would occur earlier and the effect of exogenous fetuin-B in the IVF medium may be even more pronounced. Fertilization-triggered ZP hardening is essential for embryo development but in this study the effect of fetuin-B supplementation was only studied to blastocyst stage. Any influence of added fetuin-B on later embryo development after transplantation remains to be determined. The astacin-type protease ovastacin triggers definitive ZP hardening by cleaving the zona pellucida protein 2. Animal sera are known to inhibit premature ZP hardening. The addition of rFetuB to the culture medium of oocytes could increase IVF rates by the inhibition of premature ZP hardening. In this regard, the results could be useful for clinical activity. None. The research was supported by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University. The authors ED, JF and WJD are named inventors on a patent application of RWTH Aachen University covering the use of fetuin-B in ovary and oocyte culture.

Effect of Fetal Calf Serum and Mid Estrus Cow Serum on In Vitro Maturation and Fertilization of Oocytes from Crossbred Cattle in the Tropics

The objective of the study was to examine the effect of fetal calf serum (FCS) and mid estrus cow serum (ECS) in culture media on in vitro maturation (IVM) and in vitro fertilization (IVF) of oocytes from crossbred cattle in the tropics. Oocytes from abattoir ovaries were cultured in three different maturation media at 39oC temperature, 5% CO2 tension with maximum humidity for 24 h. TCM-199 containing 25mM HEPES, 1mM glutamine L, 2.2mg/mL sodium bicarbonate, antibiotics, 22 µg/mL pyruvate, 1µg/mL estradiol-17β, 0.5µg/mL FSH and 0.06 IU hCG without serum supplement used as treatment-1 and the above media were further supplemented with 10% FCS as treatment-2. In treatment-3, instead of 10% FCS, 20% heat inactivated ECS (serum collected in mid estrum) was used as serum supplement. Oocytes with maximum degree of cumulus expansion were selected as matured and used for further IVF studies using frozen semen. The IVF medium consisted of Fert-TALP medium supplemented with 1µM epinephrine, 10µM hypotaurine, 20µM pencillamine and 0.56µg/ml heparin. Culture conditions set for IVF were 39oC temperature, 5% CO2 tension with maximum humidity. Oocytes showing sperm penetration evidence like presence of enlarged sperm head, male pronuclei with its accompanying sperm tail in the cytoplasm, oocytes with two pronuclei and a clear second polar body but without a sperm tail were considered as fertilized. Significantly higher result of cumulus expansion percentage (p&lt; 0.05) was observed when oocytes matured in media supplemented with FCS as compared to other two treatments. There was no influence of source of serum in maturation media on further IVF of matured oocytes in this study. Int J Appl Sci Biotechnol, Vol 4(3): 308-310

Protective effect of crocetin on bovine spermatozoa against oxidative stress during invitro fertilization.

Oxidative stress is one of the major factors that contribute to poor semen quality and low rates of invitro fertilization. Crocetin, a main constituent of saffron (Crocus sativus L.) possesses potent antioxidant activity, by scavenging reactive oxygen species (ROS) and/or enhancing the activity of intracellular antioxidant enzymes. The aim of this study was to investigate, for the first time, the effect of crocetin on the quality characteristics of bull spermatozoa and fertilization rate. For this reason, frozen/thawed bovine spermatozoa were incubated with crocetin (1, 2.5, and 5μm), for 120 or 240min, in the presence of a negative control, and evaluated in terms of motility, viability, acrosomal status, DNA fragmentation index, intracellular ROS, and lipid peroxidation. In order to evaluate the impact of crocetin on cleavage and blastocyst rate, the compound was added in the IVF medium at the previously identified optimal concentration (2.5μm). The results indicate that incubation of spermatozoa with 2.5μm of crocetin resulted in a statistically significant lower production of superoxide anion and hydrogen peroxide, lower lipid peroxidation, and in better maintenance of motility parameters, viability, and acrosomal integrity, with a very small number of cells with DNA fragmentation, compared to the other groups (p<0.05). The presence of crocetin (2.5μm) in the fertilization medium also resulted in a significant increase in acrosome-reacted spermatozoa and blastocyst production, compared to the control group (p<0.01). These data indicate that crocetin (2.5μm) positively affects bovine sperm quality characteristics during a 240-min incubation and improves its fertilizing ability, directly and/or indirectly, by regulating ROS concentration and lipid peroxidation.

Proof of concept: preimplantation genetic screening without embryo biopsy through analysis of cell-free DNA in spent embryo culture media

To assess whether preimplantation genetic screening (PGS) is possible by testing for free embryonic DNA in spent IVF media from embryos undergoing trophectoderm biopsy. Prospective cohort analysis. Academic fertility center. Seven patients undergoing IVF and 57 embryos undergoing trophectoderm biopsy for PGS. On day 3 of development, each embryo was placed in a separate media droplet. All biopsied embryos received a PGS result by array comparative genomic hybridization. Preimplantation genetic screening was performed on amplified DNA extracted from media and results were compared with PGS results for the corresponding biopsy. [1] Presence of DNA in spent IVF culture media. [2] Correlation between genetic screening result from spent media and corresponding biopsy. Fifty-five samples had detectable DNA ranging from 2-642ng/μL after a 2-hour amplification. Six samples with the highest DNA levels underwent PGS, rendering one result with a derivative log ratio SD (DLRSD) of <0.85 (a quality control metric of oligonucleotide array comparative genomic hybridization). The fluid sample and trophectoderm results were identical demonstrating (45XY, -13). Three samples were reamplified 1hour later and tested showing improving DLRSD. One of the three samples with a DLRSD of 0.85 demonstrated (46XY), consistent with the biopsy. Overnight DNA amplification showed DNA in all samples. We demonstrate two novel findings: the presence of free embryonic DNA in spent media and a result that is consistent with trophectoderm biopsy. Improvements in DNA collection, amplification, and testing may allow for PGS without biopsy in the future.