慢速程序化冷冻与玻璃化冷冻对第3天分裂期胚胎发育潜能的影响

目的比较慢速程序化冷冻法（慢速法）和玻璃化冷冻法（玻璃化法）对第3天分裂期胚胎发育潜能的影响。方法选择因输卵管阻塞或男性少弱精因素行体外受精（IVF）或卵母细胞胞质内单精子注射（ICSI）治疗的患者,挑选在行IVF或ICSI后第3天剩余优质胚胎数目超过4个者80例进入本研究。随机将每例患者的2个胚胎用玻璃化法冷冻,另外2个用慢速法冷冻;冷冻复苏后随机抽取40例患者移植慢速法冷冻的胚胎,另40例患者移植玻璃化法冷冻的胚胎,比较2种冷冻方法对胚胎发育潜能的影响。结果冷冻复苏后待移植的胚胎共160个,其中慢速法冷冻80个,复苏后存活73个（91％,73／80）,移植后40例患者中15例（38％,15／40）获得临床妊娠,其中3例（20％,3／15）为双胎妊娠,余为单胎妊娠,胚胎着床率为25％（18／73）;玻璃化法冷冻胚胎80个,复苏后存活71个（89％,71／80）。移植后40例患者中19例（48％,19／40）获得临床妊娠,其中9例（47％,9／19）为双胎妊娠,余为单胎妊娠,胚胎着床率为39％（28／71）,与慢速法相比,玻璃化法的临床妊娠率和双胎妊娠率均有提高,但差异无统计学意义（P〉0．05）,而胚胎着床率则显著提高,差异有统计学意义（P〈0．05）。结论玻璃化法能够更好地保存胚胎复苏后的发育潜能,更适合第3天分裂期胚胎的冷冻保存。     

Larger oocyte cohorts maximize fresh IVF cycle birth rates and availability of surplus high-quality blastocysts for cryopreservation

Research question

How does oocyte cohort size affect IVF treatment outcomes?

卵裂期发育延缓胚来源的人类胚胎干细胞系的建立

目的观察体外受精（IVF）周期中24h内不发生卵裂的第3天（D3）发育延缓胚的体外发育潜能,为建立人胚胎干细胞库提供新的材料来源。方法通过囊胚序贯培养法将卵裂期发育延缓胚培养至囊胚,观察囊胚形成率及囊胚质量。用逐步logistic回归分析囊胚形成率与卵裂球数、卵裂球对称性及卵裂球碎片比率的相关性。用免疫外科法去除滋养细胞,将得到的原代克隆接种于小鼠胚胎成纤维细胞上,体外传代并鉴定。结果120枚卵裂期发育延缓胚培养出囊胚22枚（囊胚形成率18．7％）。第6天早期囊胚、囊胚、满囊胚、扩张胚、孵化胚及孵化后囊胚的比率分别为5．9％、23．5％、35．3％、23．5％、5．9％及5．9％。内细胞团分级及滋养外胚层分级均仅有1个为A级,其余均为B或C级。发育延缓胚的囊胚形成率与卵裂球数、卵裂球对称性相关,而与卵裂球碎片比率无关。获得内细胞团7个,原代克隆3个,成功培养出2株人类胚胎干细胞系（hESC）,并具有hESC的生物学特性：碱性磷酸酶（AKP）、阶段特异性胚胎抗原（SSEA）4、肿瘤排斥抗原（TRA）1-60、TRA-1-81呈阳性;悬浮培养可形成拟胚体;在严重免疫缺陷（SCID）小鼠体内可形成畸胎瘤;分别传45及35代后核型正常。结论部分发育延缓胚可以发育为囊胚,从而可为建立人胚胎干细胞系提供新的材料来源。     

A randomized controlled trial comparing two vitrification methods versus slow-freezing for cryopreservation of human cleavage stage embryos

Purpose

To compare two different vitrification methods to slow freezing method for cryopreservation of human cleavage stage embryos. Design: Prospective randomised trial. Setting: University assisted reproduction centre. Patient(s): 568 patients (mean age 33.4 ± 5.2) from April 2009 to April 2011.

Methods

1798 supernumerary good-quality cleavage stage embryos in 645 IVF cycles intended to be cryopreserved were randomly allocated to three groups: slow freezing, vitrification with the Irvine® method, vitrification with the Vitrolife® method. Main Outcome Measure(s): Embryo survival and cleavage rates, implantation rate.

Results

A total of 1055 embryos were warmed, 836 (79.2 %) survived and 676 were finally transferred (64.1 %). Post-warming embryos survival rate was significantly higher after vitrification (Irvine: 89.4 %; Vitrolife: 87.6 %) than after slow freezing (63.8 %) (p < 0.001). No differences in survival rates were observed between the two vitrification methods, but a significant higher cleavage rate was observed using Irvine compared to Vitrolife method (p < 0.05). Implantation rate (IR) per embryo replaced and per embryo warmed were respectively 15.8 % (41/259) and 12.4 % (41/330) for Irvine, 17.0 % (40/235) and 12.1 % (40/330) for Vitrolife, 21.4 % (39/182) and 9.9 % (39/395) for slow-freezing (NS).

Conclusions

Both vitrification methods (Irvine and Vitrolife) are more efficient than slow freezing for cryopreservation of human cleavage stage embryos in terms of post-warming survival rate. No significant difference in the implantation rate was observed between the three cryopreservation methods.     

Cumulative first live birth after elective cryopreservation of all embryos due to ovarian hyperresponsiveness

OBJECTIVE: To estimate cumulative chance for first live birth after elective pronuclear stage cryopreservation of all embryos due to ovarian hyperresponsiveness. DESIGN: Retrospective analysis with longitudinal follow-up. SETTING: Academic hospital. PATIENT(S): Thirty subjects with elective cryopreservation of all embryos due to ovarian hyperresponsiveness. INTERVENTION(S): Elective cryopreservation of all embryos at the pronuclear stage (n = 30) and subsequent cryopreserved-thawed ET (n = 51). MAIN OUTCOME MEASURE(S): Cumulative chance for first live birth. RESULT(S): Cumulative chance for first live birth was 77% when analyzed by intention to treat and 82% by treatment with ET. Nearly 40% of live births were multiple. CONCLUSION(S): Cumulative first live birth increased with repetitive ET after elective pronuclear stage cryopreservation of all embryos due to ovarian hyperresponsiveness. Multiple births, however, were frequent. In the context of initial ET attempts in young women, transfer of no more than two cryopreserved-thawed embryos is advised.     

Influence of post-thaw culture on the developmental potential of human frozen embryos

Purpose

Apart from freezing/thawing related cryodamage, several additional factors have been identified as major players in the reduction of success rates after frozen embryo transfers. The post-thaw culture is particularly relevant as it may amplify environmental influences over a stressed embryo. In the present study the influence of the post-thaw culture duration on the implantation and developmental potential of cleavage stage embryos was evaluated.

Methods

In this retrospective evaluation, that spanned an 8-year period, 631 frozen-thawed embryos were allocated to one of two study groups, depending on their post-thaw culture period: 1) the long (18–24 h), or 2) the short (2–5 h) culture group. Groups were compared regarding implantation rate and live birth rate per embryo transferred. This comparison was corrected for the most common confounding factors such as maternal age at oocyte pick-up, number of transferred embryos, developmental day at freezing, blastomere survival after thawing, catheter used for transfer and year of procedure.

Results

Implantation and live birth rate per embryo transferred were inversely related to the duration of the post-thaw culture, as diminishing this period significantly increased both rates. Moreover, no advantage could be found for a long post-thaw culture period, even for embryos with observed mitotic activity.

Conclusion

This retrospective analysis indicates that a short post-thaw culture period is associated with higher implantation and live birth rates per embryo. This study supports selection of frozen-thawed embryos strictly based on blastomere cryosurvival and raises the hypothesis that environmental factors may have an important role on embryo implantation and developmental potential during post-thaw culture.     

Quality of embryos transferred and progesterone levels are the most important predictors of live birth after fresh embryo transfer: a retrospective cohort study

Purpose

To identify the independent predictors of live birth following IVF, and to assess the role of cohort-specific parameters, including antral follicle count (AFC), the number of oocytes retrieved, the total number of embryos, and the total number of good-quality embryos, in fresh IVF cycles.

Methods

A retrospective cohort study of 2,525 infertile women undergoing IVF between 2002 and 2007. The hypothesis that the number and quality of embryos transferred capture the effects previously attributed to cohort-specific variables was examined using mediation analysis and spline analysis. Independent predictors were identified by a bootstrap algorithm. Multivariable logistic regression was performed and the proportion of explained variation was measured to compare the relative importance of transfer-specific vs. cohort-specific predictors.

Results

The number of good-quality embryos transferred and progesterone level on the day of hCG administration ranked as the two most important predictors of live birth. Prospects of pregnancy started to decrease after progesterone level exceeded 0.6 ng/ml. The achievement of live birth in a fresh IVF cycle is primarily determined by the number and quality of embryos transferred, rather than by embryo cohort-specific variables.

Conclusions

The associations between cohort-specific variables and live birth in a fresh IVF cycle are completely mediated by the quality of embryos transferred. Progesterone level on the day of hCG administration is an independent predictor of pregnancy and merits further investigation.     

The effect of repeated cryopreservation and thawing using cryotip on the clinical outcomes of embryos

PurposeTo compare the clinical outcomes of embryo transfers that were cryopreserved and thawed two or three times with those cryopreserved and thawed once by CryoTip.MethodsData for 388 single cryopreserved‐thawed blastocyst transfer cycles, performed from April 2012 to March 2014, were assessed. The blastocysts were classified into three groups: blastocysts (A) cryopreserved once, (B) cryopreserved twice, and (C) cryopreserved three times.ResultsThe pregnancy rate was 43.8% (134/306) in group A and 46.3% (38/82) in group B, while the miscarriage rate was 29.1% (39/134) in group A and 23.7% (9/38) in group B. The rate of improvement/maintenance of blastocyst grade was 84.0% (257/306) in group A and 80.5% (66/82) in group B. The pregnancy and miscarriage rates of the blastocysts that showed improvement/maintenance in the grade were 45.9% (118/257) and 29.7% (35/118) in group A and 48.5% (32/66) and 21.9% (7/32) in group B, respectively. The pregnancy rate was 33.3% (2/6), while the miscarriage rate was 0.0% (0/2) in group C.ConclusionsPregnancy rates achieved with re‐cryopreserved and rethawed blastocyst transfer were comparable to those achieved with single cryopreserved‐thawed blastocyst transfer.     

The use of anti-Müllerian hormone and antral follicle count to predict the potential of oocytes and embryos

Objective

To investigate whether anti-Müllerian hormone (AMH) is better than antral follicle count (AFC) in predicting oocyte yield and embryo quality after controlled ovarian hyperstimulation for in vitro fertilization (IVF).

Study design

This is a prospective observational study involving 162 women (<40 years old) undergoing their first IVF cycle at an IVF unit within a university hospital. AMH and AFC measurements were made on day 3 of the cycle within 3 months of starting ovarian stimulation. A standard long down-regulation protocol using gonadotrophin releasing hormone agonist and recombinant follicle stimulating hormone was used. A maximum of two embryos were transferred on day 2 or 3 following oocyte retrieval. The primary outcome was the number of good quality embryos available for transfer and freezing. Embryos were graded according to the number of blastomeres, the difference in blastomere size and the degree of fragmentation, into grades 1–4. Secondary outcomes included the number of oocytes retrieved and fertilized and the live birth rate. Correlation between different parameters was calculated using Spearman's correlation coefficient. Receiver operating characteristic (ROC) curves were generated for AMH and AFC to compare ability of parameters to predict top quality or frozen embryos and the occurrence of a live birth.

Results

Of the 137 women who had fresh embryo transfer, 52 became pregnant (32.1% pregnancy rate per cycle started) and 38 had a live birth (23.5% live birth rate per cycle started). Both AMH and AFC had highly significant correlations with the number of oocytes retrieved and the number of oocytes fertilized (P < 0.001). The two markers were also significantly associated with the number of top quality embryos available for transfer and the number of embryos frozen (P < 0.01). With regard to live birth, AMH performed better than AFC (P < 0.01 and P < 0.05, respectively), but both markers were more valuable in predicting the absence rather than the occurrence of live birth (negative predictive value 84%).

Conclusions

AMH and AFC are comparable predictors of oocytes retrieved and of the number of good quality embryos available for transfer and freezing. Prediction of live birth may help clinicians selecting patients suitable for single embryo transfer.     

Developmental capacity and pregnancy rate of tetrahedral- versus non-tetrahedral-shaped 4-cell stage human embryos

Purpose

The arrangement of the blastomeres within the 4-cell stage embryo reflects the orientation of the cleavage planes during the second division. To examine their relevance, the developmental capacity and the pregnancy rate were compared between tetrahedral-shaped and non-tetrahedral-shaped 4-cell stage human embryos.

Methods

The study included 3,546 4-cell stage embryos. The arrangement of the blastomeres at the 4-cell stage was annotated as being tetrahedral or non-tetrahedral on day 2 of preimplantation development. Embryo quality was compared on day 3 and day 5. Pregnancy rates were calculated per single embryo transfer on day 3 or day 5.

Results

In total, 2,803 4-cell stage embryos (79 %) displayed a tetrahedral arrangement and 743 (21 %) displayed a non-tetrahedral arrangement. Tetrahedral-shaped embryos developed more into high-quality embryos on day 3 (p < 0.001) and day 5 (p = 0.036) and had a higher blastulation rate (p = 0.009). Though, the number of high-quality embryos selected for transfer did not differ between both groups on day 3 (p = 0.167) and day 5 (p ~ 1). Three hundred thirty single embryo transfers were analysed. No significant difference in clinical pregnancy was found between both groups after transfer on day 3 (p = 0.209) and day 5 (p = 0.653).

Conclusions

The arrangement of the blastomeres according to their previous cleavage planes was correlated to the developmental potential of the 4-cell stage embryo up to the blastocyst stage. If embryo transfers are performed on day 3 and day 5 of development using embryos of adequate quality, the blastomere arrangement at the 4-cell stage had no predictable value regarding pregnancy success.     

The application and impact of cryopreservation of early cleavage stage embryos in assisted reproduction

The contribution of cryopreserved embryos to the overall outcomes achieved by a clinical assisted reproduction programme has increased in importance with the trend towards reducing the numbers of fresh embryos transferred following in vitro fertilisation. Although cryopreservation appears to fully preserve developmental potential in early cleavage stage embryos that survive intact, it results in a reduction in potential when blastomere loss occurs during freezing and thawing. Overall, it can be estimated that cryopreservation results in approximately a 30% reduction in the potential for pregnancy in a population of embryos. Both blastomere survival and post-thaw resumption of mitosis can act as markers of implantation potential in frozen/thawed embryos. Application of strict criteria for freezing embryos and transferring thawed embryos may enhance apparent success rates, but may also result in some pregnancy potential being discarded. The role of embryo cryopreservation in minimising the incidence of multiple pregnancy must be balanced with the need for efficiency in the quest to establish pregnancy.     

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     

The influence of early cleavage on embryo developmental potential and IVF/ICSI outcome

Purpose  

To observe whether early cleavage can be a predictor of embryo developmental potential, pregnancy and implantation rates.     

Cryopreservation of human embryos at the morula stage and outcomes after transfer

OBJECTIVE: To evaluate the survival rate of human morula embryo freezing and the morphological alterations during freezing, during and after thawing, and their applications in embryo selection. DESIGN: Retrospective observational study. SETTING: Private infertility clinic. PATIENT(S): Consecutive patients under age 39 undergoing frozen morula embryo transfers from December 1999 to May 2003. INTERVENTION(S): Embryo freezing was performed at the morula stage. Embryo thaw and post-thaw ETs were conducted on the same day, which is equivalent to a day 4 ET. MAIN OUTCOME MEASURE(S): Morphological alterations during freezing and thawing and after thawing. Post-thaw embryo survival rates, transferable rates, pregnancy rates, and implantation rates. RESULT(S): Morula embryos showed reversed morphological alterations during the freezing process; these alterations were recovered during thawing or shortly after the thawing. Post-thaw survival rates showed no significant difference between any of the morula substages. However, embryos scored as grade 3, which represented good quality, had significantly higher post-thaw survival and transferable rates than grade 2 and 1 embryos. Patients who received at least one grade 3 embryo had significantly higher pregnancy rates, implantation rates, and ongoing/live birth rates than other groups. CONCLUSION(S): An acceptable survival rate can be achieved after cryopreservation of human morula embryos, and morphological alterations that occur during and shortly after an embryo thaw can be a feasible index for determining viable embryos.     

Experience with the cryopreservation of human embryos using the mouse as a model to establish successful techniques

Mouse embryos at the one-, two-, and eight-cell stages have been used to optimize the conditions for cryopreservation of human oocytes and embryos. For storage in glass vials using 1.5 M dimethyl sulfoxide (DMSO) as a cryoprotectant and slow cooling (0.3°C/min), phosphate-buffered medium was superior to Hepes-buffered medium. Termination of slow cooling at –80°C before transfer to liquid nitrogen with subsequent slow thawing (–8°C/min) resulted in more embryos surviving than when cooling terminated at –40°C and rapid thawing (–500°C/min) was employed. Dilution of DMSO upon thawing with medium containing 0.5M sucrose gave higher embryo survival rates than a stepwise (0.25 M decrements) dilution. Using these techniques, three pregnancies were established upon the transfer of 11 frozenthawed embryos to seven patients. Rates of embryo survival using the simpler cryopreservation technique of ice-free vitrification in 0.25-ml straws have been disappointing.     

Comparison of mitochondrial DNA contents in human embryos with good or poor morphology at the 8-cell stage

OBJECTIVE: To quantify the mitochondrial DNA contents in human embryos with good or poor quality at the 8-cell stage. DESIGN: Prospective study. SETTING: Private infertility clinic. PATIENT(S): Five women aged 24 to 34 years. INTERVENTION(S): Embryos obtained in standard superovulation and embryo culture procedures. MAIN OUTCOME MEASURE(S): Mitochondrial DNA (mtDNA) copy numbers in human embryos at cleavage stage were quantified by real-time polymerase chain reaction, in an effort to correlate with morphology. RESULT(S): The grade 8A embryos contained a mean mtDNA copy number at 1163937 (n = 8, from three patients); grade 8B embryos, at 939345 (n = 5, from two patients); grade 8C(+) embryos, at 637872 (n = 12, from 5 patients); and grade 8C(+) embryos derived from 3PN zygotes, at 300429 (n = 3, from a single patient). CONCLUSION(S): Great variations were found among blastomeres from a single embryo and among embryos from a single patient. The native variations of mtDNA copy number may affect developmental ability irrespective of morphology.