卵裂期活检对胚胎体外发育能力的影响

目的　探讨不同的活检方法、活检时机和活检细胞数对胚胎体外发育能力的影响。方法　选取体外受精 胚胎移植剩余的形态学分级为Ⅰ级的胚胎 1 54个 ,对其中第 1阶段的 66个胚胎分别行化学法 (2 6个 )、机械法 (2 0个 )取出 1个卵裂球 ,并设对照 (2 0个 ) ;对第 2阶段的 88个胚胎分为用化学法取出 2个卵裂球 (44个 )和对照 (44个 )。观察记录活检时胚胎细胞数、活检时间、活检后卵裂球是否退化、活检后胚胎体外发育情况及囊胚总细胞数。结果　 (1 )化学法的活检时间为 (2 31±2 0 )s,明显短于机械法的 (2 62± 2 3)s(P <0 0 1 ) ;而囊胚形成率为 65 % ,高于机械法的 35 % (P<0 0 5)。(2 ) 6 细胞胚胎的囊胚总细胞数为 (44± 4)个 ,低于 7～ 8 细胞胚胎的 (49± 5)个和≥ 9 细胞胚胎的 (50± 6)个 (P <0 0 5) ;细胞融合的≥ 9 细胞胚胎活检后不仅囊胚形成率 (2 0 % )低于对照 (67% ,P<0 0 5) ,且活检后卵裂球退化率 (50 % )高于无细胞融合的胚胎 (1 7% ,P <0 0 5)。 (3)取出 1个或 2个卵裂球胚胎的囊胚形成率和囊胚总细胞数与对照比较 ,差异均无显著性 (P >0 0 5) ,而 6 细胞胚胎取出 2个卵裂球后囊胚形成率 (1 /8)低于对照 (5/8,P <0 0 5)。结论　化学法活检比机械法更为快速、安全 ;合适的活检时     

Embryo implantation after biopsy of one or two cells from cleavage-stage embryos with a view to preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) can be offered as an alternative to prenatal diagnosis (PND) to couples at risk of having a child with a genetic disease. The affected embryos are detected before implantation by fluorescent in situ hybridisation (FISH) for sexing (X-linked diseases) and chromosomal disorders (numerical and structural) or by polymerase chain reaction (PCR) for monogenic disorders (including some X-linked diseases). The accuracy and reliability of the diagnosis is increased by analysing two blastomeres of the embryo. However, the removal of two blastomeres might have an effect on the implantation capacity of the embryo. We have evaluated the implantation of embryos after the removal of one, two or three cells in 188 PGD cycles where a transfer was done. The patients were divided into five groups: a first group which received only embryos from which one cell had been removed, a second group which received only embryos from which two cells had been removed, a third group which received a mixture of embryos from which one and two cells had been taken, a fourth group where two and three cells had been removed, and a fifth group where three cells had been removed. The overall ongoing pregnancy rate per transfer was 26.1%, the overall implantation rate per transfer was 15.2% and the overall birth rate was 14.2%. Although pregnancy rates between the groups cannot be compared because the second group (two cells removed) contains more rapidly developing and therefore 'better quality' embryos, an ongoing pregnancy rate of 29.1% and an implantation rate of 18.6% per transferred embryo in this group is acceptable, and we therefore advise analysing two cells from a > or =7-cell stage embryo in order to render the diagnosis more accurate and reliable.     

Non-invasive viability assessment of day-4 frozen-thawed human embryos using near infrared spectroscopy

This study investigated if metabolomic profiling of culture media using near infrared (NIR) spectroscopy was related to live-birth rates after single-embryo transfer of frozen-thawed embryos. Analysis of culture media of frozen-thawed embryos was performed by NIR spectroscopy. A viability score was calculated using a predictive multivariate algorithm of fresh day-5 embryos with known pregnancy outcomes. This algorithm generated with fresh day-5 embryos could help to identify the live-birth group from the no live-birth group. Multivariable regression models that tested the predictive ability of the viability score for live birth showed an odds ratio in the crude analysis of 1.50 (P=0.008), after adjustment for embryo morphology, 1.44 (P=0.022), and after adjustment for all variables, 1.71 (P=0.005); based on a 0.1 step increase in viability scores. In conclusion, higher viability scores resulted in higher live-birth rates. An algorithm generated from fresh embryos might be used to predict viability of frozen-thawed embryos. Frozen-thawed embryos have different metabolic activity which is related to implantation potential. Therefore, this method might be useful to select the best embryo for transfer within a group of embryos with similar morphology.     

Blastocyst-stage transfer of poor-quality cleavage-stage embryos results in higher implantation rates

OBJECTIVE: To determine the feasibility and success of blastocyst-stage embryo transfers in patients having only fair and poor quality cleavage-stage embryos on day 3. DESIGN: Prospective case study with historic controls. SETTING: Tertiary care private hospital IVF center. PATIENT(S): A total of 158 day 5 embryo transfer cycles in patients with grade 3 and grade 4 cleavage-stage embryos. Control group consisted of 162 day 3 transfer cycles performed with embryos of similar quality. INTERVENTION(S): In vitro culture of embryos up to the blastocyst stage. MAIN OUTCOME MEASURE(S): The percentage of cycles that culminated in the transfer of at least one blastocyst and implantation and pregnancy rate related to the day of transfer. RESULT(S): In the day 3 transfer group, a mean of 5.2 embryos were replaced per patient. This was significantly more than the mean of 2.4 embryos that could be replaced on day 5 (P <.001). The clinical pregnancy rate per embryo transfer was 27.2% and 33.5% in the two groups, respectively (P >.05). The implantation rate per embryo was significantly higher in the day 5 transfer group (15% vs. 5.9%). The multiple pregnancy and abortion rates were similar between the groups. CONCLUSION(S): Transfer of fair and poor quality embryos at the blastocyst stage is feasible and is associated with higher implantation rates as compared to transfer of similar quality embryos on day 3.     

Impact of the size of zona pellucida thinning area on vitrified-warmed cleavage-stage embryo transfers: a prospective, randomized study

Purpose

The aim of this study was to determine if the size of zona pellucida thinning area by laser assisted hatching could affect the rates of pregnancy and implantation for vitrified-warmed embryo transfers at the cleavage-stage.

Methods

A total of 120 vitrified-warmed cleavage-stage embryo transfers were randomly assigned to either quarter or half of zona pellucida thinning group.

Results

The rates of clinical pregnancy (46.7 versus 25.0%) and implantation (32.0 versus 16.2%) were significantly greater in the half thinning group than in the quarter thinning group (P?=?0.0218 and P?=?0.0090, respectively).

Conclusions

The results of this investigation show that, in vitrified-warmed embryo transfers at the cleavage-stage, the size of zona pellucida thinning area by laser assisted hatching impacts the rate of clinical pregnancy and implantation and that half of zona pellucida thinning significantly increases both of these results compared with quarter of zona pellucida thinning.     

Cryoloop vitrification of human day 3 cleavage-stage embryos: post-vitrification development, pregnancy outcomes and live births

Vitrification technology has shown great promise for cryopreservation of human embryos. The majority of this work has been with blastocyst-stage embryos. This report describes initial clinical results following vitrification of human embryos on day 3 of culture at the 6- to 8-cell stage. A total of 236 embryos were cryopreserved on cryoloops using a vitrification protocol. Warmed embryos were cultured until day 5 before transfer to the patient. The post-warming survival rate was 85%. The clinical pregnancy rate was 44% (34/77), and the implantation rate was 20% (40/201). In transfers where at least one warmed embryo reached the blastocyst stage by the day of transfer, the clinical pregnancy rate was 58% (28/48). The cryoloop was an excellent vessel for ultra-rapid cryopreservation of embryos. This study supports the effectiveness of a dimethylsulphoxide/ethylene glycol cryoprotectant combination for vitrification of human embryos at the 6- to 8-cell stage.     

Risk of ectopic pregnancy following day-5 embryo transfer compared with day-3 transfer

The incidence of ectopic pregnancy after IVF is increased approximately 2.5–5-fold compared with natural conceptions; however, the aetiology for this increased risk remains unclear. One proposed practice change to decrease the incidence of ectopic pregnancy is blastocyst embryo transfer on day 5 rather than cleavage-stage embryo transfer on day 3. A retrospective cohort study was conducted to compare the risk of ectopic pregnancy following fresh day-5 embryo transfer with day-3 embryo transfer among women who underwent IVF and achieved pregnancy from 1998 to 2011. There were 13,654 eligible pregnancies; 277 were ectopic. The incidence of ectopic pregnancy was 2.1% among day-3 pregnancies and 1.6% among day-5 pregnancies. The adjusted risk ratio for ectopic pregnancy from day-5 compared with day-3 transfer was 0.71 (95% confidence interval 0.46–1.10). Although this analysis included 13,654 cycles, with a two-sided significance level of 0.05, it had only 21.9% power to detect a difference between the low incidence of ectopic pregnancy among both day-3 and day-5 transfers. In conclusion, this study was not able to demonstrate a difference in the risk of ectopic pregnancy among day-3 compared with day-5 transfers.     

Cycle outcome can be predicted by the growth profile of untransferred cleavage-stage embryos

Excess embryos obtained from intracytoplasmic sperm injection cycles may be cultured and observed until day 5 if the couple receiving treatment do not want them to be cryopreserved. In order to investigate the correlation between blastocyst formation in extended culture and pregnancy outcome, 194 patients treated in two separate IVF units were examined retrospectively. The patients were separated into two groups: group 1 with at least one blastocyst formed in culture, and group 2 with no blastocyst formation. The pregnancy rates were 60.0% and 41.7% for groups 1 and 2, respectively. The pregnancy rate in group 1 was statistically significantly higher than in group 2 (P = 0.01). The results suggest that the developmental potential of embryos obtained from a single assisted reproduction treatment cycle may be similar and that blastocyst formation in vitro may help to predict the pregnancy outcome of that cycle.     

Increasing dehydration of human cleavage-stage embryos prior to slow cooling significantly increases cryosurvival

Increasing the proportions of embryos and blastomeres which survive cryopreservation would be expected to make a significant contribution to the outcome of assisted reproduction treatment. Despite this, the methodology used for slow cooling of human cleavage-stage embryos has remained largely unchanged for over two decades. Previous studies have demonstrated the value, in terms of cryosurvival, of increasing the extent of intracellular dehydration by increasing the concentration of non-permeating cryoprotectant prior to slow cooling of oocytes and embryos which have been biopsied for preimplantation genetic diagnosis. The present study extends the use of this approach to the slow cooling of non-biopsied day-2 embryos. Dehydration in the presence of 0.2 mol/l sucrose significantly increased the proportions of surviving embryos, surviving blastomeres and fully intact embryos (92.6%, 91.1%, and 80.4%, respectively) relative to those observed after dehydration in 0.1 mol/l sucrose (78.5%, 74.1%, and 54.6%, respectively, all P < 0.001). Post-thaw resumption of mitosis in vitro and implantation were not adversely affected by the increased prefreeze dehydration. This improved method for slow cooling of cleavage-stage embryos should have a major impact on clinical outcome.     

Influence of storage time on vitrified human cleavage-stage embryos froze in open system

Background: During in vitro fertilization, rapid growth of vitrification and liquid nitrogen storage of embryos have been well characterized. However, the effect of storage time on vitrified cleavage-stage embryos in an open system is poorly understood.

Aims: To investigate the influence of storage time on the survival and pregnancy outcomes of vitrified human cleavage-stage embryos froze and stored in an open system.

Methods: A retrospective study of 786 vitrified-warmed cycles of 735 patients was performed from January 2013 to October 2013. The cycles were divided into five groups according to storage time: 1–3 months, 4–6 months, 7–12 months, 13–24 and 25–60 months. The clinical outcomes of cycles with different storage time were analyzed.

Results: There were no significant differences of the survival rate, clinical pregnancy outcomes, birth rate, gestational weeks and singleton birthweights at various storage times.

Conclusion: For vitrified embryos froze and stored in an open system, the storage time would not influence the survival rate and pregnancy outcomes by storage time up to 5 years.     

Comparison of the transfer of equal numbers of blastocysts versus cleavage-stage embryos after repeated failure of in vitro fertilization cycles

Purpose

To evaluate the efficacy of blastocyst transfer in women with at least two previously unsuccessful in vitro fertilization-embryo transfer (IVF-ET) attempts.

Methods

Retrospective analysis of 238 couples (with previous implantation failures) had equal number (two) of cleavage-stage embryos (n = 143) or blastocysts (n = 95) transferred in the same IVF center.

Results

The clinical pregnancy rates and live-birth rates were similar in the cleavagestage embryo transfer group and the blastocyst group (35.6 % vs. 40 % and 32.1 % vs. 35.7 %; p > 0.05, respectively). Miscarriage rates (9.8 % vs. 10.5 %) and multiple pregnancy rates (15.6 % vs. 23.6 %) did not differ. Although implantation rate was higher with blastocyst transfer than that with day 3 transfer, it did not reach to a statistical significance (24.7 % and 19 %, respectively, p > 0.05).

Conclusion

Blastocyst transfer in ICSI cycles does not yield a better outcome than that obtained with cleavage-stage embryos in women who had unsuccessful IVF attempts previously.     

Chromosome mosaicism in cleavage-stage human embryos: evidence of a maternal age effect

The present study evaluated mosaicism in a large series of cleavage-stage human embryos analysed by fluorescence in-situ hybridization. Only embryos with at least three cells analysed were included (n = 1235), of which 556 were mosaics. The most common types of mosaicism were chaotic (48%), diploid/polyploid (26%), and those caused by mitotic non-disjunction (25%). The number of abnormal cells per embryo ranged from 44% in diploid/polyploid to 84% in chaotic mosaics. Chromosome 16 was most commonly involved in mitotic non-disjunction mosaics. While overall mosaicism did not increase with maternal age, the average maternal age of the embryos that had mosaics caused by mitotic non-disjunction was significantly higher than that for normal or other mosaic embryos (P < 0.001). During the cleavage stage, the embryonic genome is not yet fully activated and consequently the mRNA and protein pools are still similar to those found in the oocyte. We therefore propose that the malfunctioning of the meiosis apparatus, which is similar to the mitotic one, may cause either meiotic errors or mitotic non-disjunction at cleavage-stage embryo development.     

Pregnancy after polar body biopsy and freezing and thawing of human embryos

OBJECTIVE: To evaluate the outcome of frozen-thawed ET using embryos previously biopsied for preimplantation genetic diagnosis during a fresh ET cycle. DESIGN: Prospective evaluation. SETTING: Assisted reproductive biology program. PATIENT(s): A 31-year-old, G4, P1, TAB1, SAB2 carrier of a balanced RT 45,XX der(14;21)(q10;q10) translocation. INTERVENTION(s): Preimplantation genetic diagnosis by polar body biopsy. Excess embryos were frozen using the one-step method and then thawed. MAIN OUTCOME MEASURE(s): Embryo survival after thawing and subsequent pregnancy outcome. RESULT(s): Among the 32 mature oocytes, the results of fluorescence in situ hybridization were available for 25 polar bodies. Eleven were unbalanced, 10 were normal (8 fertilized), and 4 were balanced (3 fertilized) for the fresh IVF cycle. Two normal embryos were transferred. Four normal and 3 balanced embryos were cryopreserved. A chemical pregnancy resulted. Four months later, the 7 cryopreserved embryos were thawed; 2 survived (1 balanced and 1 normal) and were transferred. An ongoing pregnancy resulted, and a normal (46,XX) female was delivered. CONCLUSION(s): Freezing and thawing of biopsied embryos resulted in a low survival rate. However, this should not be a deterrent to the cryopreservation of extra chromosomally normal embryos because the embryos that do survive are able to implant.     

The clinical outcomes of day 3 4-cell embryos after extended in vitro culture

ObjectiveTo evaluate the development potential and clinical significance of day 3 4-cell embryos after extended in vitro culture.MethodsThis study was a retrospective cohort study for patients with infertility treatment between January 2011 and July 2013. Patients undergoing blastocyst culture in controlled ovarian hyperstimulation cycles using surplus embryos were analyzed in the study. A total of 764 women undergoing blastocyst culture with 1,522 surplus 4-cell embryos on day 3 were analyzed. An additional 2,391 patients with embryos undergoing blastocyst culture during the same period with embryos having more blastomeres were chosen as control.ResultsAfter extended culture, 253 embryos from 183 cycles in the study group which developed to blastocysts were frozen, and 118 embryos were warmed in subsequent frozen embryo transfer cycles. Implantation rates, clinical pregnancy rates (PRs) and ongoing PRs were 33.3 %, 38.4 % and 31.4 %, respectively, which were similar to those of the control group. The singleton birth weights of newborns using these blastocysts showed no significant difference to that seen in the control group.ConclusionSurplus 4-cell embryos on day 3 displayed lower blastulation rates. However, once a blastocyst is obtained, it has equivalent clinical outcomes. Embryos that are developmentally lagging on day 3 can be observed in extended culture to increase the cumulative chances of a successful pregnancy.