Effects of superoxide dismutase on mouse in vitro fertilization and embryo culture system

Purpose We recently found that, for mouse embryos fertilized in vivo, the two-cell block could be attenuated by adding superoxide dismutase (SOD), a scavenger of superoxide radicals, to the culture medium. In this study, we evaluated the effects of SOD on the process of fertilization and on the further development of the embryos fertilized in vitro.Methods We performed incubation of mouse epididymal spermatozoa, in vitro fertilization, and further cultivation in Biggers—Whitten—Whittingham's medium supplemented with various concentrations of Cu · Zn—SOD.Results High concentrations (2000 µg/ml or more) of SOD prevented loss of motility in mouse sperm over time. The addition of SOD (less than 2000 µg/ml) to the basic medium showed no significant difference in the fertilization rate. Also, no significant difference was observed in the rate of polyspermy or parthenogenesis between the basic and the SOD-supplemented media. However, 18% of the two-cell-stage embryos developed to the expanded blastocyst stage in the 500 µg/ml SOD-supplemented medium, while no blastocysts were found in the basic medium. Furthermore, the addition of SOD 7 hr after insemination increased the expanded blastocyst rate (28%). Conclusions These results indicate that the addition of SOD exerts a protecting effect from oxidative stress both on sperm viability and on the development of embryos fertilized in vitro as well as in vivo, while its addition showed no effect on the process of fertilization.     

人卵泡液对小鼠胚胎生长发育影响的研究

目的：探讨人卵泡液对小鼠胚胎生长发育的影响。方法：在培养液中分别加入人卵泡液（卵泡液组）和人血清（血清组）,观察和检测2细胞期鼠胚在体外发育至8细胞期、囊胚期以及鼠胚孵出的比率;用放射免疫方法测定人卵泡液和血清中表皮生长因子（EGF）含量。结果：卵泡液组有72.9%的2细胞期鼠胚能发育到8细胞期,血清组仅有48.0%到达8细胞期;继续培养显示,卵泡液组细胞分裂较快,有50.9%的鼠胚可发育至囊胚期,并有26.3%孵出,明显高于血清组（24.5%,6.9%),两组比较,差异有显著性（P＜0.05)。卵泡液中EGF的含量为0.50μg/L,血清中为0.26μg/L,两组比较,差异有显著性（P＜0.05）。结论：卵泡液对早期胚胎发育有促进作用,在早期胚胎培养中,添加人卵泡液比添加人血清对提高早期胚胎培养质量更为有效。     

Effect of micro-vibration culture system on embryo development

Purpose

Micro-vibration culture system was examined to determine the effects on mouse and human embryo development and possible improvement of clinical outcomes in poor responders.

Materials and methods

The embryonic development rates and cell numbers of blastocysts were compared between a static culture group (n = 178) and a micro-vibration culture group (n = 181) in mice. The embryonic development rates and clinical results were compared between a static culture group (n = 159 cycles) and a micro-vibration culture group (n = 166 cycles) in poor responders. A micro-vibrator was set at a frequency of 42 Hz, 5 s/60 min duration for mouse and human embryo development.

Results

The embryonic development rate was significantly improved in the micro-vibration culture group in mice (p < 0.05). The cell numbers of mouse blastocysts were significantly higher in the micro-vibration group than in the static culture group (p < 0.05). In the poor responders, the rate of high grade embryos was not significantly improved in the micro-vibration culture group on day 3. However, the optimal embryonic development rate on day 5 was improved in the micro-vibration group, and the total pregnancy rate and implantation rate were significantly higher in the micro-vibration group than in the static culture group (p < 0.05).

Conclusions

Micro-vibration culture methods have a beneficial effect on embryonic development in mouse embryos. In poor responders, the embryo development rate was improved to a limited extent under the micro-vibration culture conditions, but the clinical results were significantly improved.     

The temporal effects of changes in in vitro fertilization culture media on the one-cell mouse embryo system

The one-cell mouse embryo system has previously been shown to be more sensitive than the two-cell system to mild changes in in vitro fertilization (IVF) culture media. To determine whether this greater sensitivity is related to the developmental stage or to the length of exposure, one-cell embryos were collected and cultured in control media (Ham's F-10, 282 mOsm/liter), in media of altered osmolality (260, 300, and 316 mOsm/liter), or in media containing Cidex diluted 1:100,000. The one-cell embryos were exposed to control or altered media in four patterns: control group—control medium for 96 hr; Group A—altered medium for the first 24 hr followed by control medium for 72 hr; Group B—control medium for the first 24 hr followed by altered medium for 72 hr; and Group C—control medium for the first 24 hr, altered medium for the next 24 hr, and control medium again for 48 hr. The percentage of embryos developing to blastocysts in Group A (exposed to adverse conditions only for the first 24 hr of culture) was significantly lower than in the control group under all conditions studied. In contrast, the percentage of blastocysts developing in Group B was significantly lower than in the control group only in medium of 315 mOsm/liter and was not different from that in controls under the other conditions studied. There was no difference between Group C and the control group. We conclude that the higher sensitivity of the one-cell system is an inherent property of the one-cell stage, as exposure of the embryo during this critical first 24-hr period proved to have the most profound consequences.     

Effects of human sera and human serum albumin on mouse embryo culture

Human proteins normally used to supplement human in vitro fertilization—embryo transfer (IVF-ET) culture media were tested for their effects on mouse embryo development from the zygote stage. These proteins included follicular and luteal-phase maternal sera, fetal cord sera, and both human and bovine serum albumin. Our results revealed that both maternal and fetal cord sera did not permit mouse blastocyst formation. Furthermore, predialysis of the human maternal sera and removal of IgG by protein A column chromatography did not improve their support of mouse embryonic development to the blastocyst stage. Similar detrimental effects were observed with maternal sera from term-pregnant IVF-ET patients. Interestingly, these serum samples had supported the in vitro growth of the human zygotes which resulted in these patients' pregnancies. Only some batches of human serum albumin supported mouse blastocyst formation, whereas all sources of bovine serum albumin were effective in this regard. These results raise the question of the suitability of the mouse embryo culture system as a quality control for the testing of protein supplements for human IVF-ET.     

Impact of a controlled culture temperature gradient on mouse embryo development and morphokinetics

Research questionTemperature control within the IVF laboratory is an important aspect of a quality control system, helping to reduce environmental stress and ensure good-quality embryo development. Temperature fluctuations are probably more common than expected and the optimal temperature for embryo culture is not known. Modern incubators offer the opportunity to examine the impact of culture temperature on preimplantation embryo development while controlling for other variables within the system. The purpose of this study was to examine the effect of a range of temperatures during extended embryo culture on resulting mouse embryo development and morphokinetic timings.MethodsUsing a single time-lapse incubator with six individual chambers, frozen–thawed one-cell mouse embryos were cultured individually at temperatures adjusted by 0.5°C between chambers to cover the range of 35.0–37.5°C. Resulting blastocyst formation and embryo morphokinetic timings were recorded and compared.ResultsChanges in culture temperature had a significant impact on mouse blastocyst development and morphokinetic timings (P < 0.05). Under the conditions used in this study, blastocyst development was best at 37.0°C. Mouse preimplantation embryo mitotic cell divisions were generally slower at cooler temperatures and accelerated as the temperature increased from 35.0°C to 37.5°C.ConclusionIncubator culture temperature must be carefully controlled, as even slight variations of 0.5°C in the temperature used for extended embryo culture can have significant impacts on embryo development and mitotic cell divisions. These data have potential implications for application of universal morphokinetic selection algorithms and may help explain differences in mitotic errors/embryo mosaicism between laboratories.     

Establishment of an animal embryo culture system containing various embryotropins and its efficacy for culturing ICR mouse one-cell embryos derived in vivo or in vitro.

OBJECTIVE: To develop an effective ICR mouse embryo culture medium. DESIGN: In vitro model study. SETTING: University-affiliated hospital. ANIMALS: Four-week-old, superovulated mice. INTERVENTION(S): In vivo- or in vitro-derived one-cell embryos were cultured in preimplantation-1 medium (P-1). MAIN OUTCOME MEASURE(S): Preimplantation development. RESULT(S): In vivo-derived embryos were cultured in BSA-containing P-1, to which one of the following substances was added: [1] no addition, [2] amino acids (aa), [3] aa+hemoglobin (hb), [4] aa+hb+cysteine (cys), [5] aa+hb and glucose (glu) added at the four-cell, or [6] aa+hb and glu+cys added at the four-cell stage. More (P<0.05) blastocysts developed after aa or aa+hb addition than after no addition, and glu addition to such medium further stimulated the formation (54%). In P-1 with aa+glu, the addition of 1 microg/mL hb was optimal. Additional improvement of blastocyst formation (78%) was achieved by ethylenediaminetetraacetic acid (EDTA), supplementation and bovine serum albumin replacement with polyvinyl alcohol (PVA) did not inhibit the development. P-1 supplemented with aa, hb, glu, EDTA, and PVA also supported the development of in vitro-derived embryos (70%). CONCLUSION(S): A modified P-1 medium was developed, and it supported the development of both in vivo- and in vitro-derived ICR mouse embryos.     

Differential impacts of gonadotrophins,IVF and embryo culture on mouse blastocyst development

Research questionConception via assisted reproductive technology (ART) increases the risk of type 2 diabetes and cardiovascular disease in adulthood. Underlying differences between ART-conceived and in-vivo-conceived embryos that contribute to this increased risk are, however, not known.DesignThis study examined the developmental characteristics of mouse blastocysts derived from ART- compared with in-vivo-conceived embryos. To determine the effect of ovarian stimulation versus IVF versus in-vitro embryo culture on phenotype, six distinct groups of blastocysts were generated. Female mice were naturally cycling or treated with high or mild doses of gonadotrophin, followed by natural mating or IVF under clinical conditions. Embryo morphokinetics were assessed by continuous time-lapse monitoring. Cell lineage allocation to the inner cell mass (Oct4+) or trophectoderm (Cdx2+) was determined by immunohistochemistry, and mitochondrial DNA (mtDNA) copy number was measured by quantitative PCR.ResultsOvarian stimulation increased embryo number but reduced the percentage of blastocysts. Morphokinetic analysis showed that gonadotrophin treatment led to advanced development (P < 0.05) due to earlier post-pronuclear breakdown. The blastocyst rate was reduced in IVF embryos compared with those fertilized in vivo before culture (P < 0.001). Morphokinetics showed that embryo development was slower in all the IVF groups (P < <0.05), due to a delay from the 3-cell stage. A reduced total and trophectoderm cell number was observed in all groups of cultured blastocysts compared with naturally conceived blastocysts (P < 0.01). Gonadotrophin treatment did not affect the blastocyst mtDNA copy number; however, IVF embryos exhibited reduced mtDNA copy number compared with naturally conceived embryos.ConclusionOvarian stimulation, IVF and in-vitro culture differentially impair blastocyst developmental kinetics, differentiation and mtDNA copy number.     

Individual culture and atmospheric oxygen during culture affect mouse preimplantation embryo metabolism and post-implantation development

Research questionDoes single embryo culture under atmospheric or reduced oxygen alter preimplantation metabolism and post-implantation development compared with culture in groups?DesignMouse embryos were cultured under 5% or 20% oxygen, individually or in groups of 10. Spent media were analysed after 48, 72 and 96 h of culture. Blastocysts were assessed by outgrowth assay or transferred to pseudo-pregnant recipients, and fetal and placental weight, length and morphology were assessed.ResultsCompared with group culture, individually cultured blastocysts had lower net consumption of glucose and aspartate and higher glutamate production. Atmospheric oxygen reduced uptake of glucose and aspartate and increased production of glutamate and ornithine compared with 5% oxygen. Combining 20% oxygen and single culture resulted in further metabolic changes: decreased leucine, methionine and threonine consumption. Under 5% oxygen, individual culture decreased placental labyrinth area but had no other effects on fetal and placental development or outgrowth size compared with group culture. Under 20% oxygen, however, individual culture reduced outgrowth size and fetal and placental weight compared with group-cultured embryos.ConclusionsPreimplantation metabolism of glucose and amino acids is altered by both oxygen and individual culture, and fetal weight is reduced by individual culture under atmospheric oxygen but not 5% oxygen. This study raises concerns regarding the increasing prevalence of single embryo culture in human IVF and adds to the existing evidence regarding the detrimental effects of atmospheric oxygen during embryo culture. Furthermore, these data demonstrate the cumulative nature of stress during embryo culture and highlight the importance of optimizing each element of the culture system.     

The successful use of human amniotic fluid for mouse embryo culture and human in vitro fertilization, embryo culture, and transfer

The development of mouse and human embryos was assessed in human amniotic fluid to determine its suitability as a culture medium for human in vitro fertilization (IVF). Two-cell mouse embryos developed to blastocysts after 72 hours at rates similar to that in Whittingham's T6 + 10% fetal calf serum. Significantly more mouse embryos hatched in amniotic fluid. No difference was found between individual patient's amniotic fluids obtained at 16 to 21 weeks' gestation. A preliminary trial comparing amniotic fluid with T6 + maternal serum in human IVF showed no significant difference in fertilization rate and embryo development during 42 to 48 hours in vitro. Expanded blastocysts were obtained in amniotic fluid after 5 days in vitro. Four pregnancies were obtained in 9 patients' transferred embryos grown in amniotic fluid and with 2 or 12 patients' transferred embryos grown in T6 + maternal serum.     

Thoughts on embryo culture conditions

This review discusses three topics: (i) the 'back to nature' and empirical optimization approaches to the design of chemically defined media for the culture of preimplantation embryos, (ii) the evolution of the simplex optimized family of media, and (iii) adaptation and stress in preimplantation embryos when placed in chemically defined media.     

Effect of culture medium volume and embryo density on early mouse embryonic development: Tracking the development of the individual embryo

Purpose

To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly.

Methods

(1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining.

Results

The blastocyst development was significantly higher (P < 0.05) when nine embryos were cultured in 50 μl of droplet of culture medium compared with other volumes. The blastocyst development was significantly reduced (P < 0.05) in single embryo culture compared to group embryo culture with or without the WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P < 0.05) in single embryo culture with the WOW system than without.

Conclusions

Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 μl of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced embryonic development with single embryo culture cannot be ameliorated by the WOW system.     

Screening of maternal sera using a mouse embryo culture assay is not predictive of human embryo development or IVF outcome

Purpose Maternal serum is commonly added to media used for human IVF but can vary widely in its ability to support the development of human embryos in vitro.The objective of this study was to determine if the screening of maternal serum with a mouse one-cell embryo culture assay would be useful in predicting human embryo development and clinical outcome following IVF.Methods Twenty-two individual serum samples from IVF patients were used as a supplement (7.5%) to Ham's F-10 media for culturing human embryos. All embryos were evaluated at the time of transfer for stage of development and embryo quality. Each serum sample was also tested for its ability to support mouse embryo development. One-cell embryos were recovered from superovulated female mice and cultured in serum-supplemented media. Mouse blastocyst development was assessed after 96 h of incubation.Results No correlation was found between mouse blastocyst formation and human embryo development in media supplemented with maternal sera. Similarly, there was no association between the development of mouse blastocysts and clinical outcome following IVF. A subanalysis of patients whose sera tested poorly on the mouse assay again revealed no association between mouse and human embryo development.Conclusion Mouse embryo development in media containing human serum did not predict development of human embryos in vitroor clinical outcome following IVF. There would be little benefit to screening maternal sera using a mouse embryo culture system for determining its suitability for use in human IVF.Presented at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5–10, 1994.     

Effect of growth factors in culture medium on the rate of mouse embryo development and viability in vitro

The development of mouse embryos from two cells to blastocysts in Whittingham's T6 medium containing the growth factor insulin or the serum replacement NuSerum was studied and compared with development in vivo. The rate of embryo development to blastocysts was increased by the addition of Nu-Serum to T6 and there was a significant increase in the implantation rate of embryos grown in T6 + Nu-Serum compared with T6 alone or with insulin. However, the rate of embryo development in vitro was retarded compared with that in vivo and the number of normal fetuses following transfer to recipients of blastocysts grown in T6 + Nu-Serum was not different from the number for those grown in T6 alone or with insulin. The addition of neither insulin nor Nu-Serum to T6 optimizes embryo development or viability.     

Building a better mouse embryo assay: effects of mouse strain and in vitro maturation on sensitivity to contaminants of the culture environment

Purpose

The aim of this study is to compare the sensitivity of the standard one-cell mouse embryo assay (MEA) to that using in vitro-matured oocytes from hybrid and outbred mice.

Methods

The study was done by culturing embryos in the presence or absence of two concentrations (0.0005 or 0.001 %?v/v) of Triton X-100 (TX100). Embryonic development, blastocyst cell numbers (total and allocation to the trophectoderm [TE] and inner cell mass [ICM]), and blastocyst gene expression were evaluated.

Results

Neither concentration of TX100 affected (P?>?0.05) cleavage, blastocyst development, or hatching in one-cell embryos from BDF1 mice. However, all cell number endpoints were reduced (P?<?0.05) by the high concentration of TX100 and the number of ICM cells was reduced (P?<?0.05) by the low concentration of TX100. Inhibitory (P?<?0.05) effects of the high concentration of TX100 were observed in in vitro maturation (IVM) embryos from BDF1, CF1, and SW, but not ICR, mice. Cell number and allocation were negatively affected by the high concentration of TX100 in CF1 and SW embryos, but not in BDF1 or ICR embryos. The only developmental endpoints affected by the low concentration of TX100 were cleavage of BDF1 oocytes, blastocyst development of SW embryos, and cell numbers (total and inner cell mass (ICM)) of SW blastocysts.

Conclusions

The sensitivity of the MEA to TX100 is improved by using embryos from in vitro-matured oocytes, using oocytes from some outbred (SW or CF1, not ICR) strains of mice, and evaluating blastocyst cell number and allocation.

     

Alterations in mouse embryo intracellular pH by DMO during culture impair implantation and fetal growth

The preimplantation embryo is highly susceptible to in-vitro stress, and although this does not necessarily perturb blastocyst development, it can significantly affect embryo physiology and the ability to form a viable pregnancy. This study determined that the preimplantation mouse embryo is highly sensitive to a small decrease in intracellular pH (<0.2 pH units). Embryos cultured in media containing a weak acid (5,5-dimethyl-2,4-oxazolidinedione; DMO) formed blastocysts with decreased cell number and inner cell mass number, as well as increased apoptosis, even though blastocyst development and morphology were unchanged. Interestingly, the effects were similar regardless of whether the pH stress was present for a short-term ‘acute’ exposure (during the zygote to 2-cell, or 2-cell to 8-cell division) or an extended ‘chronic’ period of time (continually from the zygote to the blastocyst stage). Exposure to DMO during the first cleavage division did not alter implantation; however, fetal weight and crown–rump length were significantly decreased (P < 0.05). In contrast, continuous exposure to DMO throughout preimplantation development reduced not only implantation but also fetal weight and crown–rump length. This study highlights the importance of correct intracellular pH and demonstrates that slight deviations can significantly impact embryo development and viability.The early embryo is known to be sensitive to its environment. This study determined the effect of a small alteration to pH of the culture environment on the ability of the embryo to grow and develop into a successful pregnancy. We found that the younger the embryo was the less capable it was to cope with the stress. Also embryos that appeared normal by observation were often not and by closer examination were showing signs of stress within the cells of the embryo resulting in poor pregnancy outcomes.     

Evaluation of synthetic serum substitute versus serum as protein supplementation for mouse and human embryo culture

Purpose: Our purpose was to determine the effect of Synthetic Serum Substitute (SSS) versus serum supplementation on fertilization rates and subsequent development of embryos from patients undergoing IVF.Procedure: Experiment I compared the effects of SSS to human serum on mouse embryo development. Two hundred one-cell B6D2F1 mouse embryos were cultured in 100-µl droplets of human tubal fluid (HTF) containing either (1) no protein (control;n=37), (2) 15% serum from women with tubal infertility (n=44), (3) 15% serum from women with endometriosis (n=49), (4) 15% fertile donor serum (n=33), or (5) 15% SSS (n=37). Experiment II compared the effects of SSS to human serum on the development of embryos from patients undergoing IVF. Thirty-three women were included in this study. A total of 371 oocytes was cultured in HTF containing either (1) maternal or donor serum (n=140) or (2) 15% SSS (n=231). Embryo development was evaluated 48 hr after fertilization.Results: In Experiment I, the rate of blastocyst development was evaluated at 48, 72, and 96 hr of culture. Sixty-four and nine-tenths percent of embryos cultured in SSS were morulae at 48 hr of culture (versus 5.4, 0, 8.2, and 6.1 in Groups 1, 2, 3, and 4, respectively). By 72 hr, 29.7% of these embryos had developed into blastocysts (versus 0, 0, 8.2, and 3.0, for Groups 1, 2, 3, and 4, respectively). This percentage increased to a total of 83.7 after 96 hr (versus 27.0, 20.4, 38.8, and 39.4 for Groups 1, 2, 3, and 4, respectively). Forty-three and two-tenths percent of the blastocysts cultured in SSS had hatched from their zonae by 96 hr. With the exception of Group 5, which had a rate of 9.1%, embryo hatching was not observed in any of the groups at the termination of culture (96 hr). In Experiment II there were no differences in cell stage or quality of human embryos cultured in SSS or serum, but fertilization rates tended to be better (P=0.07) for oocytes inseminated in media containing SSS (70.0%, vs 55.0% for serum).Conclusions: SSS appears to be a superior protein source for mouse embryo growth and is as good as serum from fertile donors in promoting in vitro human embryo development.     

A detrimental effect of platelets on mouse embryo development

We reported previously that serum prepared by delayed centrifugation of whole blood (DC serum), which is used widely in human in vitro fertilization and embryo transfer programs, has a detrimental effect on embryonic development. In an attempt to understand the mode of production of embryo-toxic factors in DC serum, we developed a model in which blood coagulation is initially blocked and later induced. The serum prepared in this fashion from whole blood (WB serum) is detrimental to mouse embryo development, as is DC serum. The toxicity of WB serum appears to derive mainly from the platelet release reaction during blood coagulation. Adenosine diphosphate and/or its degradation products may act directly to impair embryonic development and appear to be the main embryo-toxic substance(s) resulting from the platelet release reaction.