No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Towards a single embryo transfer
Abstract
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.
... These diverging concepts continue today, although the increase acceptance of single-step media, especially in conjunction with time lapse imaging [49,50], has spurred more companies to develop additional single use media. Both Gardner and Lane [19,21,25], as well as Biggers and associates [34,48,51] provided us with sound science supporting their formulations and aimed to minimize embryo stress through consistency in amino acid and antioxidant supplementation, as well as using stable dipeptides of L-glutamine that minimized ammonium waste-product production [26,34,35,52]. Knowing that the original sequential media provider (Vitrolife, G-series1/2 media) and single-step media supplier (LifeGlobal ® , LG medium) applied good science and superior quality control practices in the development, formulation, and manufacturing of their products, we focused our development of an optimized embryo culture system to these products. ...
... As media products and incubator systems improved, so too did dependable in vitro blastocyst production. Most IVF programs experienced increases in implantation and live rates by converting to Day 5 ET [20,52,56,57], being significantly better than Day 3 ET as confirmed in our early analysis (Table 3). We experienced consistently good blastocyst production and high pregnancy outcomes with mLG, being equal to or better than sequential media treatments, consistent with the reported results of several other groups [51]. ...
... Following the optimization of our blastocyst culture system with mLG/TG incorporating Ovoil™ (Expt. 4), we were even more confident to strive toward a practice of single embryo transfer as advocated by Gardner and Lane [52] years earlier. Certainly, the concurrent development, validation, and implementation of highly reliable vitrification [44,45] and blastocyst biopsy [41,43] procedures has allowed us to confidently implement single ET with unprecedented success, as reflected in the outcome verification of our FET cycles in Figure 2B. ...
Objective: To develop and validate a reliable in vitro culture system for human embryos. Design: Retrospective analyses of a series of four studies were conducted between 2006 and 2010 to assess the effect of incubator type (CO2 box versus Tri-gas minibox), media type, oil type, and hyaluronate supplementation. Optimization of in vitro blastocyst development was verified by assessing our National CDC/ART Surveillance reports between 2010 and 2016. Material and Methods: All patients experienced controlled ovarian hyperstimulation, followed by egg retrieval 35 h post-hCG. Cumulus-oocyte complexes were temporarily cultured in P1 or LG Fert medium plus HSA. Eggs were moved to a more complex media (G-medium or Global®-LG medium) containing a synthetic protein and embryo adhesion supplement (SPS and EAS, respectively; mLG) post-ICSI insemination. Zygotes were assigned to group culture in 25 µl droplets under oil (light mineral oil or paraffin oil; 37 °C) and embryo development was evaluated on Days 3, 5, and 6 and transferred on Day 3 to 5 depending on the number/quality of embryos available and the IVF history of the patient. Transfers were performed under ultrasound guidance, primarily using a Sureview-Wallace catheter, and enriched ET medium containing 500 µg/mL EAS. Results: Pilot study results (Expt. 1) showed that a mLG single-step medium could be effectively used in combination with Sanyo MCO-5 tri-gas (TG) incubators. Once adapted to SCIRS Lab in 2007 (Expt. 2), the latter culture system yielded improved blastocyst production and pregnancy outcomes compared to CO2 in air sequential incubation in P1/Multi-blast medium. In Expt. 3, the mLG/TG system yielded high levels of ≥2BB quality blastocysts (51 to 66%) across all age groups, and greater (p < 0.05) pregnancy success/live birth rates using fewer embryos transferred on Day 5 versus Day 3. After validating its clinical effectiveness, mLG was then prospectively compared to a new generation G-media (1.5 & 2.5; Expt. 4) and determined that the crossover treatment using paraffin oil (Ovoil™) allowed the mLG system to be optimized. Subsequently, a compilation of our Annual CDC/ART reported data over six years verified the overall viability of in vitro cultured and vitrified blastocysts produced in the mLG/TG system. Conclusion: By systematically evaluating and implementing various components of an embryo culture system we were able to optimize blastocyst development over the last decade. Our mLG/TG culture system modified an exceptionally well designed KSOMAA LG medium using endotoxin-free EAS and SPS additives to support cellular membrane wellness under stressful in vitro conditions (e.g., culture, cell biopsy, vitrification). Our use of the mLG/TG culture system has proven to be effective, creating reliably high blastocyst production, implantation, and healthy live births.
... What followed was extremely interesting; many laboratories were able to repeat the initial studies, others found no difference between blastocyst and cleavage-stage transfers and a minority actually reported that blastocyst culture gave inferior outcomes (Gardner and Balaban, 2006). Understanding the basis for this apparent conundrum led to the concept of considering the "embryo culture system" rather than simply embryo culture media, as it was evident that all aspects of the laboratory could have an impact on the effectiveness of the culture media and hence alter outcomes (Gardner and Lane, 2003). Variables evaluated included oxygen concentration, protein source, types of laboratory ware and embryo grouping versus single culture (to name but a few variables). ...
... Variables evaluated included oxygen concentration, protein source, types of laboratory ware and embryo grouping versus single culture (to name but a few variables). In light of this "holistic approach" a request went out to the IVF community (Gardner and Lane, 2003) that all aspects of the culture system be reported in publications in an attempt to better understand and interpret the emerging data from different clinics, and to identify which factors were responsible for differences in IVF outcomes even when the same media were employed. ...
... In all mammalian species studied to date the asynchronous spatial transfer of cleavage-stage embryos to the uterus compromises resultant fetal development (Barnes, 2000;Walker et al., 2015), an observation not commonly acknowledged by the clinical IVF community. Hence, the development of more suitable and physiological culture systems that could support the development of viable human blastocysts was undertaken within the context of synchronicity, in order to increase implantation and live birth rates and to facilitate the move to single embryo transfer (Gardner and Lane, 2003). Further advantages of extended culture included the ability to identify the more viable embryos within a cohort (Gardner and Lane, 2003), the application of grading systems to quantitate both the inner cell mass and trophectoderm (TE; Gardner et al., 2000), the transfer of the embryo to the uterus at a time of decreased endometrial contractions (Fanchin et al., 2001) and the ability to undertake TE biopsy to facilitate preimplantation genetic screening (Fragouli et al., 2008). ...
Extended culture has facilitated the move to single blastocyst transfer, resulting in significant increases in implantation and live birth rate, while concomitantly reducing fetal loss during pregnancy. However, concerns have been raised regarding subsequent neo-natal outcomes following extended culture. Analysis of the literature reveals differences in outcomes according to geographical region and between individual clinics. A common factor amongst reports of potentially adverse outcomes following blastocyst transfer appears to be that atmospheric (~20%) oxygen was typically employed for embryo culture. Clinics and countries utilizing physiological concentrations of oxygen (~5%) have not reported adverse perinatal outcomes with blastocyst transfer. Atmospheric oxygen imposes significant negative effects upon the embryo's molecular and cellular physiology, and further it increases the sensitivity of the preimplantation embryo to other stressors in the laboratory. With the recent adoption of vitrification for blastocyst cryopreservation, cumulative pregnancy rates per cycle with extended culture will increase significantly. Consequently, rather than perceiving extended culture as a potentially negative procedure, it is concluded that neo-natal data need to be interpreted in light of the conditions used to culture and cryopreserve blastocysts, and that furthermore a policy of embryo culture using 20% oxygen can no longer be justified.
... These factors include patient age, genetics, aetiology of infertility, diet, smoking/drug habits and ovarian stimulation. 1 With regards to ovarian stimulation, this approach overrides the dominant follicle, thereby creating a heterogeneous pool of oocytes, which increases the variability of gamete quality. Furthermore, the use of exogenous gonadotropins has been implicated in the induction of epigenetic alterations in the resultant oocytes and embryos, 2,3 thereby contributing further variability and stress to the system, hence making it difficult to determine the precise effects of specific laboratory conditions. ...
... Extensive data from animal models have clearly shown an effect of medium composition on the kinetics of embryo development and physiology. 1,[94][95][96] The composition of current culture media used in human IVF are based around balanced salt solutions, a sodium bicarbonate/carbon dioxide buffer system, a protein source currently in the form of serum albumin (formerly in the form of serum), amino acids, vitamins and in some cases growth factors. 94,97,98 Utilizing time-lapse microscopy, two studies have considered the effects of culture media on human embryo development. ...
The phenotype of the human embryo conceived through in vitro fertilization (IVF), that is its morphology, developmental kinetics, physiology and metabolism, can be affected by numerous components of the laboratory and embryo culture system (which comprise the laboratory environment). The culture media formulation is important in determining embryo phenotype, but this exists within a culture system that includes oxygen, temperature, pH and whether an embryo is cultured individually or in a group, all of which can influence embryo development. Significantly, exposure of an embryo to one suboptimal component of the culture system of laboratory typically predisposes the embryo to become more vulnerable to a second stressor, as has been well documented for atmospheric oxygen and individual culture, as well as for oxygen and ammonium. Furthermore, the inherent viability of the human embryo is derived from the quality of the gametes from which it is created. Patient age, aetiology, genetics, lifestyle (as well as ovarian stimulation in women) are all known to affect the developmental potential of gametes and hence the embryo. Thus, as well as considering the impact of the IVF laboratory environment, one needs to be aware of the status of the infertile couple, as this impacts how their gametes and embryos will respond to an in vitro environment. Although far from straight forward, analysing the interactions that exist between the human embryo and its environment will facilitate the creation of more effective and safer treatments for the infertile couple.
... Em caprinos uma relação positiva entre redução do peso vivo e a quiescência ovariana (anestro + anovulação) induzida pela restrição alimentar tem sido demonstrada para [34]. Estes autores conforme precedentes observações Figura 2. Intervalos em dias de sensibilidade nutricional em função do diâmetro folicular. ...
... Outro estudo observou que a ação de retinóides no estímulo para o desenvolvimento embrionário pode ser por meio das células somáticas na co-cultura de embriões caprinos [21]. Meios de cultivo seqüenciais têm sido amplamente usados em humanos [34,117] e em bovinos é uma opção viável [62]. Em ovinos observou-se que o uso de um meio seqüencia comercial (G1.1/G2.2) aumentou a taxa de desenvolvimento, mas prejudicou a qualidade dos embriões, avaliada após criopreservação [33]. ...
Background: A better comprehension of the relationship between nutrition and ovarian function is a fundamental key to optimize the reproductive parameters. In ruminants it is well known and recognized that nutrition is a very important mediator at the ovary level. Thus, nutritional balancing is a critical essential condition in assisted reproduction technologies when used to support improves reproductive efficiency. In many regions of Brazil, the main drawback of ovine and caprine husbandry continues to be how to sustain the nutritional status during the prolonged seasonal food shortage. In these areas the use of hormonal treatment for both estrus and ovulation synchronization is subordinate to supplementation availability or the body condition of females. Changes of nutrient intake before mating can significantly increase in the superovulatory response and modify the number and quality of embryo produced in vivo. The objective of this paper is to review the effects of nutrition on some parameters, as follicular development, oocyte quality and embryo production with particular emphasis on effects in small ruminants. Review: It was discussed the most recent available model in ewes for nutritional stimulations of folliculogenesis and regulation of metabolic factors such as insulin-glucose, leptin and IGF. Also it was introduced a novel scheme of nutritional action (acute, dynamic and static effects). Regarding the recent progress of follicular development was illustrated the effect of nutritional restriction in utero on fetal early folliculogenesis. In goats findings showed that the initial steps of follicular growth are very sensible to the food restriction applied for short period. It has been shown that energy balance is a crucial condition for assisted reproductive tools as hormone treatments for the synchronization of estrus. Hence, using several experimental data of relationships between anoestrus and body mass reduction, it was estimated a values of body condition score between 2 and 2,5 as a crucial point for the onset of nutritional anoestrus in goats and sheep. Current researches have been demonstrated in sheep that unbalanced diet, excess or restriction of feeding, interferes in the process of oocyte capacitation and its gene expression. Specifically, such short-term nutrient restriction reduced expression of glucose transporter in oocytes, and increases the leptin receptor in granulosa cells. Reduced expression of glucose transporter is essential for post-implantation embryonic development. Extra-ovarian factors, such as nutritionally mediated changes in metabolic hormones, also directly affect follicle development and oocyte quality. Therefore it is important differentiate diets optimal for follicle growth or oocyte quality and optimal for embryo survival, because these nutritional conditions may be opposite. The current trend to increase the productive levels and to develop the husbandry production is fix the nutritional management at key stages in the reproductive process, provides an effective way to develop long term feeding strategies which enable animals to maximize their fertility. Conclusion: An expressive advance of research provide in these years useful information about the nutritional effort on reproductions in small ruminants and suggest practical aspects to be considered as fundamental prerequisite to the reproduction control in goats and sheep herd.
... Em caprinos uma relação positiva entre redução do peso vivo e a quiescência ovariana (anestro + anovulação) induzida pela restrição alimentar tem sido demonstrada para [34]. Estes autores conforme precedentes observações Figura 2. Intervalos em dias de sensibilidade nutricional em função do diâmetro folicular. ...
... Outro estudo observou que a ação de retinóides no estímulo para o desenvolvimento embrionário pode ser por meio das células somáticas na co-cultura de embriões caprinos [21]. Meios de cultivo seqüenciais têm sido amplamente usados em humanos [34,117] e em bovinos é uma opção viável [62]. Em ovinos observou-se que o uso de um meio seqüencia comercial (G1.1/G2.2) aumentou a taxa de desenvolvimento, mas prejudicou a qualidade dos embriões, avaliada após criopreservação [33]. ...
... Tiñendo los núcleos, se puede contar el número de células de la masa celular interna. (43,44) I Test de supervivencia de espermatozoides humanos Otra opción más amigable, es el Test de supervivencia de espermatozoides humanos, pero éstos no son muy sensibles a niveles bajos de toxinas, por lo que se recomienda usar al menos dos tests para probar materiales sospechosos (el de supervivencia espermática y el de embriones de ratón, por ejemplo). Esta prueba tiene la ventaja de que los espermatozoides humanos se encuentran disponibles fácilmente en los laboratorios de andrología. ...
... En el Colorado Center for Reproductive Medicine sí la hacen de rutina y han encontrado que el 25% de los materiales no pasa el Test de embriones de ratón, a pesar de tener una certificación de origen. Esto demuestra la importancia de volver a probar los materiales y reactivos al llegar al laboratorio, antes de utilizarlos para la FIV.(43,44) Enjuagar previamente con medio los insumos plásticos a utilizar disminuye los efectos tóxicos.(45)Wiemer y colaboradores desarrollaron un sistema complejo de inventario con una política estricta de introducción de insumos al laboratorio. ...
... Mammalian embryos were suffered to decreased development potential and changes in metabolism and gene expression in vitro [3,4], which may be due to some toxic substances or adverse stress [14]. Ammonium is one of the factors that have been shown to influence embryonic developmental competence [9]. ...
PurposeTo determine the association between ammonium concentration in culture medium and blastocyst development and to assess the influence of increased ammonium concentration on the expression of Bax, Bcl-2 and Oct4.MethodsA total of 254 cleavage-stage embryos were individually cultured in 30μL G2-plus medium on Day 3, and then culture media samples were collected on Day 5 for ammonium concentration determination immediately after evaluating the embryos morphology. Poor-quality blastocysts (combined score of CC) were used for gene expression analysis. The blastocyst formation rate, good-quality blastocyst rate and relative expression levels of Bax, Bcl-2 and Oct4 were analyzed.ResultsBased on receiver operating characteristic curve, the cutoff value of ammonium concentration produced by embryos was 16.07 μmol/L (AUC = 0.722, 95% CI 0.637–0.807; P = 0.000), so all embryos were assigned to two groups according to the cutoff value: normal group (< 16.07 μmol/L) and increased group (≥ 16.07 μmol/L). There was a significant difference in blastocyst formation rate (80.5% vs 59.0%, P < 0.01) between normal group and increased group, as well as for good-quality blastocyst rate (21.0% vs 3.4%, P < 0.01). A significantly higher expression level of Bax (P < 0.05) and considerably lower expression level of Oct4 (P < 0.01) were observed in increased group compared to normal group.Conclusion
Our data demonstrated for the first time that increased ammonium concentration in culture medium may promote cellular apoptosis and negatively affect pluripotency of human blastocyst.
... Despite these improvements, the birth rate per transfer remains relatively low at ∼30% (Kupka et al., 2014;Sunderam et al., 2014). The multitude of factors impacting ART encompass all components of the in-vitro culture system (Gardner and Lane, 2003) presenting compounding stresses and causing synergistic damage to embryo function and health (Gardner and Kelley, 2017;Wale and Gardner, 2015). For instance, culturing embryos at atmospheric oxygen (∼20%) is a welldocumented stress harmful to embryo development and viability (Bontekoe et al., 2012). ...
Background: Embryo culture and cryopreservation causes detrimental effects on blastocyst gene expression. While combined antioxidants (acetyl-L-carnitine/N-acetyl-L-cysteine/[Formula: see text]-lipoic acid (A3)) in culture and vitrification media have beneficial effects on embryo and fetal development, their impact on fetal gene expression has not been assessed. Therefore, the effects of antioxidants on global gene expression were examined from fetal and placental tissue of transferred blastocysts and compared to in vivo derived embryos.
Aim: To determine the effect of A3 in culture and vitrification media on mouse blastocyst, fetal and placental gene expression.
Method: Embryo transfers were conducted on in vivo flushed blastocysts or blastocysts cultured or vitrified with and without A3. Transcriptional profiles of E14 fetal liver and placental tissue in all groups were quantified using RNAseq and functional analyses (KEGG).
Results: Compared to in vivo derived embryos, in vitro culture altered the expression of 3601 fetal liver and 408 placental genes. Functional analysis showed upregulation/enrichment of oxidative phosphorylation and mitochondrial function and activity. Similarly, vitrification led to 2018 liver and 216 placental differentially expressed genes (DEGs). Upregulated KEGG pathways were enriched for cell and tissue development and cell cycle regulation. Down regulated pathways were associated with metabolism and immune response. Interestingly, exposure to A3 during development significantly reduced the number of DEGs with 1855 and 4 identified in liver and placentae respectively with no KEGG pathways identified. Correspondingly, exposure to A3 during vitrification reduced the number of DEGs to 1017 in liver and 206 in placentae. Functional pathway enrichment was similar to embryos vitrified without A3, although with decreased expression.
Conclusion: Supplementation with antioxidants reduce the number of DEGs and biological processes altered by in vitro culture and vitrification, in fetal liver and placentae, establishing a more in vivo like gene expression profile which may help maintain the viability of vitrified human embryos.
... However, what is evident is that media are but 1 aspect of the embryo culture system. 11 To support the development of a viable and healthy embryo capable of resulting in a healthy offspring, one needs to look There is an absolute necessity for an effective quality management and assurance system to ensure that variables such as equipment, laboratory air quality, oil overlay, and lot numbers of consumables do not negatively impact embryo health. 12 far beyond the formulation of the culture media employed. ...
Embryo production has proven to be a powerful technology for genetic improvement of dairy animals, primarily to propagate the genes of females with superior genetic values and lineage. Despite the Covid Pandemic, 2020 was a good year for the embryo transfer industry worldwide. 1 Number of embryos recorded increased in most countries and for all of the most representative species (cattle, horses, sheep, and goats). In cattle, overall embryo production (in vivo and in vitro) increased 7.0% compared to 2019, with more than 1.5 x 10 6 embryos recorded. Worldwide, in vitro produced (IVP) embryos accounted for 76.2% of all transferrable cattle embryos in 2020. 2 In US, since 2016, the number of IVP embryos transferred is higher than in vivo derived (IVD) embryos for dairy probably due to increased commercial use of sex-sorted semen, use of genomic evaluations, and improved success of commercial IVP (Figure 1). 3 IVP is more efficient than IVD to supply the demand for sex-sorted embryos produced from very young heifers with high genetic merit. A similar trend has started to be observed in beef breeds in 2020 (Figure 2), probably also due to increase of beef in cattle programs. Several factors need to be coordinated to obtain a live calf from an IVP embryo. Donor must provide a good quality oocyte that can be matured and be successfully fertilized. Embryo Abstract Commercial use of sex-sorted semen and genomic evaluations have revolutionized cattle breeding, and demand for sex-sorted embryos produced from very young heifers has increased over time. Worldwide, in vitro produced embryos accounted for 76.2% of all transferrable cattle embryos in 2020, probably because in vitro embryo production is a more efficient way to supply this demand. Several factors need to be coordinated to obtain a live calf from an in vitro produced embryo. Aspects related to oocyte quality, laboratory quality control, embryo quality, recipient selection, and pregnancy loss are addressed here, based on the measures that the RuAnn Genetics Laboratory (Riverdale, San Joaquin Valley, CA) adopted in the last 14 years, with the goal of improving production of live, healthy calves from in vitro produced embryos. Follicular wave synchronization and stimulation with follicular stimulating hormone is necessary to improve oocyte quality and consequently embryo production. Laboratory quality control and the use of high-quality supplies are essential to reduce variability in production and facilitate identification of other factors that might interfere with embryo production. High pregnancy rates, similar to in vivo produced embryos, can be achieved with good quality embryos selected at optimal time and stage of development, transferred by an experienced embryo transfer technician, to well managed recipients 7 or 8 days after estrus. Attention to detail at every step of the process is crucial to success.
... Despite these improvements, the birth rate per transfer remains relatively low at ∼30% (Kupka et al., 2014;Sunderam et al., 2014). The multitude of factors impacting ART encompass all components of the in-vitro culture system (Gardner and Lane, 2003) presenting compounding stresses and causing synergistic damage to embryo function and health (Gardner and Kelley, 2017;Wale and Gardner, 2015). For instance, culturing embryos at atmospheric oxygen (∼20%) is a welldocumented stress harmful to embryo development and viability (Bontekoe et al., 2012). ...
Research question
: What is the effect of combined antioxidants (acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid; A3) when used in culture media and vitrification/warming solutions, on mouse fetal gene expression?
Design
: A laboratory-based analysis of an animal model. Embryo transfers were conducted on in vivo flushed blastocysts, or blastocysts cultured or vitrified with and without A3. Transcriptional profiles of E14 fetal liver and placental tissue in all groups were quantified using RNAseq and functional analyses (GO biological processes and KEGG pathway analysis).
Results
: Both in vitro culture in the presence of 20% oxygen and vitrification of blastocysts significantly perturbed fetal liver and placental gene expression. Notably, supplementation of in vitro culture media or vitrification/warming solutions with A3 reduced the number of DEGs and biological processes altered, establishing a more in vivo like gene expression profile, particularly within the E14.5 placenta. Specifically, A3 supplementation significantly reduced the expression of genes associated with preeclampsia and intrauterine growth restriction, along with genes involved in metabolism, cell senescence and cancer associated pathways. However, despite these improvements, several biological processes remained overrepresented following both in vitro culture and vitrification, even in the presence of A3.
Conclusion
: Both in vitro culture in the presence of 20% oxygen and vitrification of blastocysts significantly perturbed fetal liver and placental gene expression, with the number of DEGs greater following vitrification. Supplementation with A3 reduced the number of DEGs and biological processes altered, establishing a more in vivo like gene expression profile, particularly in the placenta. Notably, A3 supplementation of in vitro culture significantly reduced the expression of genes associated with preeclampsia and intrauterine growth restriction.
... Improvements in the success of IVP can be attributed, in no small way, to the continued efforts of laboratories around the world to optimize media formulations. However, what is evident is that media are but one aspect of the embryo culture system (Gardner and Lane, 2003). To support the development of a viable and healthy embryo capable of resulting in a healthy offspring, one needs to look far beyond the formulation of the culture media employed. ...
Genomic evaluations have revolutionized dairy cattle breeding, and the demand for embryos produced from very young heifers with high genetic merit has increased over time. The combination of low oocyte recovery, young age of donors, and milk production status can make the in vitro embryo production (IVP) of Holstein cattle incredibly challenging. Several factors need to be coordinated to obtain a live calf from an IVP embryo, but the quality of the oocyte at the start of the process is one of the key factors. Aspects related to oocyte quality, laboratory quality control, embryo quality and recipient selection are addressed here, based on the measures that the RuAnn Genetics Laboratory (Riverdale, California, USA) adopted in the last 12 years, with the goal of improving production of live, healthy calves from Holstein embryos. Follicular wave synchronization and stimulation with follicular stimulating hormone (FSH) is necessary to improve oocyte quality and consequently embryo production. Laboratory quality control and the use of high-quality supplies are essential to reduce variability in production and facilitate identification of other factors that might interfere with embryo production. High pregnancy rates can be achieved with good quality embryos selected at optimal time and stage of development, transferred by an experienced embryo transfer (ET) technician, to well managed recipients 7 or 8 days after estrus. Attention to detail at every step of the process is crucial to success.
... From then on, essential amino acids stimulate the development of the inner cell mass, while nonessential amino acids stimulate blastocyst expansion and hatching (Lane et al., 2001). For mammalian embryos, the two-stage or sequential media (e.g., G1/G2 media for human embryos) can be used to take advantage of this to promote the viability and health of cultured embryos and improve the prospects of live births following implantation (Steeves and Gardner, 1999;Gardner and Lane, 2003;Summers and Biggers, 2003). ...
Groups of amino acids, and some selected amino acids, added to media used for culture of pre-implantation embryos have previously been shown to improve development in various ways including survival to the blastocyst stage, increased blastocyst cell number and improved hatching. In this study, we cultured 1-cell mouse embryos for 5 days to the hatching blastocyst stage in isosmotic medium (270 mOsm/kg) at high density (10 embryos/10 μL), where autocrine/paracrine support of development occurs, and low density (1 embryo/100 μL), where autocrine/paracrine support is minimized and development is compromised. When 400 μM L-Pro or 1 mM L-Gln was added to embryos at low density, the percentage of embryos reaching the blastocyst stage and the percentage hatching increased compared to low-density culture without these amino acids, and were now similar to those for embryos cultured at high density without amino acids. When L-Pro or L-Gln was added to embryos at high density, the percentage of embryos reaching the blastocyst stage didn’t change but hatching improved. Neither embryo culture density nor the presence of these amino acids had any effect on blastocyst cell number. D-Pro and the osmolytes Gly and Betaine did not improve embryo development in low- or high-density culture indicating the mechanism was stereospecific and not osmotic, respectively. L-Pro- and L-Gln-mediated improvement in development is observed from the 5-cell stage and persists to the blastocyst stage. Molar excess of Gly, Betaine or L-Leu over L-Pro eliminated improvement in development and hatching consistent with them acting as competitive inhibitors of transporter-mediated uptake across the plasma membrane. The L-Pro effect is dependent on mTORC1 signaling (rapamycin sensitive) while that for L-Gln is not. The addition of L-Pro leads to significant nuclear translocation of p-AktS473 at the 2- and 4-cell stages and of p-ERK1/2T202/Y204 nuclear translocation at the 2-, 4-, and 8-cell stages. L-Pro improvement in embryo development involves mechanisms analogous to those seen with Pro-mediated differentiation of mouse ES cells, which is also stereoselective, dependent on transporter uptake, and activates Akt, ERK, and mTORC1 signaling pathways.
... Lately, ARTs have resulted in unsuccessful and multiple pregnancies. However, these have become less prominent with sequential culture media and single embryo transfer system [8] in addition with other technologies such as microfluidics (Fig. 1). ...
With the recent development in science and technology, the advancement in in vitro fertilization to treat infertility has been one of the most revolutionary advances. However, the success rate of the IVF process depends on the efficiency of each step of the process. The physical tools used to enhance the process continue to change. Microfluidics technology is an emerging technology being used in multiple biological applications for the miniaturization and specification of laboratory techniques. Technology is used along with IVF to enhance the outcome by facilitating every step of the process. Microfluidics can be used to handle gametes, culture embryos, cryopreservation, and for many other applications. This review will highlight the applications of microfluidics in different stages of IVF, including the handling of gametes, sperm collection and isolation, sperm sorting, embryo culture, cryopreservation and the fabrication process of microfluidics, focusing on the benefits and shortcomings of these applications.
... In an attempt to improve transfer outcomes, it was typical to transfer several embryos to the uterus in a given in vitro fertilization (IVF) cycle [7,8], leading to an increase in high-order multiple gestations carrying significant risk to both mother and babies [9]. With the development of blastocyst culture, implantation and pregnancy rates have significantly increased, thereby paving the way for the routine introduction of single embryo transfer [10]. ...
An ever-increasing number of couples rely on assisted reproductive technologies (ART) in order to conceive a child. Although advances in embryo culture have led to increases in the success rates of clinical ART, it often takes more than one treatment cycle to conceive a child. Ensuring patients conceive as soon as possible with a healthy embryo is a priority for reproductive medicine. Currently, selection of embryos for transfer relies predominantly on the morphological assessment of the preimplantation embryo, however, morphology is not an absolute link to embryo physiology, nor the health of the resulting child. Non-invasive quantitation of individual embryo physiology, a key regulator of both embryo viability and health, could provide valuable information to assist in the selection of the most viable embryo for transfer, hence reducing the time to pregnancy. Further, according to the Barker Hypothesis, the environment to which a fetus is exposed to during gestation affects subsequent offspring health. If the environment of the preimplantation period is capable of affecting metabolism, which in turn will affect gene expression through the metaboloepigenetic link, then assessment of embryo metabolism should represent an indirect measure of future offspring health. Previously the term viable embryo has been used in association with the potential of an embryo to establish a pregnancy. Here we propose the term healthy embryo to reflect the capacity of that embryo to lead to a healthy child and adult.
... Ardışık kültür ortamlarının kullanılması in vivo ortamı daha iyi taklit ediyor olabilir. Bunun nedeni erken embriyo gelişimi sırasında zigotun tuba uterinadan uterusa geçişini taklit ediyor olmasıdır [3,4]. Ardışık kültür ortamları farklı günlerdeki embriyonun ihtiyaçlarını ve ideal embriyo kalitesini sağlamak için embriyo gelişiminin gününe göre içeriğinde farklılıklar göstermektedir [5]. ...
... Despite numerous advances in human IVF over the past four decades, success rates remain relatively low with live births per transfer at 30% (Kupka et al., 2014;Sunderam et al., 2014). Many documented factors have been shown to impact the success of an IVF cycle (Gardner and Lane, 2003;Wale and Gardner, 2015). One of the most characterized components of the culture system, and a documented source of stress of the embryo, is oxygen. ...
STUDY QUESTION
What is the effect of three antioxidants (acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid), when used individually and in combination, on mouse embryo development in culture, and subsequent fetal development post-transfer?
SUMMARY ANSWER
A combination of antioxidants resulted in significant increases in blastocyst cell number, maintained intracellular glutathione (GSH) levels, supported earlier cleavage times from 5-cell stage to expanded blastocyst, and improved fetal developmental irrespective of incubator oxygen concentration.
WHAT IS KNOWN ALREADY
Acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid have been shown to have beneficial effects individually in several tissues, and most recently on developing embryos, in the presence of oxidative stress.
STUDY DESIGN, SIZE, DURATION
Morphokinetics of mouse embryos were quantitated using time-lapse imaging. GSH levels in pronucleate oocytes were measured. Blastocysts underwent differential nuclear staining for inner cell mass and trophectoderm cells or were transferred to recipient females to assess implantation and fetal development.
PARTICIPANTS/MATERIALS, SETTINGS, METHODS
Pronucleate oocytes from F1 mice were cultured in 5 or 20% oxygen either individually or in groups of 10, in media G1/G2, in the presence or absence of 10 µM acetyl-L-carnitine /10 µM N-acetyl-L-cysteine /5 µM α-lipoic acid, either individually or in combination. Controls were embryos cultured without antioxidants. Intracellular levels of reduced glutathione were quantitated in pronucleate oocytes. Embryo development and viability were analysed through time-lapse microscopy and embryo transfers.
MAIN RESULTS AND THE ROLE OF CHANCE
Antioxidants significantly increased mouse blastocyst cell numbers compared with control when used individually (P< 0.05) and to a greater effect when all three were used in combination (P< 0.01) in 20% oxygen. The combination of antioxidants resulted in faster development rates to 5-cell cleavage stage, which continued until the expanded blastocyst stage when cultured in 20% oxygen. The beneficial effects of combining the antioxidants were greater for embryos cultured individually as opposed to in groups of 10 and for those embryos cultured in 20% compared to 5% oxygen. Levels of GSH were significantly decreased in control embryos that were incubated in the absence of antioxidants in 20% oxygen (P< 0.01), compared with in vivo flushed embryos. However, when embryos were cultured with antioxidants the level of GSH was not different to that of in vivo developed embryos. Embryos cultured in the presence of antioxidants in 20% oxygen and transferred resulted in significantly longer crown-rump length (11.6 ± 0.1 mm versus 11.3 ± 0.1 mm; P< 0.01), heavier fetuses (209.8 ± 11.8 mg versus 183.9 ± 5.9 mg; P< 0.05) and heavier placentas (103.5 ± 3.1 mg versus 93.6 ± 2.7 mg; P< 0.01) compared with controls (all data are mean ± SEM). Further, a post-implantation benefit of the antioxidant combination was also evident after culture in 5% oxygen.
LIMITATIONS, REASONS FOR CAUTION
Embryo development and implantation was only examined in the mouse.
WIDER IMPLICATIONS OF THE FINDINGS
These findings show that a combination of antioxidants in the culture media has a highly beneficial effect on mouse preimplantation embryo development in vitro and on subsequent fetal development post-transfer. These data indicate a potential role for the inclusion of specific antioxidant combinations in human embryo culture media irrespective of oxygen concentration. However, before application to human embryos, a proper evaluation of this approach in prospective, preferably randomized, trials will be required.
STUDY FUNDING/COMPETING INTEREST(S)
This work was funded by a research grant from Vitrolife AB (Sweden). The authors have no conflict of interest to declare.
... The increased knowledge of early embryo development (Table 1 [155]) and maternal reproductive tract micro- environment suggest the use of more than one medium during embryo culture [156]. The design of sequential media resides mainly on the switch from pyruvate metabolite (the preferred nutrient of the early cleavage stage when low levels of oxygen are consumed) to glucose during the post-compaction period, which meets the increased energy demand for blastulation, differentiation, and growth [157,158]. ...
Bovine in vitro embryo production (IVP) is an animal biotechnology important for both agriculture
and biomedical research. IVP comprises ultrasound-guided oocyte recovery from donor by
follicular aspiration, in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF) and in vitro
embryo culture (IVC). Each step of IVP has specific technicalities and the current systems are the
result of years of research. This technique is used to study the physiology of oocyte maturation
processes, fertilization and embryo development in pre-implantation stage, and enable the
development of other techniques such as cloning and gene transfer. Moreover, it has been used
commercially to produce individuals with high genetic value, becoming an alternative source in
embryo transfer programmes in cattle. It is reported that the origin of the oocyte and IVM systems
affect the blastocyst rate, and that IVC conditions influence the quality and viability of these
blastocysts; nevertheless, despite the advances in designing media and culture systems, a precise
knowledge about the nutritional and culture requirements of embryos remains still elusive. This
review provides an overview of media design and culture systems, as well as recent advances in the
embryo technology field.
Background
To investigate the effect of antioxidants (AOX) on human embryo development and clinical outcome when used during all IVF procedures.
Methods
Prospective randomized single-centre study, where cumulus-oocyte complexes and semen samples from 127 treatment cycles were equally split between study and control (with/without AOX). The primary endpoint was Day 5/6 good quality blastocyst (GQB) rate / Metaphase-II (M-II) oocyte.
Results
The GQB rate between the study group and control was almost significant (P = 0.050). An age-related analysis revealed significantly higher fertilization rates in the study group for the older age groups (P < 0.05) but not in the younger. Development of Good-quality Day 3 embryos / M-II oocytes was comparable in the younger and significantly higher in the older age group (P < 0.05). Total Day 5/6 blastocyst rate and Day 5/6 GQB rate was not significantly different in the younger groups. For older patients Day 5/6 blastocyst rate and Day 5/6 GQB rate were higher in the study group (P < 0.05). Blastocyst utilization rates were comparable in younger patients and significantly different in older patients (P < 0.05). A total of 188 single vitrified blastocyst transfers were performed; 94 in each group. We noted similar implantation and ongoing pregnancy rates between in the younger age group but higher, albeit not-significant rates, for older patients in the study group (48.2% vs. 28.6%; 46.4% vs. 28.6%). Respective live birth rates were 47.4% vs 51.9% and 42.9% vs 23.8% in the younger/older age group.
Conclusion
Using media with AOX in all IVF procedures shows age-dependent effects.
Trial regintration
Approval no: 16002
Background:
Systematic monitoring of key performance indicators (KPI) is an important component of quality management within the IVF laboratory and, as success of assisted reproduction depends on many variables, it is important to examine how each variable can be optimized to achieve the best possible outcome for patients.
Objective:
To analyze how the design of a QMS impacts homogenization, safety, and efficacy in multiple fertility centers. Study Design Multicenter, retrospective cohort study with 188,251 patients who underwent 246,988 assisted reproductive treatments at 14 private centers belonging to IVI-RMA clinics between January 2005 and December 2019. Data were stratified by year, clinic, and patient group (standard patient cycles with no PGT-A, standard patients with PGT-A, and oocyte donation patients). Unadjusted and adjusted logistic regression models with other known predictors were made to analyze the impact and the interactions of policies. Main outcomes were determined per clinic and summarized per year as the median of the rates of the clinics; each clinic had the same weight independent of the number of cycles.
Results:
Up to 188,251 patients were treated, for a total of 246,988 IVF cycles and 356,433 procedures. The introduction of standard operating procedures, trophectoderm biopsies, and blastocyst-stage transfers, coupled with an increased proportion of PGT-A cycles, led to improved outcomes while maximizing the number of single embryo transfers, driving a significant decrease in the number of multiple pregnancies while improving live birth rates. In terms of the live-birth rate per transfer, the interventions with greater impact over time in logistic regression analysis were 24-chromosome analysis and the introduction of benchtop incubators (odds ratio 1.92 [95% confidence interval 1.81 to 2.05]; p < 0.001). The odd ratios of the policies remained significant and very similar in the unadjusted and adjusted models.
Conclusions:
The greatest impact on live-birth rate per cycle was obtained with a cumulative effect of all policies, especially in egg donation patients. In patients without PGT-A changing embryo culture conditions and blastocyst stage transfer had the greatest impact; in patients with PGT-A, trophectoderm biopsy. Standardizing procedures was essential in reducing variability among clinics and implementing changes.
Over the past four decades our ability to maintain a viable human embryo in vitro has improved dramatically, leading to higher implantation rates. This has led to a notable shift to single blastocyst transfer and the ensuing elimination of high order multiple gestations. Future improvements to embryo culture systems will not only come from new improved innovative media formulations (such as the inclusion the addition of antioxidants), but plausibly by moving away from static culture to more dynamic perfusion-based systems now made a reality due to breakthroughs in 3D printing technology and micro fabrication. Such an approach has already made it feasible to create high resolution devices for ICSI, culture and cryopreservation, paving the way not only for improvements in outcomes but also automation of ART. While improvements in culture systems can lead to further increases in pregnancy outcomes, the ability to quantitate biomarkers of embryo health and viability will reduce time to pregnancy and decrease pregnancy loss. Currently artificial intelligence is being used to assess embryo development through image analysis, but we predict its power will be realised through the creation of selection algorithms based on the integration of information related to metabolic functions, cell free DNA, morphokinetics, thereby using vast amounts of different data types obtained for each embryo to predict outcomes. All of this will not only make ART more effective, but it will also make it more cost effective, thereby increasing patient access to infertility treatment worldwide.
Multiple pregnancies are the most frequent and serious complication of assisted reproduction. Both high-order multiple and twin pregnancies entail a number of medical and economic outcomes that affect the children, the mother, the parents, the families, and society as a whole. Limiting the number of embryos to transfer is the only method available to decrease the incidence of multiple pregnancies. Single Embryo Transfer reviews the advantages and limitations of this approach to assisted reproduction. The crucial issue of selecting the best embryo will be reviewed in detail. All clinical issues involved in setting up and running an SET programme will be covered, including important topics such as cryopreservation of embryos, embryo donation, and patient counselling. The final chapters on future SET trends in Europe and North America are written by leading figures in the IVF world. The book is of interest to physicians, embryologists, nurses, insurers, politicians, ethicists and patients.
To address the proposition that ‘the way to improve ART outcomes is through the introduction of more technologies in the laboratory’, it is prudent to first define what is considered to be improved outcomes. Evidently, this equates to an increase in the live birth rate but it should also include parameters such as time to pregnancy, cumulative pregnancy per oocyte retrieval and health of the resultant child. Furthermore, being able to maintain clinical results week in, week out through quality management also contributes to the overall success of a clinic, and hence can be considered an improved outcome. With regards to these outcomes, it is offered that not only does the introduction of several new technologies (defined here as instrumentation, techniques and enhanced computer utilization and analysis) have the potential to improve outcomes, but also some of them have the capacity to facilitate automation and standardization in the ART laboratory. Although the automation of procedures can be perceived as a justifiable goal itself, in this contribution the emphasis is on how new technologies could help more patients become parents of healthy children in the shortest possible time.
Triiodothyronine (T3) plays an essential role in different animal species’ embryonic development. The present research was designed to identify the effect of triiodothyronine on the in vitro ovine embryonic development and the expression of apoptotic genes.A total of 436 immature cumulus-oocyte complexes (COCs) were cultured for 24 h in the oocyte maturation medium supplemented with two concentrations of T3 (T-10 and T-100 ng/mL) or without T3(T-0: control group). Oocyte maturation, cleavage, and blastocyst rates were assessed under an inverted microscope as crucial indicators of embryo development.The relative mRNA abundance of BCL-2-associated X protein (BAX) and anti-apoptotic B-cell lymphoma-2 (BCL2) were determined at blastocysts (day 8 after IVF on day 0)by quantitative reverse transcription PCR.The data were analyzed by logistic regression using the GLIMMIX procedure followed by Chi-Square, and one-way ANOVA tests. The higher concentration of T3(100 ng/mL) significantly decreased cumulus expansion and blastocyst rate compared to controls (P<0.001). Additionally, a significantly higher expression level of BAX(P<0.001) and a dramatically lower expression level of BCL2 (P<0.01) were detected in the T-100ng/mL group compared to the controls.However, the relative mRNA level of BCL-2 was significantly higher in the T-10 ng/mL group compared to the control group (P<0.01).It appears that the supplementation of ovine oocyte maturation medium with T3 at high concentration (100 ng/mL) suppresses the ratio of blastocyst formation.
Embryos undergo chaotic division and decrease in quality on day 3 with a reduction in the rates of subsequent blastocyst formation. Disordered cleavage causes a deterioration in embryonic quality, here we assessed the relationship between an cleavage model in first mitosis and the chromosomal status of human embryos, and discuss the potential biological and clinical implications for the cleavage model as a single parameter that can be used to assess embryonic quality. Thirty-two infertile couples, with normal karyotypes and who underwent their first IVF cycle were recruited to donate one normal two-cell-stage embryo each for this study between 2019 and 2020. Twenty-eight two-cell embryos underwent preimplantation genetic testing of each blastomere, and four chaotic-division embryos were stained with Hoechst and cultured in a confocal laser-scanning microscopy incubator system. This system showed high specificity and PPV but low sensitivity and NPV using the CM in the prediction of euploidy, indicating that CM could be considered a screening method for embryo selection; additional observational studies using the CM to select transferable embryos are needed before it can be used in clinical practice.
Study question:
Can vascular endothelial growth factor (VEGF)-loaded silica supraparticles (V-SPs) be used as a novel mode of delivering VEGF to the developing preimplantation embryo in vitro?
Summary answer:
Supplementation of embryo culture media with V-SPs promoted embryonic development in a manner equivalent to media supplemented with free VEGF.
What is known already:
VEGF is a maternally derived growth factor that promotes preimplantation embryonic development in vitro. However, its use in clinical media has limitations due to its low stability in solution.
Study design, size, duration:
This study was a laboratory-based analysis utilising a mouse model. V-SPs were prepared in vitro and supplemented to embryonic culture media. The bioactivity of V-SPs was determined by analysis of blastocyst developmental outcomes (blastocyst development rate and total cell number).
Participants/materials, setting, methods:
SPs were loaded with fluorescently labelled VEGF and release kinetics were characterised. Bioactivity of unlabelled VEGF released from V-SPs was determined by analysis of embryo developmental outcomes (blastocyst developmental rate and total cell number) following individual mouse embryo culture in 20 µl of G1/G2 media at 5% oxygen, supplemented with 10 ng/ml recombinant mouse VEGF in solution or with V-SPs. The bioactivity of freeze-dried V-SPs was also assessed to determine the efficacy of cryostorage.
Main results and the role of chance:
VEGF release kinetics were characterised by an initial burst of VEGF from loaded spheres followed by a consistent lower level of VEGF release over 48 h. VEGF released from V-SPs resulted in significant increases in total blastocyst cell number relative to the control (P < 0.001), replicating the effects of medium freely supplemented with fresh VEGF (P < 0.001). Similarly, freeze dried V-SPs exerted comparable effects on embryonic development (P < 0.05).
Large scale data:
N/A.
Limitations, reasons for caution:
In this proof of principle study, the effects of V-SPs on embryonic development were only analysed in a mouse model.
Wider implications of the findings:
These findings suggest that SPs represent a novel method by which a targeted dose of therapeutic agents (e.g. bioactive VEGF) can be delivered to the developing in vitro embryo to promote embryonic development, an approach that negates the breakdown of VEGF associated with storage in solution. As such, V-SPs may be an alternative and effective method of delivering bioactive VEGF to the developing in vitro embryo; however, the potential use of V-SPs in clinical IVF requires further investigation.
Study funding/competing interest(s):
This work was funded by the University of Melbourne. The authors have no conflict of interest to declare.
NMR offers the potential to holistically screen hundreds of metabolites and has already proved to be a powerful technique able to provide a global picture of metabolic changes in a wide range of biological systems underlying complex and multifactorial matrixes. This review covers the literature until May 2020 centered on the early prediction of the viability of in vitro developed embryos using several analytical techniques, including NMR. Nowadays, the predominant non‐invasive technique for selecting viable embryos is based on morphology, where variables associated with the rate of cleavage and blastocyst formation are evaluated by the embryologist following standardized criteria that are somewhat subjective. This morphological approach is therefore inadequate for the prediction of embryo quality, and several studies have focused on developing new non‐invasive methods using molecular approaches based particularly on metabolomics. This review outlines the potential of NMR as one of these non‐invasive in vitro methods based on the analysis of spent embryo culture media. The present work reviews some of the most relevant studies up to May 2020 based on the early prediction of the viability of in vitro developed embryos using NMR. This review outlines its potential as a non‐invasive in vitro method based on metabolomics.
Research question
Does the inclusion of three antioxidants (A3), Acetyl-L-Carnitine (ALC), N-Acetyl-L-Cysteine (NAC) and α-Lipoic Acid (ALA) improve human embryo development and pregnancy potential?
Design
Prospective randomized multicentre comparison on sibling oocytes. A total of 1,563 MII oocytes from 133 patients in 2 IVF centres. Day 3 embryo and day 5/6 blastocyst quality were assessed. Good embryo quality on day 3 was defined as 8 to 10-cells with even cells and low fragmentation; good quality blastocysts as equal or greater than 3BB. Clinical outcome was assessed on transfers of a single fresh or vitrified-warmed embryo on day 5.
Results
Of the 2PN, 40.7% (G-Series) and 50.3% (G-Series with A3 group) resulted in good quality embryos on day 3 (P<0.05). The implantation rate by fetal sac was 39.2% and 50.6%, and by fetal heart beat was 37.8% and 47.1% for the G-Series and G-Series with A3 group respectively. When stratified by female patient age patients 35 to 40 years had an implantation rate by fetal sacs and hearts of 23.5% in the G-Series compared to 57.5% (P<0.05) and 50.0% (P<0.05) in the A3 group. The ongoing pregnancies in patients 35 to 40 years were significantly higher in the A3 group (50%) compared to the control (25.8%).
Conclusions
The presence of antioxidants during IVF and embryo culture for patients 35 to 40 years resulted in a significant increase in implantation and pregnancy rate. Supplementation of antioxidants to IVF and culture media may therefore improve the viability of human embryos in ART, plausibly through the reduction of oxidative stress.
Blastocyst culture and transfer for human IVF has resulted in an increase in implantation and live birth rates and successfully facilitated the move toward single embryo transfer. Initially this was attained through the development and introduction of sequential media, designed to mirror the changing nutritional environments within the oviduct and uterus. Sequential media underwent years of development using several animal models and were validated by the transfer of thousands of embryos prior to its clinical evaluation and subsequent use in prospective randomized trials. Such media have proved themselves to be highly effective in clinical IVF and have facilitated the move to trophectoderm biopsy and vitrification of the embryo. However, successful embryo culture goes beyond the choice of media and requires an holistic approach, whereby all facets of the embryo culture system, especially the oxygen concentration, are optimized.
This study reports the results of a 2-year long IVF programme (‘One by One’) in which all patients (median age 40 years; range 27–45 years) were offered preimplantation genetic testing for aneuploidy (PGT-A) and had all blastocysts vitrified (freeze-only), followed later by single vitrified-warmed blastocyst transfer (vSET) in managed cycles. Between January 2016 and December 2017, a total of 155 patients started 222 treatment cycles and 99 (45%) cycles resulted in one or more vitrified blastocysts (untested or with normal copy number for all chromosomes) available for transfer. Seventeen patients (11%) aged ≤35 years opted out of PGT-A. Over this period, 85 vSETs in 74 patients resulted in an implantation rate of 80% (68/85) and a singleton clinical pregnancy rate of 66% (56/85). Cumulative live birth rates will not be known for 1–2 years. Nevertheless, these high success rates with vSET confirm larger studies using selected patients and are likely to deliver similar, if not higher, live birth rates per cycle started than rates typically reported in national registries with conventional IVF and transfer of one or more fresh and/or frozen embryos.
Abstract Unveiling the transcriptome of human blastocysts can provide a wealth of important information regarding early embryonic ontology. Comparing the mRNA production of embryos with normal and abnormal karyotypes allows for a deeper understanding of the protein pathways leading to viability and aberrant fetal development. In addition, identifying transcripts specific for normal or abnormal chromosome copy number could aid in the search for secreted substances that could be used to non-invasively identify embryos best suited for IVF embryo transfer. Using RNA-seq, we characterized the transcriptome of 71 normally developing human blastocysts that were karyotypically normal vs. trisomic or monosomic. Every monosomy and trisomy of the autosomal and sex chromosomes were evaluated, mostly in duplicate. We first mapped the transcriptome of three normal embryos and found that a common core of more than 3,000 genes is expressed in all embryos. These genes represent pathways related to actively dividing cells, such as ribosome biogenesis and function, spliceosome, oxidative phosphorylation, cell cycle and metabolic pathways. We then compared transcriptome profiles of aneuploid embryos to those of normal embryos. We observed that non-viable embryos had a large number of dysregulated genes, some showing a hundred-fold difference in expression. On the contrary, sex chromosome abnormalities, XO and XXX displayed transcriptomes more closely mimicking those embryos with 23 normal chromosome pairs. Intriguingly, we identified a set of commonly deregulated genes in the majority of both trisomies and monosomies. This is the first paper demonstrating a comprehensive transcriptome delineation of karyotypic abnormalities found in the human pre-implantation embryo. We believe that this information will contribute to the development of new pre-implantation genetic screening methods as well as a better understanding of the underlying developmental abnormalities of abnormal embryos, fetuses and children.
This monograph, written by the pioneers of IVF and reproductive medicine, celebrates the history, achievements, and medical advancements made over the last 40 years in this rapidly growing field.
This chapter describes the procedures performed in the in vitro fertilization (IVF) laboratory. Ideal laboratory conditions are discussed to highlight the enormity of the undertaking required to create an environment for optimal clinical outcomes. Procedures performed in the laboratory are introduced in sequence. The conceptual framework underlying each procedure and the details surrounding their execution are delineated. In addition, the current status of preimplantation genetic testing (PGT) is described.
Noninvasive methods for evaluation of the quality and prediction of developmental competence of in vitro-produced preimplantation-stage embryos are of crucial importance in human-assisted reproduction. Currently, one of the most demanding tasks in this field is to find the optimal solution between two partially contradicting requirements: to achieve the highest pregnancy (and live birth) rates and to keep the proportion of multiple pregnancies at a minimum level. The most feasible approach to meet these requirements is the extended culture and decreased number of transferred embryos; the former may result in the survival of the best embryos, while the latter decreases the chance of multiple implantation. The ultimate realization of this strategy is the single blastocyst transfer. At present, the only routine method for this prediction and selection is the bi-daily light microscopic evaluation of the morphology. The limits of this approach are widely acknowledged and are related to the snapshot type analysis of dynamic processes. Alternate possibilities include specific investigation of certain structures or combined evaluation of morphological features, and sophisticated indirect analysis of intracellular processes by measuring changes in certain parameters in surrounding media. A third approach is the real-time light microscopic monitoring of embryo development by using time-lapse techniques.
With the advent of culture media based upon the nutrient composition of the human oviduct and uterus, and specifically designed to support the human embryo in vitro, it has become feasible to culture the pronucleate oocyte to the blastocyst stage as a routine procedure in human IVF. The potential advantages of extended culture go beyond increases in implantation and pregnancy rates and include lower miscarriage rates and the ability to perform comprehensive genetic and physiological analysis of the embryo proper prior to transfer. The capacity to better select a viable embryo will continue to improve the safety of IVF to the mother and child without a compromise in efficacy. The ability of blastocysts to undergo successful cryopreservation with vitrification will culminate in the transfer of genetically analyzed embryos to a naturally cycling uterus. The transfer of vitrified blastocysts in a nonstimulated cycle may ultimately be the way all future IVF is performed.
Introduction, Over the past three decades basic research has culminated in many significant advances in human assisted conception (Edwards 2004). An example of this is the relatively recent development of more physiological culture conditions. By employing such conditions, it has become possible to culture the human embryo to the blastocyst stage as a matter of routine (Gardner and Lane 1997). Clinics now have more options regarding the day of embryo transfer, giving increased flexibility in scheduling. Subsequently, the question raised is, is there an optimal day for embryo transfer following IVF in the human? In this chapter, the probable advantages of blastocyst transfer are discussed and the clinical data reviewed. There are numerous potential benefits of blastocyst transfer in human IVF, which are summarized in Table 56.1. Not all sperm or oocytes are destined to give rise to a viable embryo (Tesarik 1994). By culturing the human embryo past the cleavage stages, that is, past embryonic genome activation (Braude et al. 1988), it is feasible to study the embryo properly. A key factor in determining transfer outcome in animal models is the synchronization of the embryo with the female reproductive tract. In all mammalian species studied to date, including nonhuman primates (Marston et al. 1977), the transfer of embryos to the uterus prior to compaction (and, therefore, before the generation of the first transporting epithelium) results in greatly reduced pregnancy rates compared to the transfer of morulae or blastocysts.
The need for non-invasive marker of developmental competence It is a tribute to the success of in vitro fertilization (IVF) as a primary treatment of human infertility that the subject of an entire volume is devoted to the notion that single embryo transfers can and perhaps should become the worldwide standard of practice. The intent of this chapter is to provide a general survey of research that has sought to identify cellular, molecular and developmental (i.e., performance in vitro) characteristics of hyperstimulated ovarian follicles, oocytes and cultured preimplantation stage embryos that may relate to or predict competence and outcome after uterine transfer. The need for predictors of competence is related to the basic clinical strategy of this treatment; namely, induce large numbers of follicles to develop coordinately by managed or “controlled” ovarian hyperstimulation, so that a corresponding number of aspirated oocytes will be meiotically mature (i.e., meiosis arrested at metaphase II, MII), fertilizable and capable of normal development in vitro. Selection for transfer is usually made at a set time after insemination (program-specific protocol) from a cohort of embryos in which those deemed stage- and performance-appropriate are selected for transfer or cryopreservation. While the first human IVF birth in 1978 was conceived during a “natural cycle” [1] and sporadic reports of positive outcomes with this approach were reported in subsequent years [2, 3], it soon became evident that the efficacy of this process could be significantly increased if more oocytes were available to produce more than a single embryo for transfer.
Introduction Over the past several decades the success rates of in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) assisted reproductive technology (ART) procedures using fresh non-donor oocytes with or without ICSI have increased. Quinn reported a linear increase in viable rates for IVF/ICSI procedures in Australia and New Zealand from 1992 to 2001 [1]; rates more than doubled from 10% to 24% per transfer (Figure 2.1). Pregnancy rates in gamete intrafallopian transfer (GIFT) remained constant at 21% over the same period, indicating that the primary reason for the increase in the IVF/ICSI patients during this time was due to improvements in the culture systems being used. Similar increases over the same time span were reported for US data [2]. These increases in success rates in the USA have continued with live births per embryo transfer for fresh embryos from non-donor eggs significantly increasing from 35.8% to 36.4% to 38.5% for the years 2002 to 2006 to 2010, respectively, with the average number of embryos transferred decreasing from 3.0 to 2.6 to 2.4 over this period [3]. These data taken as a whole can be interpreted to indicate that improvements in the laboratory system are responsible for the majority of the increased success rates. Various aspects of these improvements, both in the culture media and in the important interacting aspects of the culture systems are discussed below. ART media formulations This topic is one of the oldest, most studied and discussed in preimplantation mammalian embryology so the discussion below will be more of a summary of the situation as seen by this author. There are many reviews on the topic that provide further details [4-10] and, of course, several chapters in this book.
This chapter considers the parameters important for embryo culture and analysis, and looks at the future of embryo culture systems. In contemplating the future of embryo culture systems, a very valuable initial exercise is to consider the constraints that are likely to shape future developments. One of the fundamental guiding principles for culture systems is to provide conditions that promote both optimal development to the blastocyst stage and maximal pregnancy rates. Another consideration for current and future culture systems is to avoid or minimize the exposure of embryos to conditions that are toxic or stressful. From the advent of the culture of preimplantation, mammalian embryos analysis has been an integral part of the process. One of the most exciting prospects for microfluidic culture platforms is that many mature analytical technologies are available that can be readily incorporated into chips.
Introduction The introduction of amino acids to the culture media for the mammalian oocyte and embryo is arguably the most important advance made in regards to the successful culture of viable embryos. Currently all media for the culture of human embryos contain amino acids as a core component of their formulation. Amino acids are molecules containing an amine group (NH2) as well as a carboxylic acid group (COOH) and a variable side chain. In mammals there are 20 proteinogenic amino acids that are naturally incorporated into polypeptides within the body, while some other organisms have 22 (Table 8.1). Traditionally amino acids have been shown to be important in cellular metabolism and in many organisms as energy substrates and osmolytes. However, there is growing evidence in other tissues that amino acids are able to control many cellular functions including regulation of cell signaling and gene expression. Interestingly, although it is clear that their addition to a culture medium formulation improves embryo development and viability, the cellular function of amino acids in regulating embryo development is largely unknown. This chapter will review the current knowledge and history of the use of amino acids in culture media for the mammalian embryo as well as the in vitro artifact of by-product accumulation of ammonium in the medium.
Regulation of energy production is fundamental to the survival and propagation of any cell type. What makes the preimplantation mammalian embryo so fascinating to study is the fact that the embryo undergoes major changes in its physiology and gene expression profiles during development. As the fertilised oocyte develops and differentiates into the blastocyst, embryonic genes are successively turned on (with the concomitant destruction of maternally derived mRNAs). Subsequently there is a growing energy demand as mitoses and biosynthesis increase post-embryonic-genome activation and as the blastocoel subsequently forms (through the activity of Na/K ATPase in the trophectoderm). Concomitantly, there are major changes in the regulation and relative activities of energy generating pathways. Of clinical interest is the fact that should an embryo at any stage of development have substantially altered energy production, i.e. if the flux of a specific nutrient through a metabolic pathway alters to a significant degree, even for a brief period, then this is associated with significantly impaired development in culture and reduction of viability post-transfer. Clearly it is in our interest to understand how the preimplantation embryo regulates its energy production, and to develop culture systems that best support an 'optimal' metabolism. Furthermore, it is evident that analysis of embryo metabolism is an appropriate means of assessing embryonic health and predicting subsequent viability. The task ahead is to determine the optimal range of metabolic functions that reflect viability at successive stages of embryo development. © 2013 Springer Science+Business Media New York. All rights reserved.
The field of modern embryo culture spans several decades. However, in the last decade of the 20th century a resurgence of interest in embryo physiology and metabolism led to highly significant improvements in embryo culture conditions. It also became apparent that for successful development of viable mammalian embryos in vitro, one needs to understand that culture conditions go far beyond the composition of the media used. Rather, it is imperative to appreciate the many variables of the culture system, each of which is capable of impacting the development and viability of the preimplantation embryo. By taking a more holistic approach to embryo culture, it is not only possible to attain high rates of viable embryo development, but also feasible to ensure consistent success of laboratory performance over time. Finally, it is proposed that further analysis of the physiology of cloned embryos will greatly assist in the development of more specific and effective culture systems.
The mammalian pre-implantation embryo, perhaps more than any other cell type, has proved difficult to culture successfully. A primary reason for this is that during the period, spanning fertilization to implantation, the embryo undergoes remarkable changes in gene expression, biosynthesis, physiology, and metabolism. In effect, each day of development is analogous to trying to culture a different cell type. Understanding the underlying metabolic changes during development has, therefore, been of great value in optimizing culture conditions for the pre-implantation embryo. In this chapter, the dynamics and regulation of metabolism are considered, and the development and application of novel culture systems for the human embryo described.
Background: IVF is limited by low success rates and a confounding high multiple birth rate contributing to prematurity, increased neonatal mortality and child handicap. These problems could be overcome if single embryos of known developmental competence could be selected for transfer on day 2/3 of development, but current methods, which rely on morphological appearance, are poor predictors of viability. Methods: We have measured non-invasively the depletion/appearance (i.e. turnover) of a physiological mixture of 18 amino acids by single human embryos during in-vitro culture using high performance liquid chromatography. Results: From the time of transfer (day 2/3), embryos with future competence to develop to the blastocyst stage (day 5/6) exhibit amino acid flux patterns distinct from those of embryos with similar morphological appearance which arrest. Significantly, the profiles of Ala, Arg, Gln, Met and Asn flux predict blastocyst potentiality at >95%. The amino acid most consistently depleted throughout development by those embryos which form blastocysts; was leucine. Of the amino acids which were produced, the most striking was alanine, which appeared in increasing amounts throughout development. Conclusions: Non-invasive amino acid profiling has the potential to select developmentally competent single embryos for transfer, thereby increasing the success rate and eliminating multiple births in IVF.
The role of amino acids, ethylenediaminetetraacetic acid (EDTA), transferrin, oxygen, glucose, glutamine, taurine and ammonium
in CF1 mouse zygote development in culture was examined. Non-essential amino acids and glutamine were shown to alleviate the
2-cell block in culture, and acted in synergy with EDTA to facilitate development to the blastocyst stage. In the presence
of amino acids and EDTA, transferrin conferred no beneficial effect Development of zygotes was significantly impaired if amino
acids were removed from the collection medium, even when they were subsequently cultured in the presence of amino acids. Zygote
development to the blastocyst stage was significantly improved when modular incubator chambers were used compared to using
a conventional incubator, and when an oxygen concentration of 7% was used as opposed to 20%. Addition of taurine to medium
containing non-essential amino acids had no effect on embryo development, whereas the removal of glutamine and/or glucose
from the culture medium significantly reduced blastocyst cell number. Removal of glucose from the culture medium also resulted
in a significant decrease in implantations. Ammonium, generated from the breakdown of amino acids, significantly reduced blastocyst
development EDTA was found to confer its beneficial effects during the first 48 h of culture, and indeed was inhibitory during
the second 48 h, resulting in loss of subsequent viability. In summary, the data demonstrate that development of CF1 zygotes
to the blastocyst stage is readily achievable. In the presence of non-essential amino acids and glutamine the removal of glucose
is detrimental to CF1 mouse embryo development in culture and reduces subsequent viability. Optimal development and maintenance
of viability requires more than one culture medium to support the preimplantation period.
The culture conditions for the development in vitro of (C57BL/6 x CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85% of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2: 5% CO2: 90% N2. The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.
The effects of amino acids and ammonium on the postimplantation development of cultured preimplantation mouse zygotes were assessed. Development after transfer revealed that the mouse embryo undergoes a switch in nitrogen requirements during the preimplantation period. Although Eagle's nonessential amino acids and glutamine supported the highest implantation and fetal development rates per embryo transferred when zygotes were cultured for 48 h, by 93 h of culture the highest implantation rate was observed when all 20 amino acids were in the culture medium. Furthermore, fetal development per implantation at 69 and 93 h of culture was increased only in the presence of essential amino acids without glutamine. The beneficial effects of amino acids on postimplantation development when embryos were cultured for 4 days required that the medium be renewed after 48 h (at the 6-8-cell stage) to alleviate the build-up of ammonium. Ammonium was shown to induce fetal retardation and exencephaly in a time- and concentration-dependent manner. Renewal of amino-acid-free culture medium reduced fetal mass, providing indirect evidence for the production of an embryo-derived growth factor capable of stimulating postimplantation development. These data demonstrate that inclusion of amino acids in the culture medium for preimplantation embryos significantly increases postimplantation development the preimplantation mouse embryo changes its nitrogen requirement as development proceeds, nonessential amino acids increase the implantation rate while the essential amino acids enhance fetal development, and ammonium in the medium retards fetal development and induces the neural tube defect exencephaly.
The regulation of 1-cell mouse embryo development in culture by amino acids was investigated. When the 20 amino acids in Eagle's medium were present, blastocyst formation at 72 h (9%; p < 0.01), and blastocyst cell number (66; p < 0.05) and hatching (45%; p < 0.05) after 96 h of culture were significantly increased, compared to control embryos grown in the absence of amino acids (0%, 60, and 23%, respectively). The beneficial effect of Eagle's amino acids was attributed primarily to the non-essential group. In the presence of non-essential amino acids, blastocyst formation (54%; p < 0.001) and cell number after 72 h of culture (33; p < 0.05), and blastocyst cell number (69; p < 0.01) and hatching (68%; p < 0.01) after 96 h of culture were all significantly greater than for embryos cultured with all amino acids (9%, 26, 66, and 45%, respectively). In the absence of glutamine, essential amino acids significantly reduced blastocyst cell number after 96 h (53; p < 0.05). Continual culture in the presence of amino acids reduced the cleavage rate after around 72 h of in vitro culture; this decrease was not observed in the absence of amino acids. Transfer of embryos to fresh medium after 48-72 h of culture resulted in increases in the percentage of blastocysts formed and in blastocyst cell numbers. These data are consistent with the build-up of an inhibitory compound in the medium, possibly ammonium, an end-product of amino acid metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)
It has previously been reported that ovine embryos cultured in Synthetic Oviduct Fluid medium supplemented with 20% human serum (SOF+HS) develop into lambs with a high birth weight. We have investigated this phenomenon by culturing ovine zygotes in SOF+HS or a serum-free version of Synthetic Oviduct Fluid with BSA and amino acids (SOFaaBSA) in place of serum. Zygotes were either obtained from superovulated and naturally mated ewes or produced in vitro. Embryos were subsequently transferred to synchronized recipient ewes (n = 63). An additional group of ewes (n = 16) served as flock fertility and lambing controls. Development of zygotes to stages suitable for transfer (i.e., good to excellent compact morulae or blastocysts) was not affected by medium (SOFaaBSA = 53 +/- 5% vs. SOF+HS = 59 +/- 5%) but was affected by source (in vivo-derived = 74 +/- 5% vs. in vitro-derived = 35 +/- 5%, p < 0.001). Embryos incubated in SOF+HS were morphologically different from those incubated in SOFaaBSA, having abundant lipid droplets. Pregnancy rate (65%) and embryo survival (48%) of recipients determined by ultrasonography on approximately Day 60 of pregnancy did not differ between medium treatments or source of embryo. Mean weight of lambs from embryos cultured in SOF+HS (4.2 +/- 0.2 kg) was significantly heavier than that of controls (3.4 +/- 0.2 kg, p < 0.01) or of lambs from embryos cultured in SOFaaBSA (3.5 +/- 0.2 kg, p < 0.05). Furthermore, mean gestation length was longer in recipients receiving embryos incubated in SOF+HS (147 +/- 1 days) than in SOFaaBSA (145 +/- 1 day, p < 0.05). Reasons for this birth weight and gestation length difference are unclear, but our data suggest that different culture conditions can produce embryos with differing morphology, apparent chemical composition, and rate of development, resulting in lambs with differing gestation length and birth weight.
The role of amino acids, ethylenediaminetetraacetic acid (EDTA), transferrin, oxygen, glucose, glutamine, taurine and ammonium in CF1 mouse zygote development in culture was examined. Non-essential amino acids and glutamine were shown to alleviate the 2-cell block in culture, and acted in synergy with EDTA to facilitate development to the blastocyst stage. In the presence of amino acids and EDTA, transferrin conferred no beneficial effect. Development of zygotes was significantly impaired if amino acids were removed from the collection medium, even when they were subsequently cultured in the presence of amino acids. Zygote development to the blastocyst stage was significantly improved when modular incubator chambers were used compared to using a conventional incubator, and when an oxygen concentration of 7% was used as opposed to 20%. Addition of taurine to medium containing non-essential amino acids had no effect on embryo development, whereas the removal of glutamine and/or glucose from the culture medium significantly reduced blastocyst cell number. Removal of glucose from the culture medium also resulted in a significant decrease in implantations. Ammonium, generated from the breakdown of amino acids, significantly reduced blastocyst development. EDTA was found to confer its beneficial effects during the first 48 h of culture, and indeed was inhibitory during the second 48 h, resulting in loss of subsequent viability. In summary, the data demonstrate that development of CF1 zygotes to the blastocyst stage is readily achievable. In the presence of non-essential amino acids and glutamine the removal of glucose is detrimental to CF1 mouse embryo development in culture and reduces subsequent viability. Optimal development and maintenance of viability requires more than one culture medium to support the preimplantation period.
In human in-vitro fertilization (IVF) embryos are routinely transferred to the uterus on day 2 or day 3 of development. Resultant implantation and pregnancy rates are disappointingly low, with only approximately 10% of embryos transferred leading to a live birth. The ability to culture embryos to the blastocyst stage should help to resolve this problem by synchronizing the embryo with the female reproductive tract, and by identifying those embryos with little developmental potential. Co-culture has offered a possible means of producing blastocysts capable of high implantation rates. However, recent developments in the field of embryo physiology and metabolism have led to the formulation of new sequential serum-free culture media capable of supporting the development of viable blastocysts in several mammalian species, including the human. It is therefore proposed that blastocyst transfer should be considered for routine use in human IVF. The high viability of blastocysts cultured in the appropriate sequential media means that fewer embryos are required for transfer to achieve a pregnancy, culminating in fewer multiple births. Furthermore, the development of suitable non-invasive tests of embryo viability should further increase the overall success of human IVF by the ability to select before transfer those blastocysts most able to establish a pregnancy.
Culture of in-vivo-developed mouse blastocysts in a simple culture medium based on a balanced salt solution supplemented with carbohydrates for 3 h significantly perturbed embryo metabolism. Maximal perturbation occurred after just 6 h of culture. Similarly, culture of rat blastocysts in a simple culture medium for 3 h also resulted in perturbed metabolism. Cultured mouse and rat blastocysts both had an abnormally elevated rate of glycolysis of approximately 100% after culture (P < 0.05). Rates of pyruvate oxidation by mouse blastocysts were also significantly reduced after culture in a simple medium for 6 h (P < 0.01). Furthermore, the developmental competence of mouse blastocysts after transfer was significantly reduced by just 6 h of culture in a simple medium (P < 0.05). Addition of Eagle's amino acids or vitamins to the culture medium reduced the perturbation of both the glycolytic activity and oxidative capacity of cultured mouse blastocysts and acted in synergy to further the inhibition. Importantly, culture with amino acids and vitamins prevented any loss of viability of mouse blastocysts after culture for 6 h. It can be concluded that the mouse blastocyst is sensitive to its environment and that culture-induced stress results in the loss of normal cellular function, as manifested in this case by an abnormal pattern of glucose utilization and loss of viability.
In human in-vitro fertilization (IVF), embryos are routinely transferred to the uterus on either day 2 or day 3 of development, resulting in a 10-15% implantation rate. However, in other mammalian species, the transfer of cleavage stage embryos, which normally reside in the oviduct, to the uterus results in a significantly lower implantation rate compared with blastocysts. It is therefore proposed that, in order to increase implantation rates in human IVF, one has to move to extended culture and transfer at the blastocyst stage. The transfer of blastocysts will not only help synchronize the embryo with the female tract but will facilitate the identification of those embryos with little or no developmental potential. In order to culture viable blastocysts it is important to use more than one culture medium to cater for the changing requirements of the preimplantation embryo as it develops and differentiates. If sequential culture media are not used, one can obtain blastocysts but their resultant viability is low. The use of sequential serum-free media in human IVF has resulted in > 50% of embryos becoming blastocysts with an implantation rate of approximately 50%. Further advances in human embryo culture should come from the replacement of protein with the glycosaminoglycan hyaluronate, which is more suitable than albumin in supporting implantation in the mouse, and which will eliminate biological variation and possible contamination from blood products. With the routine culture of human blastocysts will come the introduction of non-invasive tests of embryo viability, capable of identifying those blastocysts most likely to develop from a given cohort. As the implantation rate of blastocysts is higher than that of the cleavage stage embryo, fewer embryos will be required for transfer in order to establish a successful pregnancy, thereby reducing the number of multiple gestations and increasing the overall efficiency of human IVF.
The inclusion of specific amino acids in conventional culture media has been shown to enhance mammalian embryo development in vitro. Amino acids have been shown to confer their benefits to the preimplantation embryo in a number of different ways. However, their ability to buffer intracellular pH (pHi) has not been investigated. Thus, the aim of this study was to determine if amino acids regulate pHi in the mouse preimplantation embryo. pHi was determined using carboxy-seminaphthorhodafluor-1 (SNARF-1) and confocal microscopy. Incubation with 5,5-dimethyl-2,4-oxazol-idinedione (DMO), a non-metabolizable weak acid, resulted in a significant intracellular acidification in the zygote, 2-, 4- and 8-16-cell embryo. However, in the presence of groups of amino acids, the degree of acidification due to DMO was markedly reduced in the mouse embryo up to the 4-cell stage. Specifically, non-essential amino acids and glutamine had the greatest capacity to buffer pHi in the early embryo. The ability of amino acids to buffer pHi was not apparent from the 8-16-cell stage onwards. In contrast to the precompacted embryo, the morula did not undergo a significant decrease in pHi until exposed to DMO concentrations > or = 10 mM in the absence of amino acids. This may be due to the generation of a permeability seal during compaction, thus enabling the morula to regulate its own pHi. This regulatory ability could either be reversed by causing the morula to decompact, or created by inducing premature compaction in the 8-16-cell embryo. Data presented in this study indicate that amino acids act as buffers of pHi in the early embryo and play a key role in regulating cell physiology. Further evidence for this was provided by the result that only those embryos cultured in 30 mM DMO in the presence of non-essential amino acids and 1 mM glutamine did not block at the 2-cell stage, but grew on to develop into expanded blastocysts.
The effectiveness of blastocyst culture and transfer in human in-vitro fertilization (IVF) was evaluated in a prospective randomized trial in patients having a moderate to good response to gonadotrophin stimulation. Embryos were transferred either on day 3 after culture to around the 8-cell stage in Ham's F-10 medium supplemented with fetal cord serum, or on day 5 after culture to the blastocyst stage in the sequential serum-free media G 1.2 and G 2.2. The pregnancy rates after transfer on day 3 or day 5 were equivalent, 66 and 71% respectively; however, significantly more embryos were transferred on day 3 (3.7) than on day 5 (2.2). The number of blastocysts transferred did not affect the implantation rate, and pregnancy rates when either two or three blastocysts were transferred were 68 and 87% respectively. The implantation rate of the blastocysts (50.5% fetal heart beat) was significantly higher compared to the cleavage stage embryos transferred on day 3 (30.1%). The percentage of blastocyst development was not affected by the number of 2-pronuclear embryos, or by maternal age. Irrespective of the number of blastocysts formed, pregnancy rates were similar. Furthermore, the pregnancy rate following blastocyst transfer in patients with 10 or more follicles at the time of human chorionic gonadotrophin administration was not affected by patient age. More than 60% of patients having blastocyst culture and transfer had supernumerary embryos for cryopreservation. The establishment of a pregnancy following thaw and transfer confirmed the viability of cryopreserved blastocysts cultured in the absence of serum or co-culture. The ability to transfer just two blastocysts while maintaining high pregnancy rates will therefore help to eliminate high order multiple gestations and improve the overall efficiency of human IVF.
In most in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) programmes approximately one ongoing pregnancy in three is multiple. The need to characterize embryos with optimal implantation potential is obvious. We retrospectively examined all of 23 double transfers resulting in ongoing twins, occurring between January 1, 1996 and May 19, 1997. Characteristics of these top quality embryos were absence of multinucleated blastomeres, four or five blastomeres on day 2, seven or more cells on day 3, and </=20% anucleated fragments. In a subsequent series of 400 IVF/ICSI cycles (out of which 372 were selected for embryo transfer) from May 20, 1997 to July 31, 1998, only women <38 years of age had multiple pregnancies: after 221 transfers of two embryos, 45/116 (39%) were multiple, and after 77 transfers of >2 embryos, 11/31 (35%) were multiple. We applied our top quality criteria to the 221 double transfers: 106 transfers with two top embryos resulted in 65 (63%) ongoing pregnancies with 37 (57%) twins, 65 transfers with one top embryo in 38 (58%) ongoing pregnancies with eight (21%) twins. In the group without top embryos, 12/52 (23%) ongoing singletons occurred, with no twins. The corresponding ongoing implantation rates were 49, 35 and 12%. This analysis suggests that single embryo transfer with an acceptable pregnancy rate might be considered if a top quality embryo is available.
A prospective randomized study comparing single embryo transfer with double embryo transfer after in-vitro fertilization or intracytoplasmic sperm injection (IVF/ICSI) was carried out. First, top quality embryo characteristics were delineated by retrospectively analysing embryos resulting in ongoing twins after double embryo transfer. A top quality embryo was characterized by the presence of 4 or 5 blastomeres at day 2 and at least 7 blastomeres on day 3 after insemination, the absence of multinucleated blastomeres and <20% cellular fragments on day 2 and day 3 after fertilization. Using these criteria, a prospective study was conducted in women <34 years of age, who started their first IVF/ICSI cycle. Of 194 eligible patients, 110 agreed to participate of whom 53 produced at least two top quality embryos and were prospectively randomized. In all, 26 single embryo transfers resulted in 17 conceptions, 14 clinical and 10 ongoing pregnancies [implantation rate (IR) = 42.3%; ongoing pregnancy rate (OPR) = 38.5%] with one monozygotic twin; 27 double embryo transfers resulted in 20 ongoing conceptions with six (30%) twins (IR = 48.1%; OPR = 74%). We conclude that by using single embryo transfer and strict embryo criteria, an OPR similar to that in normal fertile couples can be achieved after IVF/ICSI, while limiting the dizygotic twin pregnancy rate to its natural incidence of <1% of all ongoing pregnancies.
A prospective randomised study was performed to evaluate stimulated versus natural oviductal environment in comparison with
in-vitro culture for the developmental capacity of mouse embryos. Therefore, embryos of superovulated F1 hybrid CBAxC57Bl
females were collected at 17, 22, 41 and 46 h after human chorionic gonadotrophin treatment and randomly divided into five
groups. They were either transferred immediately to untreated pseudopregnant females, cultured in vitro for 5, 24 or 29 h before transfer, or cultured in vitro for 96 h to blastocysts. The transfers resulted in an impaired implantation (P < 0.001) and a lower numbers of living fetuses (P < 0.001) when embryos had been exposed longer to the stimulated oviductal environment. Similar results were obtained after
a longer period of in-vitro culture (P < 0.05). However when embryos were flushed earlier from the superovulated mice and cultured longer in-vitro until the transfer
was performed, the implantation rate was improved (P < 0.01). Blastocyst development, however, was better (P < 0.001) when embryos were flushed later. In conclusion, the stimulated oviductal environment impairs the developmental capacity
of embryos in comparison with untreated pseudopregnant females. In-vitro culture is also suboptimal but better than the stimulated
oviductal environment.
Polycystic ovary syndrome (PCOS) is closely associated with high miscarriage rates and, following in-vitro fertilization (IVF), with decreased fertilization rates, suggesting that oocytes and embryos are of poor quality. In this prospective study, we examined the development, metabolic activity and blastocyst cell number of embryos following IVF from 51 patients with either anovulatory PCOS, ovulatory PCOS or tubal disease. The number of oocytes retrieved and the fertilization rates were similar for patients with PCOS and tubal disease. Following embryo transfer, 46% of the patients with PCOS and 36% of patients with tubal disease became pregnant. A similar proportion of surplus embryos from patients with PCOS and tubal disease developed to the blastocyst stage (38% and 43% respectively). Patients with anovulatory PCOS had embryos with less fragmentation which cleaved faster, cavitated earlier and had more cells at the blastocyst stage than embryos from patients with tubal disease. While the profile of glucose uptake and lactate production was similar for all groups throughout preimplantation development, patients with tubal disease who underwent ovulation induction using the ‘titrated’ regimen optimized for PCOS patients resulted in embryos with reduced pyruvate uptake, in addition to low blastocyst cell numbers. This study demonstrates that with an optimized ovulation induction regimen, embryos from PCOS patients are of good quality and developmental potential.
During the preimplantation period of mammalian embryo development, the conceptus undergoes significant changes in its physiology, metabolism, and genetic control. These changes are so dramatic that the starting point of development—the zygote—and the final stage—the blastocyst—can be likened to two very different somatic cell types. The zygote, like the oocyte from which it is derived, has a low metabolic activity and exhibits low levels of oxygen consumption and low QO2, and therefore can be likened to relatively quiescent adult tissue such as the brain. Prior to compaction, the embryo in culture benefits from the presence of specific amino acids such as alanine, aspartate, asparagine, glycine, glutamate, glutamine, proline, and serine. This chapter focuses on the utility of sequential media in optimizing mammalian embryo development in culture. The conditions that are good for the early stage are not optimal for blastocyst differentiation, and the conditions that do support good blastocyst differentiation and maintain embryo viability are not optimal for the development of the zygote. Supplying the embryo with gradients of carbohydrates and amino acids not only provides the changing nutrients required but also reduces intracellular stress. The ability to minimize intracellular stress is a significant factor in being able to maintain embryo viability in culture. This premise is supported by the fact that the sequential culture media, G1 and G2, can support the development of viable blastocysts of the primate, rodent, and ruminant, despite the species differences in embryo physiology. With the use of sequential media, blastocyst developmental rates in vitro and the subsequent implantation rates are very close to those observed in vivo.
Many companies have fallen, not because they ignored their customers or lacked superior management skills, but because business conditions shifted beneath them. In an environment of fluctuating markets, proliferating technologies, and changing political frontiers, the management challenge is to cope with breakpoints, or sudden shifts in the rules of the game. Paul Strebel provides a framework for dealing with radical business change. Strebel shows that breakpoints may be as enterprising as Drexel Burnham Lambert's promotion of the junk bond market - or as dramatic as its collapse. They may result from changes in industry conditions such as the emergence of new technologies or price wars. When business conditions shift, the formula for success inevitably changes. This book presents a way of managing different types of radical business change. Strebel describes a simple framework that allows managers to adapt to diverse conditions, especially those that lead to breakpoints. He suggests how to develop radical change scenarios that will help managers better anticipate or catch up with discontinuities. Further, Strebel reveals what managers must do to create competitive discontinuities - and become true market leaders.
The success of the present experiments confirms that the techniques employed have not affected adversely either the eggs or the conditions for conception. The results also prove that the environment provided by the contralateral oviduct, away from the immediate influence of the corpus luteum, is adequate for the survival of the embryo. Uterine transfer of the egg can be used to mimic the conditions of accelerated tubal transport, and in the present experiments it seems likely that the eggs were placed in the uterus at least 1 day ahead of their normal schedule. It may be significant that 2 of the 3 animals receiving uterine transfers each showed one positive sign of early conception. Our results suggest that perfectly synchronized transfers of tubal eggs between rhesus monkeys should have a good chance of being successful. Kraemer et al. (1976) obtained one pregnancy in their series of 10 synchronized, between animal transfer of uterine eggs in the baboon (Papio cynocephalus) using techniques similar to ours. Their results may reflect the greater technical difficulty of making uterine transfers, but are perhaps more likely to be due to the difficulties of synchronizing the donor and recipient animals. It may therefore be equally difficult to obtain successful heterologous tubal transfers in the rhesus monkey.
In a study of 29 cycles of IVF, ET was performed on day 5 after oocyte recovery when embryos had developed to the morula/blastocyst stage. Three preclinical pregnancies and three live births resulted (2 singleton and 1 twin), giving a viable PR per ET of 10%. It is concluded that while day 5 ET may well be important in terms of embryo biopsy for the preimplantation diagnosis of genetic disease, day 2 ET remains preferable for therapeutic IVF. Although these data would not support the introduction of day 5 ET into routine therapeutic IVF, delayed ET should be considered as an alternative approach to preimplantation diagnosis. Indeed, because the latter will generally involve the treatment of normal, fertile couples, it might be predicted that embryo survival rates, and thus the rate of pregnancy after day 5 ET, would be better than those presented here.
Culture media were developed for pronuclear-stage mouse embryos using simplex optimization, which has the benefit of being able to optimize several components simultaneously. Initially, several different media were generated. All media contained the same components, yet each medium was characterized by having a different component at a high concentration. The simplex procedure identified 4 components (NaCl, pyruvate, KH2PO4 and glucose) which at high concentrations were detrimental to embryo development, compared to the other components tested. For example, all embryos cultured in a medium with high NaCl blocked at the 2-cell stage. The optimization method then adjusted each medium by lowering the concentration of the component or removing it entirely, which resulted in a significant increase in development. In an experiment comparing 8 media generated from the simplex optimization, along with 7 other media, removal of KH2PO4 resulted in the largest increase in development; 88% of embryos were greater than or equal to 4 cells on Day 3 after hCG, and 53% developed into blastocysts by Day 5. Another experiment compared 4 of the best media generated from the simplex optimization. In 3 out of the 4 media, 90% or more of the embryos were greater than or equal to 4 cells on Day 3. In 3 of the media, approximately 60% or more of the embryos developed into blastocysts. The simplex optimization procedure is an efficient method for developing culture media and determining requirements for development in vitro.
The earliest stages of development in most animals, including the few mammalian species that have been investigated, are regulated by maternally inherited information. Dependence on expression of the embryonic genome cannot be detected until the mid two-cell stage in the mouse, the four-cell stage in the pig (J. Osborn & C. Polge, personal communication), and the eight-cell stage in the sheep. Information about the timing of activation of the embryonic genome in the human is of relevance not only to the therapeutic practice of in vitro fertilization and embryo transfer (IVF), but more importantly for the successful development of techniques for the preimplantation diagnosis of certain inherited genetic diseases. We describe here changes in the pattern of polypeptides synthesized during the pre-implantation stages of human development, and demonstrate that some of the major qualitative changes which occur between the four- and eight-cell stages are dependent on transcription. In addition, it appears that cleavage is not sensitive to transcriptional inhibition until after the four-cell stage.
Using uterine lavage performed 5 days after the luteinizing hormone peak, we collected 25 uterine ova from five fertile donors who had had a single, periovular artificial insemination. After examination, all recovered ova were transferred to recipient uteri and resulted in three intrauterine and one tubal pregnancy. Morphologic development ranged from degenerating single-cell ova to mature blastocysts. Ages of the ova at recovery ranged from 93.5 to 130.0 hours postovulation. Mean age of the five blastocysts, 109.1 hours, was not significantly different from the mean age of the 20 less mature ova, 108.1 hours. Neither the mean interval from insemination to recovery for blastocysts nor the mean interval from insemination to ovulation for blastocysts was significantly different from the mean intervals of the other ova. The five blastocysts resulted in intrauterine pregnancies in three recipients. There were no intrauterine pregnancies from the other 20 transfers (p less than 0.004). A transferred 12-cell ovum with degenerating blastomeres was associated with tubal pregnancy in the recipient. The large variability in the state of intrauterine ova observed in the relatively fixed postovulatory interval of this study appears to be due to differences in viability and maturation, not differences in ovum age. Maturational state of the ova at the time of transfer appears to be a significant determinant of the likelihood of ensuring pregnancy.
Simplex optimization has generated several media that have improved the development of mouse preimplantation embryos in vitro. One objective of this study was to compare the development of preimplantation mouse embryos in one of these computer-optimized media, KSOM, with embryos that developed in vivo, in terms of the relative abundances of specific mRNAs involved in metabolism, transcription, and cell proliferation. First, however, since studies have indicated an improvement of other simple embryo culture media by addition of amino acids, the effects of the addition of amino acids to KSOM (KSOM/AA) on preimplantation development were assessed. We find that addition of both essential and nonessential amino acids to KSOM augments development in vitro, as compared to development supported by KSOM without amino acids. This augmentation is observed starting at the blastocyst stage, and is associated with increased rate of development to the blastocyst stage, increased frequency of hatching, and increased number of cells in the blastocysts. Reverse-transcription PCR was then used to assess the relative abundance of mRNAs for actin, glyceraldehyde-3-phosphate dehydrogenase, Na+, K(+)-ATPase, Sp1, TATA box-binding protein TBP, IGF-I, IGF-II, IGF-I receptor, and IGF-II receptor in embryos that developed in vivo and in vitro using KSOM/AA. Eight out of 9 of these mRNAs were present in the 8-cell embryos and blastocysts raised in KSOM/AA in amounts that were indistinguishable from those in embryos that developed in vivo. It is concluded that KSOM/AA provides an environment in which preimplantation mouse embryos can undergo development that is quantitatively similar to that occurring in vivo.
An enzymatic method for removing embryo-toxic ammonium from culture medium has been developed. Ammonium, produced by both embryo metabolism and spontaneous breakdown of amino acids at 37 degrees C, is transaminated by glutamate dehydrogenase to nontoxic glutamate. Initially, the individual components of the transamination reaction were titrated against mouse embryo development in vitro to determine embryo-safe levels. ADP, an allosteric activator of glutamate dehydrogenase, was found to inhibit embryo development and was therefore omitted from the final formulation (alpha-ketoglutarate, 0.44 mM; glutamate dehydrogenase, 0.375 U; NADH, 0.12 mM). It was found that 0.30 mM ammonium could be removed from the culture medium in situ in 3 h. In situ removal of ammonium significantly increases both blastocyst cell number, implantation, fetal development, and fetal weight after transfer. Removal of ammonium by the conventional method of renewing the culture medium also increased blastocyst cell number but did not affect postimplantation development. In conclusion, it is possible to alleviate the toxic effects of ammonium in vitro on pre- and postimplantation mouse embryo development by its transamination in situ, thereby facilitating the continual exposure to embryo-derived factor(s) which stimulates both pre- and postimplantation development.
To determine the levels of metabolites surrounding the human oocyte and embryo in vivo.
Oviduct and uterine fluids were collected throughout the menstrual cycle. Cumulus cells were collected at oocyte retrieval and their production of metabolites was assessed. Samples were analyzed for pyruvate, lactate, and glucose by microfluorimetry.
Luminal fluids were collected from naturally cycling patients at the time of routine clinical investigation. Patient consent and hospital ethics approval were obtained for this study.
Pyruvate in the oviduct did not vary with the day of cycle, the mean value was 0.24 mM. Lactate and glucose concentrations varied with the day of cycle; lactate increasing from 4.87 mM in the follicular phase to 10.50 mM at the time of ovulation, whereas glucose decreased from 3.11 mM in the follicular phase to 0.50 mM midcycle and subsequently increased to 2.32 mM in the luteal phase. The concentrations of pyruvate, lactate, and glucose in uterine fluid remained constant throughout the cycle (0.10, 5.87, and 3.15 mM, respectively). All metabolite concentrations in uterine fluid were significantly different from those in the oviduct midcycle. Cumulus cells readily consumed glucose in vitro, with lactate being the major metabolite produced.
These data indicate that lactate and glucose concentrations in the oviduct change with day of cycle and that the human embryo is exposed to different metabolite concentrations as it passes along the tract. Furthermore, cumulus cells readily consume glucose, producing lactate. Therefore, the early human embryo is exposed to low glucose and high lactate levels in vivo.
Polycystic ovary syndrome (PCOS) is closely associated with high miscarriage rates and, following in-vitro fertilization (IVF), with decreased fertilization rates, suggesting that oocytes and embryos are of poor quality. In this prospective study, we examined the development, metabolic activity and blastocyst cell number of embryos following IVF from 51 patients with either anovulatory PCOS, ovulatory PCOS or tubal disease. The number of oocytes retrieved and the fertilization rates were similar for patients with PCOS and tubal disease. Following embryo transfer, 46% of the patients with PCOS and 36% of patients with tubal disease became pregnant. A similar proportion of surplus embryos from patients with PCOS and tubal disease developed to the blastocyst stage (38% and 43% respectively). Patients with anovulatory PCOS had embryos with less fragmentation which cleaved faster, cavitated earlier and had more cells at the blastocyst stage than embryos from patients with tubal disease. While the profile of glucose uptake and lactate production was similar for all groups throughout preimplantation development, patients with tubal disease who underwent ovulation induction using the 'titrated' regimen optimized for PCOS patients resulted in embryos with reduced pyruvate uptake, in addition to low blastocyst cell numbers. This study demonstrates that with an optimized ovulation induction regimen, embryos from PCOS patients are of good quality and developmental potential.
To compare the implantation rate of embryos after 3 and 5 days of IVF culture.
Prospective randomization of ET depending on the weekday of ovum pick-up (OPU).
University Department of Endocrinology and Reproduction.
All women entering an outclinic IVF program.
Two hundred thirty-three ETs performed on day 3 after OPU and 410 performed on day 5 were analyzed. When blastocysts with a clear inner cell mass were available, a maximum of two were replaced.
On day 3 after OPU, 60 pregnancies per 233 ET (26%) and on day 5, 102 pregnancies per 410 ET (25%) were induced. The average implantation rate per embryo was 13% and 12%, respectively. After subdivision according to embryo morphology, pregnancy rate per ET (n = 59) and implantation rate per embryo on day 3 with exclusively unfragmented embryos were 32% and 18%, respectively, not significantly different from ET (n = 73) exclusively with embryos containing > 0% and < 20% fragments: 27% and 12%. After transfer on day 5, when one or more cavitating embryos were available (n = 227), pregnancy and implantation rates were 40% and 23%, statistically different from ET on day 3. On day 5, ET exclusively with morula stages showing signs of starting blastulation (n = 26), pregnancy rate and implantation rate were 12% and 11%, respectively, from ET (n = 157) with embryos not reaching the latter stage: 6% and 3%.
Overall ET results after 3 and 5 days are comparable. After 5 days of culture, one to two embryos can be replaced with an average implantation rate of > 23% per embryo, minimizing the incidence of triplets.
The transition between dependence on maternal transcripts and proteins inherited in the oocyte and embryonic gene expression in the human preimplantation embryo occurs at the four- to eight-cell stage. Recently, studies using reverse transcriptase polymerase chain reaction (RT-PCR) have detected paternal transcripts for the Y-linked genes, ZFY and SRY, and the myotonic dystrophy associated protein kinase gene, DK, as early as the late pronucleate one-cell stage. However, expression at the protein level has not been demonstrated and its function at these early stages is unknown. Using coding sequence polymorphisms to distinguish maternal and paternal transcripts, we have examined the transcription of two ubiquitously expressed genes: X-linked glucose-6-phosphate dehydrogenase (G6PD) and adenosine deaminase (ADA). Both G6PD and ADA are housekeeping genes with TATA-less promoters which, because of their roles in metabolism and ubiquitous expression, may provide a more reliable indication of the timing of activation of the embryonic genome. They also each have biallelic polymorphisms with a high heterozygosity ratio which can be detected by restriction digestion. Couples undergoing in vitro fertilization (IVF) were screened for these polymorphisms. Individual spare oocytes and embryos at different stages of preimplantation development were analyzed by RT-PCR and appropriate restriction digestion in those cases in which the male partner carried a different allele to the female partner. In addition, since only female embryos inherit the paternal allele of X-linked G6PD, cDNA was also analyzed for ZFX/ZFY transcripts to identify the sex of each embryo. One hundred and twenty three individual oocytes and embryos were analyzed by RT-PCR and restriction digestion to detect the paternal transcripts from the polymorphic alleles. Maternal transcripts for G6PD, ADA, and ZFX were detected in all oocytes and embryos and at all stages.
A retrospective analysis of results from 114 initiated in-vitro fertilization cycles utilizing pronuclear embryo transfer is presented. Patients were unselected for age or infertility criteria, constituted a continuous series and were grouped according to response to stimulation (Group 1, ideal; Group 2, suboptimal) or ovarian reserve (Group 3, poor). At 16-18 h post-insemination, embryos were scored for alignment of pronuclei and nucleoli and the appearance of the cytoplasm, generating an embryo score (ES). Transfers were performed 24-26 h post-insemination using two to six embryos with the highest ES. A corrected score was calculated (total score/number of embryos; CS). A total of 114 initiated cycles resulted in 97 oocyte retrievals with 38 clinical pregnancies (39%; 15% implantation). Pregnancy rates were significantly different between the three groups; 37 pregnancies in Group 1 (55% clinical pregnancy; 20% implantation), none in Group 2 and one in Group 3 (6%; 2% implantation: P < 0.001). The ES of transferred embryos correlated with groups. There was a strong correlation between CS and implantation and delivery rates. CS >15 resulted in a 28% implantation; 65% delivery rate. CS <14 resulted in four pregnancies, one delivered. The data show that oocyte quality and pronuclear embryo morphology are related to implantation and that pronuclear embryos can be successfully selected for embryo transfer.
To analyze the effect on uterine receptivity of a decrease in E2 levels during the preimplantation period with the use of a step-down regimen in high responders undergoing IVF.
Prospective controlled clinical study.
The Instituto Valenciano de Infertilidad.
High responders in whom at least one previous IVF attempt failed in which 3-4 good-quality embryos were transferred and E2 levels were >3,000 pg/mL on the day of hCG administration.
Gonadotropins were administered according to two different protocols. Blood samples were collected and IVF was performed.
Serum E2 levels and reproductive outcome of IVF.
Estradiol levels on the day of hCG administration and throughout the preimplantation period and the number of oocytes collected were significantly lower with the use of the step-down regimen than during the previous failed cycle in which the standard protocol was used. The fertilization rate was similar and the number of good-quality embryos transferred was comparable. However, the implantation and pregnancy rates were significantly improved in patients who underwent the step-down regimen compared with those who received the standard protocol.
With the use of a step-down regimen with FSH in high responders, our clinical results demonstrate that uterine receptivity can be improved when E2 levels are decreased during the preimplantation period.
To investigate the possible consequences of uterine contractions (UC) as visualized by ultrasound (US) on in-vitro fertilization (IVF)-embryo transfer outcome, we studied prospectively 209 infertile women undergoing 220 cycles of controlled ovarian stimulation. Inclusion criteria were age < or = 38 years, a morphologically normal uterus, and at least three good quality embryos transferred. Just before embryo transfer, women underwent 5 min digital recordings of the uterus using US image analysis software for UC assessment. Plasma progesterone and oestradiol concentrations were measured. Four groups were defined according to UC frequency: < or = 3.0 (n = 53), 3.1-4.0 (n = 50), 4.1-5.0 (n = 43), and > 5.0 (n = 74) UC/min respectively. Patients, controlled ovarian hyperstimulation and embryology characteristics were comparable in all groups. A stepwise decrease in clinical and ongoing pregnancy rates as well as in implantation rates occurred from the lowest to the highest UC frequency groups (53, 36, 21; 46, 32, 20; 23, 19, 10; and 14, 11, 4%; P < 0.001). Plasma progesterone and UC frequency were negatively correlated (r = -0.34, P < 0.001). Direction of UC did not affect embryo transfer outcome. As this study was controlled strictly for confounding variables and UC were assessed objectively by a computerized system, its results indicate that high frequency UC on the day of embryo transfer hinder IVF-embryo transfer outcome, possibly by expelling embryos out of the uterine cavity. The negative correlation between UC frequency and progesterone concentrations supports the uterine relaxing properties of progesterone.
To determine whether blastocyst transfer is of benefit to patients with multiple IVF failures.
Retrospective cohort study.
The George Washington University Medical Center.
Patients undergoing IVF between October 1, 1997, and November 30, 1998, who had previously undergone three or more unsuccessful IVF cycles. Patients who had at least three embryos at the 8- to 12-cell stage available on day 3 were eligible for the study.
Patients were given the option of day 3 ET (group A) or blastocyst transfer (group B).
Blastocyst-formation rate, clinical pregnancy rate (PR) per transfer, and implantation rate per transfer.
Groups A and B were similar in terms of age, the number of previous failed IVF cycles, fertilization rate, and the number of fertilized oocytes per cycle. The blastocyst-formation rate was 51.0%. Clinical pregnancy and implantation rates per transfer were statistically significantly higher in the blastocyst-transfer group. There were no multiple pregnancies after blastocyst transfer.
Blastocyst transfer increases implantation rates and PRs in patients with multiple failed IVF cycles, without increasing the risk of multiple pregnancy.
To determine whether previously described advanced blastocyst development and high implantation rates are confirmed in an expanded multicenter trial.
Retrospective review.
Two private assisted reproductive technology units.
One hundred seventy-four patients who underwent blastocyst culture and transfer.
Culture of all pronucleate embryos in sequential media to the blastocyst stage (day 5) followed by ET.
The number and percentage of blastocysts developed, implantation rates, pregnancy rates, and parameters that affected outcome were analyzed.
Only 3 of 174 patients failed to achieve blastocyst-stage ET. The mean blastocyst development rate was 48%. The ongoing pregnancy rate was 66.3% per oocyte retrieval, with a mean (+/-SE) of 2.2 +/- 0.05 blastocysts transferred and an implantation rate of 48% per blastocyst transferred.
Blastocyst culture and transfer is an effective means of treating patients who respond well to gonadotropins. High pregnancy rates can be accomplished with low numbers of embryos transferred. Patients who failed to achieve ET were rare.
To evaluate the nonselective application of extended embryo culture on the outcome of IVF.
Retrospective analysis.
Private practice assisted reproductive technology center.
Seven hundred ninety nonselected patients undergoing IVF with controlled ovarian stimulation.
For day 3 ET, multicell embryos were cultured in human tubal fluid medium and 12% synthetic serum substitute. For day 5 ET, embryos were cultured for 48 hours in S1 medium and then for 48 hours in S2 medium.
Implantation rate (determined by total no. of visualized gestational sacs), ongoing pregnancy rate, and number of embryos available for ET.
Respective day 3 and day 5 implantation rates for patients aged <35 years (29.5% and 38.9%), patients aged 35-39 years (20.7% and 28.2%), and all patients combined (23.3% and 32.4%) were statistically significantly different. Significantly more embryos were transferred on day 3 than on day 5 for patients aged <35 years (2.9 vs. 2.4), patients aged 35-39 years (3.1 vs. 2.6), and all patients combined (3.0 vs. 2.5). The difference in ongoing pregnancy rates per retrieval was statistically significant for day 3 compared with day 5 transfers for all patients combined (35.9% vs. 43.8%). Cancellation rates for transfer after retrieval increased significantly for day 3 compared with day 5 transfer (2.9% vs 6.7%).
These results demonstrate the feasibility of using extended embryo culture in a nonselective manner for couples undergoing IVF. Overall, extended embryo culture was associated with a significant increase in pregnancy rates and implantation rates and a significant decrease in the number of embryos transferred. The rate of multiple implantation among patients aged <35 years warrants consideration of single blastocyst transfers for this group.
To compare implantation and pregnancy rates (PRs) achieved with blastocyst transfer (BT) and day 3 ET in similar patient populations.
Retrospective analysis.
Academic infertility center.
One hundred consecutive patients <40 years undergoing IVF, each with more than three eight-cell embryos on day 3.
Patients used their own eggs for IVF or IVF and intracytoplasmic sperm injection. Embryos were cultured in P1 medium (Irvine Scientific, Santa Ana, CA) until day 3, when they were either transferred or, in the case of embryos for BT, incubated in Blastocyst Medium (Irvine Scientific), followed by transferring on day 5.
Implantation and PRs.
There were no statistically significant differences in patient age, FSH level, or number of oocytes or zygotes. The BT group had fewer embryos transferred (mean, 2.4) compared with the day 3-ET group (mean, 4.6). The viable PR (cardiac activity at 6-7 weeks was considered indicative of a viable pregnancy) was higher with BT (68%, 34/50) than with day 3 ET (46%, 23/50). The implantation rate was increased with BT (47%, 56 sacs/120 embryos) compared with day 3 ET (20%, 46 sacs/231 embryos).
The BT group in our study had higher implantation and PRs compared with the day 3-ET group. Better embryo selection, improved embryo-uterine synchrony, and decreased cervical mucus on day 5 may have accounted for the enhanced outcome. Our data support the use of BT to limit the number of embryos transferred while improving PRs.
To evaluate implantation and pregnancy rates in patients undergoing IVF after the transfer of a maximum of two embryos that had been cultured for 3-5 days.
Prospective study.
An IVF laboratory at a tertiary referral university hospital.
One thousand seven hundred eighty-seven couples who underwent their first IVF cycle between January 1995 and December 1997.
In vitro fertilization and transfer of embryos after 3, 4, or 5 days of culture using a single medium without coculture.
Implantation and pregnancy rates.
Overall implantation and pregnancy rates were not significantly different with different culture periods. Forty-one percent of all available embryos developed into blastocysts on day 5. The transfer of at least one good-quality blastocyst could be performed in 62% of patients. Blastocysts had an implantation rate of 26% per embryo, whereas the implantation rate of eight-cell embryos on day 3 was 18%. Implantation rates for retarded, normal, and advanced embryos were not significantly different with an extended culture period.
Under the study conditions, the transfer of embryos after 5 days rather than 3 days of embryo culture did not change the overall implantation and pregnancy rates. The implantation potential of embryos available for transfer can be assessed better after an extended culture period. Five days of culture allows the transfer of a reduced number of embryos without decreasing overall pregnancy rates.
Along with the transition from maternal to embryonic genome control the mammalian preimplantation embryo undergoes significant changes in its physiology during development. Concomitant with these changes are altering patterns of nutrient uptake and differences in the subsequent fate of such nutrients. The most significant nutrients to the developing mammalian preimplantation embryo are carbohydrates and amino acids, which serve not only to provide energy but also to maintain embryo function by preventing cellular stress induced by suboptimal culture conditions in vitro. It is subsequently proposed that optimal development of the mammalian embryo in culture requires the use of two or more media, each designed to cater for the changing requirements of the embryo. Importantly, culture conditions that maintain the early embryo are not ideal for the embryo post-compaction, and conditions that support excellent development and differentiation of the blastocyst can actually be inhibitory to the zygote. A marker of in vitro-induced cellular stress to the embryo is the relative activity of the metabolic pathways used to generate energy for development. Quantification of embryo energy metabolism may therefore serve as a valuable marker of embryo development and viability.
The H19 gene is imprinted with preferential expression from the maternal allele. The putative imprinting control region for this locus is hypermethylated on the repressed paternal allele. Although maternal-specific expression of H19 is observed in mouse blastocysts that develop in vivo, biallelic expression has been documented in embryos and embryonic stem cells experimentally manipulated by in vitro culture conditions. In this study the effect of culture on imprinted H19 expression and methylation was determined. After culture of 2-cell embryos to the blastocyst stage in Whitten's medium, the normally silent paternal H19 allele was aberrantly expressed, whereas little paternal expression was observed following culture in KSOM containing amino acids (KSOM+AA). Analysis of the methylation status of a CpG dinucleotide located in the upstream imprinting control region revealed a loss in methylation in embryos cultured in Whitten's medium but not in embryos cultured in KSOM+AA. Thus, H19 expression and methylation were adversely affected by culture in Whitten's medium, while the response of H19 to culture in KSOM+AA approximated more closely the in vivo situation. It is unlikely that biallelic expression of H19 following culture in Whitten's medium is a generalized effect of lower methylation levels, since the amount of DNA methyltransferase activity and the spatial distribution of Dnmt1 protein were similar in in vivo-derived and cultured embryos. Moreover, imprinted expression of Snrpn was maintained following culture in either medium, indicating that not all imprinted genes are under the same stringent imprinting controls. The finding that culture conditions can dramatically, but selectively, affect the expression of imprinted genes provides a model system for further study of the linkage between DNA methylation and gene expression.