Blastocyst culture in human IVF: the final destination or a stop along the way?

In the field of human IVF, culturing embryos to the blastocyst stage has gained popularity within the past few years. The impetus to transfer blastocysts has been spurred by several factors: 1) the desire to improve implantation rates in infertility patients, 2) a desire to reduce the multiple pregnancy rate by transferring fewer embryos, 3) the desire to perform pre-implantation genetic diagnosis, and 4) the advent of sequential media. Although culturing human embryos to the Hastocyst stage has improved implantation rates and reduced the incidence of multiple pregnancies in some patient populations, it has not worked for all populations of infertility patients. Factors that may affect the ability of a human embryo to reach the blastocyst stage include the patient's age, cohort of ova retrieved, the use of intracytoplasmic sperm injection of blastomere biopsy, culture conditions, or intrinsic factors within the embryo itself. Culture of human embryos to the blastocyst stage can be an effective method for improving implantation rates and reducing the high order multiple pregnancy rates seen in human IVF clinics when more than three embryos are transferred.     

Successful pregnancy after transfer of rabbit blastocysts grown in vitro from single-cell zygotes

To establish successful pregnancy in rabbits after the transfer of blastocysts cultured in vitro for 72 h, pregnancy rates were compared according to synchronization methods of recipient and embryo transfer sites. Also, the effect of RDH (1:1:1 mixture of RPMI, DMEM and Ham's F10) medium with additives such as BSA and taurine was evaluated for developmental capacity and cell number. Developmental capacity and cell number were considered important for implantation. When we evaluated the relative survival of rabbit one-cell embryos after culture in Ham's F10, in RD or in RDH for 72 h, embryos cultured in RDH and RD developed much better than in Ham's F10. When the effects of BSA and taurine in RDH medium were tested for rabbit embryo development, BSA or taurine promoted transition to the blastocyst stage and increased cell numbers of cultured embryos in RDH medium. The BSA and taurine together in RDH medium had a synergistic effect on embryo development. By transferring cultured blastocysts to the oviduct of the recipient doe synchronized one day behind the donor, live-born pups were obtained successfully. These results demonstrated that rabbit blastocysts can develop to normal pups after in vitro culture and embryo transfer.     

The culture of human cleavage stage embryos alone or in groups: effect upon blastocyst utilization rates and implantation

The effect of cleavage-stage group culture (CGC; embryos cultured in groups of three or more for the first 3 days and then individually to blastocyst) was compared to extended single embryo culture (ESC; embryos cultured individually to the blastocyst stage). While implantation and ongoing pregnancy rates were similar between groups, the blastocyst utilization rate (number of blastocysts suitable for freezing and thawing/total number of embryos cultured to Day 5 and 6) was significantly higher when embryos were cultured in CGC for women ≤35 yrs thereby increasing the number of embryos available for clinical use for the younger women. This strategy of group culture to Day 3 would seem an ideal protocol to capitalize on an overall embryo quality in two particular settings, namely programmes wishing to (i) undertake Day 3 transfers, and (ii) keep embryos separate from Day 3 to Day5/6 for the purposes of selection. The culture system can also be applied to the embryos of older women without adverse effect, enabling the same system to be used for all embryos.     

Comparison of day 2 and overnight day 3 frozen embryo transfers: A prospective randomized controlled trial

In certain patients cleavage stage embryos may be preferred. The relationship between an additional day in culture and pregnancy outcomes is not well established. We aimed to compare outcomes of day 2 versus overnight day 3 frozen embryo transfer (FET). In this randomized controlled trial, patients with day 2 cryopreserved embryos were allocated to two groups. In group A embryos were transferred on day 2, the same day of thawing. In group B embryos were transferred one day after thawing, on day 3 after overnight incubation. Out of 410 patients eligible, 92 were recruited. Finally, 72 patients participated, 39 in group A and 33 in group B. No significant difference in implantation (11 % in group A and 14 % in group B, p = 0.81), clinical pregnancy (18 % in group A and 21 % in group B, p = 0.73) or live birth rates (13 % in group A and 18 % in group B, p = 0.53) was found. To conclude, no significant difference in reproductive outcomes was found when comparing patients with day 2 or overnight day 3 FET. Considering published data on blastocyst transfer, cleavage stage ET may still be a relevant option and the decision between day 2 or overnight day 3 ET depends on patients’ and physicians’ preference and recommendation.     

移植胚胎数目对高龄不孕患者IVF—ET结局的影响

目的：探讨移植胚胎数目对高龄不孕患者IVF—ET结局的影响。方法：根据移植胚胎个数将年龄超过35岁的不孕患者338个周期分为单胚胎移植组（I组）,2个胚胎移植组（Ⅱ组）,3个胚胎移植组（Ⅲ组）。分析患者IVF治疗情况并按年龄分层比较三组患者的IVF-ET结局。结果：高龄不孕患者行IVF,随年龄增加,获卵数、优质胚胎率和临床妊娠率降低,流产率呈增高趋势。Ⅰ组的妊娠率9．43％,显著低于Ⅱ组（24．24％）和Ⅲ组（31．37％）（P〈0．05）。对于40岁以下的患者,移植3个胚胎的妊娠率与移植2个胚胎差无异,但显著高于移植1个胚胎（P〈0．05）。增加40岁以上患者移植胚胎的数目,妊娠率未出现有统计学意义的升高。三组的胚胎种植率分别为9．43％,12．12％和12．2％,无统计学差异。Ⅲ组中多胎率12．5％（6／48）,其中35-36岁年龄段多胎率16．67％（5／30）。结论：高龄不孕患者可移植的胚胎数目随年龄增加和获卵数目降低而降低。其中较年轻者（35-36岁年龄段）,移植3个胚胎,对妊娠率提高无明显效果,但多胎发生显著增加。     

Metabolite availability as a window to view the early embryo microenvironment in vivo

A preimplantation embryo exists independent of blood supply, and relies on energy sources from its in vivo environment (e.g., oviduct and uterine fluid) to sustain its development. The embryos can survive in this aqueous environment because it contains amino acids, proteins, lactate, pyruvate, oxygen, glucose, antioxidants, ions, growth factors, hormones, and phospholipids—albeit the concentration of each component varies by species, stage of the estrous cycle, and anatomical location. The dynamic nature of this environment sustains early development from the one‐cell zygote to blastocyst, and is reciprocally influenced by the embryo at each embryonic stage. Focusing on embryo metabolism allowed us to identify how the local environment was deliberately selected to meet the dynamic needs of the preimplantation embryo, and helped reveal approaches to improve the in vitro culture of human embryos for improved implantation rates and pregnancy outcome.     

子宫内膜容受性检测技术在反复种植失败患者冻融胚胎移植中的应用价值及其临床妊娠的影响因素分析

摘要 目的：探讨子宫内膜容受性检测（ERT）技术在反复种植失败患者冻融胚胎移植（FET）中的应用价值,并分析其临床妊娠的影响因素。方法：回顾性分析2019年10月~2022年4月期间海南省妇女儿童医学中心收治的150例反复种植失败患者的临床资料,根据是否接受ERT技术分为ERT组（n=78,接受ERT技术）和无ERT组（n=72,未接受ERT技术）。按照反复种植失败患者是否临床妊娠分为临床妊娠组和未临床妊娠组。采用单因素和多因素Logistic回归模型分析临床妊娠的影响因素。结果：两组异位妊娠率组间对比未见统计学差异（P>0.05）。ERT组临床妊娠率、活产率高于无ERT组,移植日内膜厚度大于无ERT组,移植胚胎数少于无ERT组,流产率低于无ERT组（P<0.05）。所有患者按照是否临床妊娠分为临床妊娠组（n=85）和未临床妊娠组（n=65）。单因素分析结果显示：临床妊娠与年龄、移植胚胎类别、移植胚胎数量、总周期数、FSH、子宫内膜厚度、子宫内膜类型有关（P<0.05）,而与体质量指数（BMI）、不孕年限、不孕类型、胚胎冷冻保存时间无关（P>0.05）。多因素Logistic回归分析结果显示：年龄偏大、FSH偏高是临床妊娠的危险因素,而移植胚胎类别为囊胚、移植胚胎数量偏多是临床妊娠的保护因素（P<0.05）。结论：ERT技术用于反复种植失败患者FET中,可有效改善患者的临床妊娠。年龄、FSH、移植胚胎类别、移植胚胎数量是临床妊娠的影响因素。     

Mitochondrial DNA in the mouse preimplantation embryo

Total DNA was extracted from mouse embryos that were collected from CD-1 random-bred females on Day 1 of pregnancy and cultured for up to 4 days in vitro, or from the reproductive tracts of pregnant females on Days 1, 3, 4 and 5 of pregnancy. Southern blot analyses with a cloned mouse mitochondrial DNA probe were performed to determine the relative levels of mitochondrial DNA in the zygote, morula, blastocyst and early egg cylinder stage embryos. The results indicated that the total amount of mitochondrial DNA does not change during development of the mouse embryo up to the egg cylinder stage and is not altered during in-vitro culture of the fertilized one-cell embryo to the blastocyst stage.     

Equine cloning: in vitro and in vivo development of aggregated embryos

The production of cloned equine embryos remains highly inefficient. Embryo aggregation has not yet been tested in the equine, and it might represent an interesting strategy to improve embryo development. This study evaluated the effect of cloned embryo aggregation on in vitro and in vivo equine embryo development. Zona-free reconstructed embryos were individually cultured in microwells (nonaggregated group) or as 2- or 3-embryo aggregates (aggregated groups). For in vitro development, they were cultured until blastocyst stage and then either fixed for Oct-4 immunocytochemical staining or maintained in in vitro culture where blastocyst expansion was measured daily until Day 17 or the day on which they collapsed. For in vivo assays, Day 7-8 blastocysts were transferred to synchronized mares and resultant vesicles, and cloned embryos were measured by ultrasonography. Embryo aggregation improved blastocyst rates on a per well basis, and aggregation did not imply additional oocytes to obtain blastocysts. Embryo aggregation improved embryo quality, nevertheless it did not affect Day 8 and Day 16 blastocyst Oct-4 expression patterns. Equine cloned blastocysts expanded and increased their cell numbers when they were maintained in in vitro culture, describing a particular pattern of embryo growth that was unexpectedly independent of embryo aggregation, as all embryos reached similar size after Day 7. Early pregnancy rates were higher using blastocysts derived from aggregated embryos, and advanced pregnancies as live healthy foals also resulted from aggregated embryos. These results indicate that the strategy of aggregating embryos can improve their development, supporting the establishment of equine cloned pregnancies.     

Preimplantation embryo development in the mouse: Role of histidine decarboxylase

The present study determines the effect of a specific and an irreversible inhibitor of histidine decarboxylase (HDC), α-fluoromethylhistidine (α-FMH) on the mouse preimplantation embryo development in vitro. The embryo culture technique was used to assess the effect of α-FMH. Embryos recovered at 0800–0900 hr (AM) on day 3 of pregnancy were 4–8 cells, whereas those recovered at 1600–1630 hr were mostly 8-cell compacted embryos. Of the day 3-AM embryos, 81.3 ± 4.3% developed to blastocysts within 48 hr when cultured in the medium alone, but addition of α-FMH (0.19 or 0.38 mM) drastically reduced the blastocyst formation to 26.6 ± 7 or 16.8 ± 4.3%. Most of them were arrested before the compaction stage. Addition of L-histidine, the substrate for HDC, did not alter the inhibition of blastocyst formation in the presence of α-FMH (37.2 ± 10.9%). Of the day 3-PM embryos, 99.3 ± 0.7% developed to blastocyst stage when cultured in the medium alone and addition of α-FMH (0.19 or 0.38 mM) did not affect the embryo development (92.1 ± 4.3 or 81.9 ± 9.9% developed to blastocysts). The birth of healthy young following transfer of these blastocysts into pseudopregnant mice indicates normal development of the embryos under this condition. The results suggest that histamine synthesis may be required for the process of compaction and thus the formation of blastocyst.     

Parthenogenic blastocysts cultured under in vivo conditions exhibit proliferation and differentiation expression genes similar to those of normal embryos

Parthenote embryos offer multiple possibilities in biotechnological investigation, such as stem cell research. However, there is still a dearth of knowledge of this kind of embryo. In this study, development and ploidy were analysed in parthenotes under in vitro and in vivo culture conditions. Subsequently, using real-time PCR, the expressions of factor OCT-4, Vascular Endothelial Growth Factor, Epidermal Growth Factor Receptor 3 and Transforming Growth Factor β2 genes were analysed to compare the embryo types at the blastocyst stage. Development and implantation of parthenote embryos were described after transfer at day 10 of pregnancy. Parthenotes showed similar blastocyst development for both culture conditions and most of the parthenotes produced were diploid. However, parthenotes developed under in vivo conditions showed similar mRNA expression of OCT-4, VEGF and TGF-β2 to 5 and 6 day old blastocysts. In contrast, parthenotes developed under in vitro conditions had altered the expression pattern of these genes, except for erbB3 mRNA. Finally, transferred parthenotes had the ability to implant but showed severe growth retardation and lesser size. This is the first demonstration of the influence of culture conditions on parthenote mRNA expression. Our study highlights the importance of culture conditions in subsequent uses of parthenotes, such as the production of stem cell lines.     

Use of two replacements of serum during bovine embryo culture in vitro

This study evaluated the effect of two commercial serum replacements (Ultroser G and CPSR-3 on in vitro bovine embryo culture. In Experiment 1, zygotes were cultured in SOF+Ultroser G (2, 4 and 6%), SOF+CPSR-3 (2, 4 and 6%), and SOF+5% FCS (control). Blastocyst rates obtained after culturing with Ultroser G were lower than those with FCS. However, blastocyst rates for CPSR-3 were similar to those for serum. In addition, embryos produced in SOF+CPSR-3 had the same proportion inner cell mass number and total cell number as embryos cultured with FCS. In Experiment 2, a combination of serum replacements during different periods showed that treatment before the five-to eight-cell stages had no effect on further embryo development. However, treatments up to the morula stage affected blastocyst formation. The concentration of supplement and the timing of its inclusion in culture markedly affected embryo development. The serum replacement CPSR-3 can supplement embryo culture with blastocyst rates and quality similar to those for serum.     

Logistic regression analysis of pregnancy rate following transfer of Bos indicus embryos into Bos indicus x Bos taurus heifers

Factors affecting pregnancy rate of 5627 Zebu embryos in crossbred females with unknown proportions of Holstein and Zebu breeding were examined. After evaluation for developmental stage, quality, and viability, embryos were immediately transferred to recipients. Pregnancy diagnosis was conducted approximately 53 d after transfer; pregnancy rate was coded as a binomial event and analyzed using logistic regression models. Maximum likelihood methodology and the likelihood ratio statistic were used to estimate regression coefficients and test hypotheses. Explanatory variables were year of transfer (1992-1999), season of transfer (summer, autumn, winter and spring), breed of the embryo (Guzerat, Gyr or Nellore), stage of the embryo (morula, early blastocyst, blastocyst, expanded blastocyst, and hatching blastocyst), quality of the embryo (excellent, good or regular), and donor-recipient synchrony (estrus in the recipient occurred 2-3 d before, 1 d before, the day of, 1 d after, or 2-3 d after estrus in the donor). Average pregnancy rate was 63.7%. Pregnancy rates were not significantly affected by breed of embryo. The best multiple-logistic model to explain the pregnancy result included the effects of year and season of transfer, embryo stage and quality, and estrous synchrony between donor and recipient (P相似文献   

Embryo culture conditions are significantly improved during uninterrupted incubation: A randomized controlled trial

A parallel group superiority prospective randomised controlled trial was devised to compare the culture characteristics of human pre-implantation stage embryos during uninterrupted culture in a time lapse incubator (TLI) versus the conventional model of interrupted culture in a standard incubator (SI) under low oxygen tension using a single step medium. 221 patients aged 35-and-under, 124 patients aged between 36 and 39 and 86 patients aged 40-and-over years were randomised and cultured either in a SI or in a TLI. Patients in the three age groups were distributed between the TLI and SI in a 1:1 ratio. The development of embryos on days 2, 3 and 5, and the clinical pregnancy and implantation rates were recorded. The fertilisation rate, development of day 2 and clinical pregnancy rates were similar in both treatments but the 8-cell development rate in all age groups combined (p?=?0.016), blastocyst development rate (p?=?0.0022) and the implantation rate (p?=?0.0022) was significantly higher for the uninterrupted culture. These findings demonstrated significant differences between the two incubation groups. It also indicated less efficacious embryonic development with age in both treatments which appeared more pronounced in the conventional incubator. In conclusion uninterrupted culture is superior compared to the interrupted incubation culture system.     

Maternal age effect on early human embryonic development and blastocyst formation

In humans, age-related decline in female fertility can be explained by a reduction in quality either of the older uterus or of the embryos arising from aging oocytes. The aim of this study was to examine the latter hypothesis, using in vitro fertilization (I.V.F.) and coculture of embryos until the blastocyst stage. We determined the blastocyst formation rate ([expanded blastocysts/blastocysts]*100) according to the patient's age the day of I.V.F. With increase in age, the number of retrieved oocytes decreased, without alteration of the cleavage rate. In patients above age 30 years, preimplantation development to blastocysts declined due to an increase in embryo arrest at the morula stage. If blastocyst stage was reached, a negative linear relationship between blastocyst expansion rate and patient age was observed. Drops in gamete production and embryo development with increasing age led to a drastic decrease in patients having at least one expanded blastocyst (<30 years, 82%; ≥40 years, 36%). A high delivery rate per oocyte retrieval (25.8%) was observed in patients above age 40 years after embryo transfer at the blastocyst stage. These results give a clear indication of decline in the quality of human embryos arising from aging oocytes. The origin of this alteration is discussed in terms of chromosome abnormalities, role of maternally-inherited products from the oocyte, timing of genomic activation, and temporal pattern of gene expression during initial development of the human embryo. © 1996 Wiley-Liss, Inc.     

Probing morphological,genetic and metabolomic changes of in vitro embryo development in a microfluidic device

Assisted reproduction technologies for clinical and research purposes rely on a brief in vitro embryo culture which, despite decades of progress, remain suboptimal in comparison to the physiological environment. One promising tool to improve this technique is the development of bespoke microfluidic chambers. Here we present and validate a new microfluidic device in polydimethylsiloxane (PDMS) for the culture of early mouse embryos. Device material and design resulted embryo compatible and elicit minimal stress. Blastocyst formation, hatching, attachment and outgrowth formation on fibronectin-coated devices were similar to traditional microdrop methods. Total blastocyst cell number and allocation to the trophectoderm and inner cell mass lineages were unaffected. The devices were designed for culture of 10–12 embryos. Development rates, mitochondrial polarization and metabolic turnover of key energy substrates glucose, pyruvate and lactate were consistent with groups of 10 embryos in microdrop controls. Increasing group size to 40 embryos per device was associated with increased variation in development rates and altered metabolism. Device culture did not perturb blastocyst gene expression but did elicit changes in embryo metabolome, which can be ascribed to substrate leaching from PDMS and warrant further investigation.     

Gene expression regulating blastocyst formation

Development of embryos to the blastocyst stage is a critical event in the early lives of all eutherian mammalian species. Blastocyst formation is essential for implantation and is the principal morphological determinant of embryo quality prior to embryo transfer. The physiological events and roles of specific gene families that regulate blastocyst formation are subjects of intense research Recent findings have demonstrated that bovine embryos express multiple members of the Na/K-ATPase ion transporter gene family. Two members of this family have been co-localized to bovine trophectoderm, but each becomes largely confined to opposing cell membrane margins. Bovine blastocysts display a greater sensitivity to ouabain (potent inhibitor of the Na/K-ATPase) than murine blastocysts, and enzyme activity (ouabain sensitive 86Rb+ uptake) undergoes a 9-fold increase from the bovine morula to the blastocyst stage. Disruption of Na/K-ATPase gene expression by antisense oligodeoxynucleotide inhibition abolishes blastocyst formation. These results have implicated the Na/K-ATPase as a key regulator of bovine blastocyst formation and have provided insights necessary for the production of healthy bovine embryos by the application of in vitro maturation, in vitro fertilization and in vitro culture methods.     

Effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation

Mouse embryo implantation depends on the complex interaction between the embryo trophoblast cells and the uterine environment, which deposits an extracellular matrix with abundant amounts of laminin. Intrauterine injection and blastocyst or ectoplacental cone culture models were used to study the effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation in vivo and in vitro. Intrauterine injection of 32/67 kDa laminin-binding protein antibody (0.4 mg in 1 ml Ham's F-10 medium, 5 microl per mouse) into the left uterine horns of mice (n = 22) on day 3 of pregnancy inhibited embryo implantation significantly (P < 0.001) compared with the contralateral horns that had been injected with normal rabbit IgG. A continuous section study on day 5 after injection showed that the embryos in the control uteri implanted normally and developed healthily, but there were no embryos or the remaining embryos had disintegrated in the uteri injected with 32/67 kDa laminin-binding protein antibody. Blastocysts or ectoplacental cones were cultured in media containing 32/67 kDa laminin-binding protein antibody (0.2 mg ml(-1)) on laminin-coated dishes with normal rabbit IgG at the same concentration as in the controls. The 32/67 kDa laminin-binding protein had no effect on blastocyst or ectoplacental cone attachment, but prohibited the blastocyst or ectoplacental cone outgrowth and primary or secondary trophoblast giant cell migration. These results indicate that 32/67 kDa laminin-binding protein antibody blocked mouse embryo implantation by preventing embryo trophoblast cell invasion and migration through the uterine decidual basement membrane-like extracellular matrix which has a high laminin content.     

EGF increases expression and activity of PAs in preimplantation rat embryos and their implantation rate

Background

Embryo implantation plays a major role in embryogenesis and the outcome of pregnancy. Plasminogen activators (PAs) have been implicated in mammalian fertilization, early stages of development and embryo implantation. As in-vitro developing embryos resulted in lower implantation rate than those developed in-vivo we assume that a reduced PAs activity may be involved. In the present work we studied the effect of EGF on PAs activity, quantity and embryo implantation.

Methods

Zygotes were flushed from rat oviducts on day one of pregnancy and grown in-vitro in R1ECM supplemented with EGF (10 ng/ml) and were grown up to the blastocyst stage. The control groups were grown in the same medium without EGF. The distribution and quantity of the PAs were examined using fluorescence immunohistochemistry followed by measurement of PAs activity using the chromogenic assay. Implantation rate was studied using the embryo donation model.

Results

PAs distribution in the embryos was the same in EGF treated and untreated embryos. Both PAs were localized in the blastocysts' trophectoderm, supporting the assumption that PAs play a role in the implantation process in rats. EGF increased the quantity of uPA at all stages studied but the 8-cell stage as compared with controls. The tissue type PA (tPA) content was unaffected except the 8-cell stage, which was increased. The activity of uPA increased gradually towards the blastocyst stage and more so due to the presence of EGF. The activity of tPA did not vary with the advancing developmental stages although it was also increased by EGF. The presence of EGF during the preimplantation development doubled the rate of implantation of the treated group as compared with controls.