Towards a single embryo transfer

The delivery of a single, healthy child is the desired outcome of human assisted reproduction techniques. To attain this goal, there is an increasing movement toward single embryo transfer. The question is, therefore, at what stage to transfer the human embryo back to the uterus? Maximal implantation rates reported to date have come from the transfer of blastocysts (70% fetal heart rate). In any given cycle of treatment the probability of conceiving a child will be further increased by the ability to cryopreserve those embryos not transferred. It is therefore proposed that the transfer of a single blastocyst is the best treatment for most patients, given the high implantation rates of fresh transfers, and that it is now possible to cryopreserve supernumerary blastocysts effectively. The next decision is how to culture the human embryo to the blastocyst stage. The use of sequential culture media, designed not only to allow for changes in nutrient requirements and metabolism as development proceeds, but also to minimize intracellular trauma, can facilitate the development of highly viable blastocysts. Sequential culture media have been evaluated against a single-step culture system. It has been shown that sequential media (G1/G2) produce more viable blastocysts than those embryos cultured in a single medium formulation (simplex optimized medium with elevated potassium and with amino acids, KSOM(AA)) throughout the preimplantation period. Furthermore, even if KSOM(AA) is used for embryo culture, it is essential that the medium be renewed after 48 h to alleviate the toxicity associated with ammonium build-up. Of great significance, embryos cultured in sequential media G1 and G2 have the same rate of development as embryos developed in vivo.     

人类体外受精培养液内毒素水平的评价

目的:检测人类体外受精使用的商品性培养液的内毒素水平,评价人精子存活试验和小鼠2-细胞发育试验测试内毒素的差异。方法:36批次的商品性体外受精培养液,使用前(A组,n=36),及使用后的其中25份样品(B组,n=25)采用鲎试验法检测内毒素水平。另对比人精子存活试验和2-细胞胚胎发育试验的敏感性。结果:A组样品无内毒素阳性检出,B组有2份样品检出内毒素。A组和B组样品24h精子活动率改变与对照组比无显著性差异(P>0.05),但A组中有3份样品抑制小鼠2-细胞胚胎的发育。结论:体外培养环境和实验操作须防止内毒素污染。虽然商品性培养液未检出内毒素,仍须对其作严格的质量控制。人精子存活试验测试培养液质量和低内毒素水平的敏感性低于小鼠2-细胞发育试验。     

A comparative study on the diagnostic sensitivity of rodent sperm and embryos in the detection of endotoxin in earle's balanced salt solution

Purpose The bioassay used by most IVF units to assess culture media is the mouse embryo test. The limitations of this assay are well known. The objective of this study was therefore to evaluate the in vitro bioassay potential of rodent sperm, in terms of relative sensitivities to endotoxins, and to compare the results with the routine mouse embryo and human sperm tests.Results The greater sensitivity of rodent sperm than of mouse embryos was evident in this study. A further advantage in using the mouse sperm test was the time (4–6 hr) in which endotoxins could be detected.Conclusion This rapid sperm test proved to be inexpensive, convenient, and invaluable for detecting potential sources of cytotoxicity.     

Individual demands of human embryos on IVF culture medium: influence on blastocyst development and pregnancy outcome

The elucidation of the metabolic requirements of human embryos in vivo or in vitro remains, despite being intensively investigated, a work in progress. The adoption of extended embryo culture to the blastocyst stage during the last decade has entailed new challenges. With the increased attention to culture media formulations, more evidence on the sensitivity of embryos to their early environmental conditions is accumulating which might affect phenotype and developmental potential. A retrospective study was conducted that comprised 286 IVF cycles to evaluate the effect of two different culture media on blastocyst development and pregnancy outcome. Embryos were either cultured in a one step or a sequential medium. Higher fertilization rates and augmented blastocyst rates as well as higher implantation rates were observed when embryos were cultured in one step medium (P<0.05). Interestingly, the transfer of two embryos where one embryo was cultured in either medium resulted in a significantly higher rate of twin pregnancies. Although multiple pregnancies should be avoided in assisted reproduction treatment to reduce risks for offspring and mother, this higher frequency of twin pregnancies resulting from the transfer of embryos derived from different culture media suggests that each embryo makes individual demands on its early environment.     

Embryo nutrition and energy metabolism and its relationship to embryo growth, differentiation, and viability

Over the past decade there has been a resurgence of interest in the culture media used in clinical in vitro fertilization. Unfortunately, during this time more confusion than consensus appears to have developed regarding the composition of these media. In order to facilitate a clearer understanding of this field, it is important to understand the role of specific medium components and how their use is regulated by the embryo. The roles of the key nutrients glucose, pyruvate, lactate, and amino acids during the preimplantation period have therefore been presented. Analysis of how the embryo regulates the utilization of such nutrients has led to a clearer understanding of the embryo's requirements during the dynamic period of preimplantation development. From such information, sequential culture media have been developed along with novel noninvasive tests of embryonic viability. It is proposed that continued studies on the human embryo will lead to further improvements in embryo culture conditions and the optimization of viability assays, culminating in the ability to transfer single embryos for the majority of, if not all patients.     

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.     

Embryo culture medium: which is the best?

With the growing move in in-vitro fertilization (IVF) clinics to transfer fewer embryos to women, there is an increasing reliance on the IVF laboratory to maximize embryo viability. Subsequently, there is justified scrutiny on the culture system and the media used to sustain the human embryo in vitro. The transfer of fewer embryos to patients also creates an increased dependence on the ability to cryopreserve embryos successfully. Therefore, in addition to the ability of a culture system to produce a single top-quality embryo for transfer, it is also necessary to enhance the cryotolerance of sibling embryos so that they can survive freezing or vitrification. Therefore, when examining which culture media is the best, it is prudent to not only examine the ability of a culture system to produce a pregnancy with the one or two highest-grade embryos, but also to determine how many embryos from the entire cohort (both fresh and frozen embryos) are capable of producing a live birth. Additionally, research on animal models has demonstrated that stress, and the resultant adaptation to conditions during pre-implantation stages, can affect pregnancy loss and fetal growth. It is therefore important to understand the role of each medium component and to identify possible sources of cellular stress to the embryo that will ultimately affect the function and viability of the conceptus.     

人精子存活试验对培养液内毒素敏感性的评价

目的:探讨人精子培养试验检测培养液内毒素的敏感性。方法:活动精子分别与6个内毒素浓度(0.5 ng/mL,1 ng/mL,10 ng/mL,1 000 ng/mL,10 000 ng/mL,50 000 ng/mL;n=10)共培养,检测培养后的精子活动率,比较实验组与对照组的精子活动率改变,并观察不含白蛋白培养液的3种内毒素浓度(1 000 ng/mL,10 000 ng/mL,50 000 ng/mL;n=6)对精子活动率的影响。另对比小鼠1-细胞胚胎和2-细胞胚胎培养试验对内毒素(0.5 ng/mL,1 ng/mL,10 ng/mL)的敏感性。结果:精子培养24 h后,在含白蛋白的内毒素浓度为0.5-1 000 ng/mL的4组中,实验组的精子活动率与对照组的比较没有显著性差异(P>0.05),但在内毒素浓度为10 000 ng/mL和50000ng/mL组,精子活动率则显著降低(P<0.01)。在不含白蛋白的内毒素浓度为50 000 ng/mL组和10 000 ng/mL组中,培养2 h和8 h的精子活动率分别显著降低(P<0.01)。在培养液内毒素浓度为0.5 ng/mL组和1 ng/mL组,小鼠1-细胞胚胎和2-细胞胚胎各发育阶段的发育率均显著减少,内毒素对胚胎发育的抑制呈剂量效应。结论:人精子存活试验可检测培养液中较高的内毒素水平,其敏感性比小鼠1-细胞胚胎和2-细胞胚胎培养试验的低。培养液中不含白蛋白可提高精子对内毒素的敏感性。     

What does an embryo need?

This review argues that the question "What does an embryo need?" cannot be adequately answered in quantitative terms to allow the formulation of media for culturing early mammalian embryos. It can be shown experimentally that "needs" in terms of the nutrients an embryo chooses to consume, and their rates of consumption, vary widely, as they are determined by the concentration of the nutrients under consideration and other constituents in the culture medium. Similarly, it is impossible to define "needs" from knowledge of the kinetic properties of nutrient transport systems. Measurements of nutrient consumption, are, however, valuable in determining overall metabolic activity and the balance between oxidative and glycolytic metabolism, in demonstrating qualitative requirements for specific nutrients and in providing markers of normality or abnormality against which to devise methods for diagnosing embryo health. On the basis of these and other considerations, a strategy is proposed for the formulation of embryo culture media that promotes metabolism that is "quiet" rather than "active", reduces the concentrations of nutrients to match those in the Fallopian tube, selects the "quietest" embryos for transfer, and trusts the autonomy of the embryo.     

Quality control in IVF with mouse bioassays: A four years' experience

Objective: We analyzed retrospectively the relevance of 4 years of quality control on homemade culture medium with the mouse IVF and zygote bioassay. Design: In vitro or in vivo fertilized mouse oocytes were cultured in each batch of medium. Two-cell-stage and expanded blastocyst development was recorded for each batch of medium. Data on fertilization and embryo quality obtained in human in vitro fertilization were recorded for each batch. IVF treatment cycles for male infertility and cycles with sperm microinjection were excluded. Results: Human oocyte fertilization dropped from 60 to 54%, respectively, from 57 to 41% in a significant way (P<0.05 resp. P<0.01) and the human mean embryo score decreased from 4.17±1.21 to 3.69±1.06 (P<0.05) when media were used with a low two-cell-stage development (75%) for the mouse zygote or mouse IVF bioassay. The pregnancy rate was not affected. Conclusions: Media with high scores in mouse bioassays show higher fertilization rates and better embryo quality when used for human IVF.     

The use of synthetic culture medium and patient serum for human in vitro fertilization and embryo replacement

The use of heat-inactivated patient serum as both fertilization and embryo replacement medium was compared in a prospective randomized study with a fully synthetic culture medium containing human serum albumin without serum addition (B₃ INRA Menezo). Another series of the author's IVF program was analyzed retrospectively when a commercially available synthetic medium with bovine serum albumin (B₂ INRA Menezo) or B₃, as mentioned above, was used without serum addition for fertilization but with 50–100% patient serum as embryo replacement medium. Fertilization rates were significantly higher in the synthetic culture media (70%) than in serum (57%). The rate of polyploid fertilization was significantly lowest in B₃ medium. There was a clear trend toward better pregnancy rates when high-percentage or 100% patient serum was used for embryo replacement, no matter if one, two, three, four or more embryos were replaced. We conclude that there should be no need for any kind of serum addition to fertilization media. The present study proves our previous observation that the use of serum seems to be beneficial as embryo replacement medium. This might well be explained by a protein stick effect due to the high macromolecular contents of serum rather than by viscosity measurements, since no significant increase in viscosity was observed when a high percentage of serum was added to culture media.     

Estimation of embryo transfer media viscosity and consideration of its effect on media and uterine fluid interactions

Research questionWhat are the viscosities of media used for human embryo transfer and what is the possible effect of viscosity as it relates to interactions between transfer media and uterine fluid.DesignChamber slide filling times, in seconds, were used to calculate viscosity for each commercial and in-house modified medium, with 12 or 24 replicates per medium under standard operating procedure temperature and gas equilibration conditions used for embryo transfer. Means, standard deviations and coefficients of variation were calculated, and each viscosity was estimated using a regression equation; viscosities for each medium were presented for comparative purposes.ResultsComplete culture media (G1-Plus, G2-Plus, G-TL, 1-Step, Global Total, Global Total HEPES, and Sperm Wash Medium) had viscosity estimates of 1.65 cP, 1.77 cP, 1.68 cP, 1.29cP, 1.18 cP, 1.15 cP, and 1.20 cP, respectively. Complete transfer media (EmbryoGlue, UTM), had viscosity estimates of 3.59 cP and 1.28 cP, respectively. Global HEPES medium with 10%, 20%, 30%, and 50% synthetic serum substitute (SSS) volume per volume had viscosity estimates 1.16 cP, 1.23 cP, 1.25 cP, and 1.34 cP, respectively. For reference, water had a viscosity estimate of 1.06 cP.ConclusionsA relatively narrow distribution of viscosities was observed across several transfer media despite the various commercial or in-house modifications. These data demonstrate the vast difference between viscosities of embryo transfer media and the assumed viscosity of uterine fluid (1000 cP). Contemporary embryo transfer media may be well-suited for IVF, but evaluation of all variables, e.g. media viscosity in the context of embryo transfer, adds to the knowledge base that should be available to practitioners.     

Andrology in 2010

This review argues that the question ‘What does an embryo need?’ cannot be adequately answered in quantitative terms to allow the formulation of media for culturing early mammalian embryos. It can be shown experimentally that ‘needs’ in terms of the nutrients an embryo chooses to consume, and their rates of consumption, vary widely, as they are determined by the concentration of the nutrients under consideration and other constituents in the culture medium. Similarly, it is impossible to define ‘needs’ from knowledge of the kinetic properties of nutrient transport systems. Measurements of nutrient consumption, are, however, valuable in determining overall metabolic activity and the balance between oxidative and glycolytic metabolism, in demonstrating qualitative requirements for specific nutrients and in providing markers of normality or abnormality against which to devise methods for diagnosing embryo health. On the basis of these and other considerations, a strategy is proposed for the formulation of embryo culture media that promotes metabolism that is ‘quiet’ rather than ‘active’, reduces the concentrations of nutrients to match those in the Fallopian tube, selects the ‘quietest’ embryos for transfer, and trusts the autonomy of the embryo.     

Parameters of the Mouse Embryo Assay that affect detection of peroxides in mineral oil

Research questionCan culture conditions influence the sensitivity of a Mouse Embryo Assay and its potential to detect peroxide-related toxicity in mineral oil samples?DesignProtein type and concentration, embryo density and culture dish design were selected as the variables in the culture system with the potential to influence the assay's sensitivity. Fresh 1-cell mouse embryos were cultured under mineral oil samples with known peroxide concentrations. Protein type (human serum albumin [HSA] + α/β-Globulins versus HSA versus bovine serum albumin [BSA]), concentration (5 mg/ml versus 0.5 mg/ml), embryo density (25 versus 3 µl/embryo) and culture dish (Petri versus micro-well dish) were adjusted to define the culture conditions with the highest sensitivity.ResultsHigh concentrations of peroxides can be easily detected by current quality control standards. However, for oil samples with a lower concentration of peroxides, supplementing the culture medium with 5 mg/ml of HSA + alpha/beta-globulins or with HSA resulted in an increased detection of embryo toxicity compared with when BSA was used as the protein supplement. The sensitivity of the assay was greatly reduced when embryos were cultured in groups and when certain micro-well dishes were used.ConclusionsCurrent quality control protocols may not be sensitive enough to identify low concentrations of peroxides, which, if undetected, can increase over time and become potentially harmful during gamete and embryo culture. The different parameters established in this study allow the sensitivity of the Mouse Embryo Assays to be optimized to specifically detect peroxides in mineral oil samples prior to their release into the market and their broad use in human IVF.     

Effects of culture medium on HCG concentrations and their value in predicting successful IVF outcome

The hypothesis was tested that the medium used to culture embryos affects the concentration of human chorionic gonadotrophin (HCG) early in pregnancy. The value of these concentrations in predicting successful outcome was also assessed for each medium studied. Patients undergoing IVF between January 1998 and December 2004 and having a day 3 embryo transfer were stratified into one of four groups according to the medium in which their embryos were cultured (P1, IVF500, G1.2, and G1.3). Using receiver operating characteristic (ROC) curve analysis, cut-off values for serum HCG concentrations on day 15 after embryo transfer were calculated for optimal discrimination between cycles resulting in implantation failure and success for each medium. Cut-off points were chosen to maximize sensitivity and specificity. For viable singleton pregnancies, mean HCG concentrations were greater for G1.3 and lower for IVF500 compared with the other media. Discriminatory HCG cut-off concentrations for predicting implantation success were lowest for IVF500, intermediate for P1 and G1.2 and highest for G1.3. The data support the hypothesis that the medium used to culture embryos significantly affects the concentrations of HCG early in pregnancy. Furthermore, when using HCG cut-off concentrations to assess pregnancy outcome, medium type should be taken into consideration.     

Embryo-derived platelet activating factor,a marker of embryo quality and viability following ovarian stimulation for in vitro fertilization

Embryo-derived platelet-activating factor (PAF) may be an important mediator of early maternal recognition of pregnancy. PAF was higher in media associated with clinical pregnancies when compared to preclinical pregnancies but not higher in pregnant vs nonpregnant groups. The production of PAF by the preimplantation embryo was not related to follicle size or embryo morphology. However, differences in PAF concentrations in the culture media were related to the age of the embryo culture medium and the developmental stage of the embryo.     

Improvement in early human embryo development using new formulation sequential stage-specific culture media

OBJECTIVE: To determine whether altering selected components of sequential culture media can improve early development variables of human embryos. DESIGN: Prospective, randomized, sibling oocyte split trial. SETTING: Private ART center. PATIENT(S): Two hundred eight undergoing treatment with in vitro fertilization or microinjection. INTERVENTION(S): Oocytes from each patient were randomly allocated to fertilization and cleavage media of a control and a trial culture medium formulation. MAIN OUTCOME MEASURE(S): Rates of fertilization, cleavage, and uncontrolled division; average embryo morphology score; blastomeres per embryo; embryo score parameter (number of blastomeres x embryo morphology grade); and embryo utilization.The trial media resulted in a higher fertilization rate, higher cleavage rate, lower rate of uncontrolled division, higher number of blastomeres per embryo, higher average embryo morphology score, a higher embryo score parameter, and higher embryo utilization rate compared to the control media. All differences were statistically significant. CONCLUSION(S): Improved sequential stage-specific culture media can reduce the occurrence of severe human embryo fragmentation and improve developmental variables in early IVF- and ICSI-generated embryos.     

序贯培养在体外受精-胚胎移植技术中的应用

目的: 试用序贯培养方法延长胚胎体外培养时间,以获得８细胞胚及囊胚进行移植,提高体外受精－胚胎移植（ＩＶＦ－ＥＴ）的成功率,方法：将行ＩＶＦ－ＥＴ的１６１例患者１８２３个卵子分为序贯培养（使用Ｐ１液、Ｂ液系列培养液,４７例,４７个]周围）与传统培养（１１例,１１４个周期）两组进行培养,分析受精率,卵裂率,卵裂速率,优质胚胎比率及率及临床妊娠率,采用x^2体验进行统计学分析,结果：序贯培养组卵裂率,优质胚胎比率,胚着床率及妊娠率分别为９７．９％,６４．４％,１９．１％和４６．７％,结论：采用适事的培养组分别为８９．７％,４０．２％,１１．０％和２８．２％,两组比较,差异有显著性（Ｐ＜０．０５）,结论：采用适合的培养液进行贯培养,更适合于卵子及胚胎的体外发痛,方法安全可行。     

Connections between preimplantation embryo physiology and culture

Purpose

To review the history of experimental embryo culture and how culture media that permitted complete preimplantation development in vitro were first discovered, and the physiological insights gained.

Methods

This article reviews the history of in vitro mammalian embryo culture, in particular the efforts that led to the current generation of successful culture media and how these reflect embryo physiology, highlighting the contributions of Dr. John D. Biggers and his colleagues and students.

Results

The culture of mammalian embryos began about a century ago. However, defined media without biological fluids were only developed in the late 1950s, and the first live young born from cultured embryos, using these media, were produced by McLaren and Biggers in 1958. It wasn’t until the late 1980s, however, that preimplantation mammalian embryos could generally be cultured in vitro from fertilized eggs to blastocysts. These new media led to insights into embryo physiology, including the importance of cell volume homeostasis to early embryo viability.

Conclusions

The development of successful preimplantation embryo culture media has had a profound effect on assisted reproduction technologies and on research into early embryo physiology.     

Human amniotic fluid for fertilization and culture of human embryos: Results of clinical trials in human in vitro fertilization (IVF) programs

We report the outcome of clinical trials carried out in two IVF programs, comparing the use of human amniotic fluid (HAF) as a complete medium to Whittingham's T6 medium containing human serum T6+10% HS) for egg incubation, insemination, embryo culture, and embryo transfer. There were no significant differences in the clinical trials between HAF used alone as a complete medium and T6+10% HS in fertilization rates of eggs, cleavage rates of embryos up to 48 hours in culture, pregnancy success rates after embryo replacement or the outcome of pregnancies. There was no advantage in using T6+10% HS for fertilization of eggs and HAF as a complete medium for embryo culture and transfer in any of the parameters examined. We conclude that HAF does not meet the complete requirements of human eggs and embryos in vitro and further developments of culture media are required to obtain embryo development equivalent to that in vivo.