Abstract
The aim of this study was to evaluate the influence of in vitro fertilization (IVF) versus intracytoplasmic sperm injection (ICSI), fertilization mode embryonic morphology at day 3, and female age on blastocyst development, on the clinical outcomes of pregnancy after blastocyst transfer. A total of 471 cycles were retrospectively investigated. The rates of blastocyst formation and of good blastocyst morphology were higher in IVF than in ICSI cycles but there were no significant differences in the clinical pregnancies or in the miscarriage rates. The rates of formation of blastocyst and of blastocysts with good morphology were significantly higher from good-morphology embryos than from poor-morphology embryos. Nevertheless, 16.9% of the poor-morphology embryos reached the blastocyst stage. The total rates of blastocyst formation, and rates of clinical pregnancy and implantation were statistically similar in the age <35, 35–39, and >39 year groups, although tending to decrease with increasing age. When equal numbers of embryos were transferred on day 3, the rates of clinical pregnancy and implantation after blastocyst transfer were significantly higher in the <35 year age group than in the 35–39 and >39 year age groups, which were not significantly different. The miscarriage rates after embryo or blastocyst transfers were not statistically different in groups of similar age. Therefore, extended embryo culture up to the blastocyst stage could be implemented for women aged younger than 35 years to increase the pregnancy rate. For older women, transfer and vitrification of available embryos at day 3 and extended culture of morphologically poor embryos to the blastocyst stage for cryopreservation may improve the clinical outcome.
Introduction
The universal goal of assisted reproductive technology (ART) is the delivery of a healthy singleton full-term baby. Therefore, it is important to establish an embryo screening method that allows the transfer of the minimum number of embryos without affecting the chance of pregnancy. The blastocyst, which is the terminal stage of embryo culture in vitro, is usually formed 5–6 days after fertilization of the oocyte. Extending embryo culture to the blastocyst stage might allow effective screening for viable embryos with the greatest potential for implantation; those that develop initially but are arrested later, of which more than 50% involve multiple aneuploidy, could be identified [Munne Citation2006]. Extended embryo culture and the greater availability of blastocysts suitable for transfer have led to increased rates of implantation and live birth, and decreased early abortion rates compared with the transfer of cleavage-stage embryos [(Papanikolaou et al. Citation2008; Papanikolaou et al. Citation2006]. Other advantages of blastocyst transfer include: 1) better temporal synchronization between embryonic and endometrial development at the time of embryo transfer; 2) reduced multiple pregnancies through transferring fewer embryos; and 3) higher implantation rates [Gardner et al. Citation2004; Ryan et al. Citation2007; Tsirigotis Citation1998].
Advances in our understanding of the physiological dynamics of early human embryos have led to the development of culture systems capable of yielding viable blastocysts with greater consistency. However, reported rates of blastocyst formation vary from 31% [Sepúlveda et al. Citation2009] to 67% [Langley et al. 2001] in embryos cultured in sequential media, and from 47% [Rijnders and Jansen Citation1998] to 73–78% [Westphal et al. Citation2003] following assessment of day 3 embryos. This variability in the rate of blastocyst development results from many factors, including the laboratory environment and embryo culture conditions, differences among patient populations, and different protocols for controlled ovarian stimulation. A potential drawback of blastocyst culture is the risk of cancellation of the transfer if no blastocyst is formed at day 6, despite the availability of embryos on day 3. It is generally accepted that blastocyst culture is not an appropriate program for all patients. The prevailing challenge is to determine prospectively, how to select appropriate patients for blastocyst culture program and for each patient whether this technology will increase the likelihood of a healthy baby compared with cleavage-stage transfer.
In recent years, non-invasive, computer-automated, time-lapse image analysis, and routine developmental markers have been available to select the most viable embryos [Conaghan et al. Citation2013]. However, their value in the assessment of blastocyst developmental potential remains unclear. Most assisted-reproduction laboratories still select embryos for transfer based on their morphology because this is the simplest tool available. There is a significant literature on the influence of the in vitro fertilization (IVF) laboratory and the culture conditions on blastocyst formation, and on the predictive value of pronuclear and cleavage-stage morphology. In contrast, there is little published information concerning the combined effects of embryonic morphology at day 3, the age of the patient, and extended culture of discarded embryos to blastocysts, on this outcome. The aim of the present study was to evaluate the influence of fertilization methods, cell number, the quality score of embryos at day 3, and female age, on blastocyst development and the clinical features of pregnancy after day 5 transfer. This will provide information to determine whether an individual patient should be assigned to the blastocyst culture program.
Results
Total rates of blastocyst formation and clinical pregnancy after blastocyst transfer
A total of 2,637 blastocysts were obtained from 5,287 cleavage-stage embryos among 471 IVF or intracytoplasmic sperm injection (ICSI) cycles. The rates of blastocyst formation and blastocysts showing good morphology were 49.9% and 64.1%, respectively. There was no significant difference in the total rate of blastocyst formation between patients with primary infertility (47.8%) and secondary infertility (51.7%), and no significant difference between IVF and ICSI indications.
No blastocysts were formed in seven of the cycles. Of the 464 blastocyst transfer cycles, the rates of clinical pregnancy and implantation were 58.41% and 39.89%, respectively, which were significantly higher than the values following embryo transfer at day 3 during the same period (44.1% and 27.9%, respectively) (p < 0.05). There was no significant difference in miscarriage rates between the day 3 and day 5 transfers.
The influence of fertilization method on subsequent blastocyst development and clinical pregnancy
shows features of blastocyst development and clinical pregnancy in the IVF and ICSI cycles. There was no significant difference between the two groups in the rates of good-morphology embryos on day 3 (p > 0.05) but their total rates of blastocyst formation (52.2% vs. 43.0%) and the percentages blastocysts formed from good-morphology embryos at day 3 (67.4% vs 59.8%) were significantly different. The good-morphology blastocyst rate in the IVF group was significantly higher than in the ICSI group (64.7% vs. 61.7%) (p < 0.05). There were no significant differences between the two groups in the rates of clinical pregnancy (59.1% vs. 56.3%), implantation (40.4% vs. 38.3%), and miscarriage (6.9% vs. 6.0%) (p > 0.05).
Table 1. Blastocyst development and clinical pregnancy in IVF and ICSI cycles.
The influence of the cell number and the quality score of embryos at day 3 on blastocyst development
illustrates the predictive value of the cell number and the quality score of embryos at day 3 on blastocyst development. The rates of blastocyst formation (65.6% vs. 16.9%) and of good-morphology blastocysts (68.3% vs 29.8%) from good-morphology embryos at day 3 were significantly higher than those from embryos exhibiting poor morphology (p < 0.05). However, there were no significant differences between 6–8 cell embryos and ≥9 cell embryos in their blastocyst formation rates (66.0% vs. 64.4%) or in the rate of formation of good-morphology blastocysts from good-morphology embryos (67.6% vs. 71.0%) (p > 0.05). The blastocyst formation rate was significantly dependent on the cell number of poor-morphology embryos; the highest rate was observed in the ≥6 cell group (25.7%); the highest good-morphology blastocyst rate was 31.7% derived from 4 to 6 cell embryos.
Table 2. Blastocyst development from good-morphology and poor-morphology embryos and embryos with different cell numbers at day 3.
The relationship between blastocyst development and the number of good-morphology embryos on day 3 is shown in . A significant positive correlation was found between the number of good-morphology embryos and blastocyst formation rate (r = 0.789; p < 0.01).
Table 3. Relationship between blastocyst formation rate and the proportion of good-morphology embryos at day 3.
The influence of female age on blastocyst development and clinical pregnancy
Patients were divided into three groups according to age: <35 years, 35–39 years, and >39 years ( and ). There were no significant differences in ICSI cycle proportion, normal fertilization rate, and good-morphology embryo rate at day 3 in the three groups (p > 0.05). Total blastocyst formation rate, and blastocyst formation rate at days 5 and 6 were similar in the three groups of patients but with a non-significant tendency to decrease with increasing age (p > 0.05). The rate of blastocyst formation from good-morphology embryos in the <35 year age group (66.9%) was significantly higher than in the >39 year age group (53.0%). Likewise, the good-morphology blastocyst rate in the <35 year age group (65.1%) was significantly higher than that in the >39 year age group (49.4%) (p < 0.05). The clinical pregnancy rates for the <35 years, 35–39 years, and >39 years (60.1%, 3.3%, 42.8%, respectively) and implantation rates (41.6%, 35.3%, 33.3%, respectively) were not significantly different in the three groups, although tending to decrease according to age (p > 0.05). The miscarriage rate in the >39 year old age group (33.3%) was higher than that in the <35 year old age group (5.2%) (p < 0.05).
Table 4. Blastocyst development according to female age.
Table 5. Clinical outcomes of blastocyst transfer according to female age and comparison with embryo transfer at day 3.
When compared with the transfer of the same number of embryos at day 3, the rates of clinical pregnancy and implantation after blastocyst transfer were significantly higher in the <35 year age group (p < 0.05) than in the other age groups. There was no significant difference in these parameters between the 35–39 year age group and the >39 –year age groups (). The miscarriage rate was not statistically different between the two transfer protocols for groups of similar age (p > 0.05).
Discussion
It remains unclear whether ICSI technology has an adverse effect on embryonic development and blastocyst formation. Some studies have demonstrated that the rate of blastocyst formation by surplus embryos obtained during IVF cycles was significantly higher than those obtained from ICSI cycles [Dumoulin et al. Citation2000; Xu et al. Citation2009; Shoukir et al. Citation1998]. Other reports have also indicated that the blastocyst formation rate in IVF cycles was slightly, but non-significantly, higher than that in ICSI cycles [Van Landuyt et al. Citation2005; Westphal et al. Citation2003]. In this study, the rates of blastocyst formation and of good-morphology blastocysts obtained from good-morphology embryos in IVF cycles were significantly higher than those in ICSI cycles. This suggests that the mode of sperm-oocyte combination might affect blastocyst development. It is known that sperms with normal morphology and intact acrosomes combine more readily with the zona pellucida, and have superior DNA integrity than abnormal sperms [Jiang et al. Citation2011], suggesting that the the zona pellucida-bound sperm appeared to be superior or more competent than the general population of motile sperm [Liu et al. 2011]. In the IVF procedure, the zona pellucida itself appears to be relatively selective for sperm in the process of natural sperm-oocyte combination. ICSI technology circumvents this process and may allow fertilization by damaged sperm, subsequently interfering with embryonic development. DNA fragmentation of morphologically normal sperm negatively impacted embryo quality and probability of pregnancy in ICSI cycles [Avendaño et al. 2010]. Additionally, the process of ICSI may damage the cytoskeleton and the spindle of the oocyte [Rienzi et al. Citation2003]. Early embryonic development is mainly regulated by maternal genetic information. Therefore, delayed cleavage could be associated with impaired internal regulation of oocytes. Additionally, delayed or arrested embryonic development could be related to mitochondrial dysfunction or to the generation of reactive oxygen species [Betts and Maden Citation2008]. It is known that the degree of fragmentation in cleavage-stage embryos is proportional to the incidence of chromosomal abnormality, which degrades the capacity of embryos to develop to the blastocyst stage. Another study demonstrated that anucleate fragmentation initiates apoptosis in embryos by activating programmed cell death pathways [Jurisicova et al. Citation1996]. The most important criteria for selecting blastocysts for culture relate to understanding the relationship between embryo assessment on day 3 and blastocyst development. In the present study, high-quality cleavage embryos on day 3 formed more blastocysts, and a greater proportion of good-morphology blastocysts, compared with low-quality embryos. It was also found that there was no significant difference in the rates of formation of blastocysts and good-morphology blastocysts from high-quality embryos of 6–8 cells and ≥9 cells. These findings are consistent with an earlier study, comparing embryos of ≤6 cells, 7–9 cells, and ≥10 cells, which observed that the rates of formation of blastocysts and good-quality blastocysts were significantly higher in rapid-cleavage embryos than in slow-cleavage embryos [Luna et al. Citation2008]. Those results suggested that the rate of blastocyst formation was closely related to day- 3-embryo morphology and cell number. That is, high-quality embryos with a lower degree of fragmentation had a higher developmental potential to reach the blastocyst stage. Furthermore, more rapidly cleaving embryos were more likely to form good-morphology blastocysts than were slowly cleaving embryos.
Although the poor-morphology embryos with delayed cleavage were still able to develop to the blastocyst stage, their developmental potential was significantly reduced compared with normal-cleavage embryos. This suggests that the speed of cleavage is an important factor in embryonic development. Our data also showed that only 4.9% of embryos with ≤4 cells developed to the blastocyst stage and that they produced the lowest proportion of good-morphology blastocysts. Poor-morphology embryos with 4–6 blastomeres tended to form good-morphology blastocysts because this group included some grade I and II embryos with little fragmentation. Consequently, although some batches of low-grade embryos at day 3 may not be suitable for vitrification, extended culture to day 5 or 6 may yield a small number of suitable blastocysts. Transfer or vitrification of these blastocysts could reduce the rate of transfer cancellations and maximize use of the embryo resource. Additionally, they could provide materials for embryonic stem-cell research [Wang et al. Citation2012].
Multivariable logistic regression analysis showed that fertilization mode, cell number, the quality of embryos at day 3, and female age were independently associated with blastocyst development [Dessolle et al. Citation2010]. These authors developed a cycle-based model to predict clinically relevant factors to reduce the incidence of cancellation of day-5 transfers but this has not been confirmed in other centers. Our data showed a positive linear correlation between the number of good-morphology embryos and blastocyst formation rate. The blastocyst formation rate increased and the transfer cancellation rate decreased progressively with increasing number of good-morphology embryos. Provided there are more than two good-morphology embryos per IVF/ICSI cycle, at least one blastocyst would be obtained based on the 50.7% blastocyst formation rate in our results; thus the success of blastocyst culture is predictable.
Maternal age is one of the most important factors affecting the pregnancy rate of IVF embryo transfer and is closely related to oocyte quality, high-quality embryo rate, chromosomal abnormality rate of embryos, intrauterine environment, and psychological state. It is still unclear whether female age has an adverse effect on blastocyst formation. Sepúlveda et al. [Citation2011] demonstrated no statistical difference in the rate of blastocyst formation among the age groups <35, 35–39, and >39 years although there was a reducing trend with increasing maternal age (40.2%, 37.2%, and 34.2%, respectively; p > 0.05). In contrast, another study reported a negative correlation between maternal age and blastocyst development [Thomas et al. Citation2010]. In the present study, the blastocyst formation rate showed no obvious difference among ages <35, 35–39, and >39 years (p > 0.05) but the rate of good-morphology blastocysts obtained from high-quality embryos decreased significantly in ages 35–39 and >39 years than in ages <35 years (p < 0.05). This observation may result in part from increased abnormal chromatin and mitochondrial genome D-loop loci mutations in oocytes of older women [Li et al. Citation2010]. Our findings are strengthened by the observation of Wu et al. [Citation2011] of a significant negative correlation between the degree of embryo fragmentation and blastocyst formation rate. However, age had no effect on the degree of fragmentation within embryos with appropriate cleavage status, nor did it modify the significant effect of embryo fragmentation on blastocyst formation [Wu et al. Citation2011]. Thus, the total blastocyst formation rate did not decrease significantly with the maternal age. However, older women may have abnormally low levels of stored maternally transcribed mRNA, which is involved in trophectoderm function and maintenance of the blastocoel [Jedrusik et al. Citation2010]. That might explain the lower good-morphology blastocyst rate in older women [Harton et al. Citation2013]. It is noteworthy that, although there was no clear difference in the rates of clinical pregnancy and implantation in the three age groups, there was a significant difference in the <35 year age group which favored blastocyst transfer rather than cleavage-stage embryo transfer.
A meta-analysis of the Cochrane Database evaluated 23 randomized controlled studies and reported a significant small difference in live-birth rate for transfers on day 5 compared with transfers on day 3. However, cumulative clinical pregnancy rates from cleavage stages (derived from fresh and thaw cycles) were higher than rates from blastocyst cycles [Glujovsky et al. Citation2012]. Blastocyst culture as an embryo selection tool does not improve embryo viability but it could help patients to achieve pregnancy more quickly [Zhu et al. Citation2013]. Our observations showed that blastocyst transfer increased pregnancy and implantation rates compared with transfer of cleavage-stage embryos only in patients of <35 years of age, which is consistent with the opinion of the ASRM Practice Committee [ASRM Citation2013].
Embryo morphology at day 3, fertilization mode, and female age were strongly associated with blastocyst development. The blastocyst formation rate was lower in ICSI cycles than that in IVF cycles but there were no significant differences in clinical pregnancy and implantation rates. Good-morphology embryos with few fragments usually developed to the blastocyst stage and formed high-quality blastocysts. With larger numbers of good-morphology embryos, the blastocyst formation rate increased and the transfer cancellation rate decreased. Among the poor-morphology embryos, those with fewer cells exhibited lower developmental potential. With increasing maternal age, the blastocyst formation rate did not decrease significantly but the rate of good-morphology blastocysts decreased significantly. Nevertheless, the clinical pregnancy rate and implantation rate were not significantly different among the different age groups. However, compared with embryo transfer at day 3, blastocyst transfer resulted in increased pregnancy and implantation rates only in patients of <35 years of age. Therefore, blastocyst culture and transfer may be suitable for the patients of <35 years of age with more than two good-morphology embryos at day 3. For infertile women of advanced age, we recommend transfer and vitrification of available embryos at day 3. Extended culture of discarded embryos to the blastocyst stage for cryopreservation may optimize the clinical outcomes. The information obtained in this study may be helpful in choosing appropriate patients for blastocyst culture and transfer, and in counseling our infertile patients.
Materials and Methods
Subjects
The study was performed in the reproductive center of the 105th Hospital of PLA (the Chinese People’s Liberation Army) between December 2012 and October 2013. Data from 471 consecutive patients undergoing blastocyst culture in IVF or ICSI treatments were analyzed retrospectively. Couples were selected for extended culture if they had at least four normal zygotes on day 1 after fertilization. The age range of the female patients was 21–45 years (mean 30.1 ± 4.8 years); 210 cases were for primary infertility and 261 cases were for secondary infertility. A total of 5,287 cleavage-stage embryos with normal cellular division were analyzed, including high- and low-quality embryos. Good-morphology embryos were defined as normal fertilized embryos with more than six blastomeres and a fragmentation score of I or II on day 3. The remaining normal cleavage embryos were defined as poor-morphology embryos (also referred to as discarded embryos unsuitable for vitrification at day 3). All patients provided written informed consent for the procedures. The study was approved by the Ethics Committee of the 105th Hospital of PLA, Hefei, China.
IVF and laboratory procedures
Embryo culture
For conventional IVF and ICSI, fertilization (day 0) was performed in G-IVFTM medium (Vitrolife, Gothenburg, Sweden). The following morning (day 1), fertilized oocytes were individually cultured in 20-μL microdrops of pre-equilibrated G1-plusTM medium (Vitrolife) under mineral oil until day 3. On day 3, the embryos were transferred to 20-μL microdrops of pre-equilibrated G2-plusTM (Vitrolife) under mineral oil until days 5–7. All cultures were incubated at 37°C in an atmosphere of 6% CO2.
Embryo score
All embryos and blastocysts were observed using an inverted microscope with Hoffman modulation contrast (magnification 200× or 400×). On day 3, assessment of individually cultured embryos was based on the number of blastomeres, blastomere evenness, and the degree of fragmentation. The embryos were classified into four grades: grade I, embryos with regular blastomeres, translucent cytoplasm, and <5% fragmentation; grade II, blastomeres slightly uneven or irregular, cytoplasmic granules present, and fragmentation between 6% and 20%; grade III, blastomeres obviously uneven or irregular, cytoplasmic granules present, and fragmentation between 21% and 50%; grade IV, blastomeres severely uneven or irregular, cytoplasmic granules present, and fragmentation rate >50% [Edwards Citation1996]. Blastocysts were evaluated on the basis of the expansion of the blastocoel and the number and cohesiveness of the inner cell mass (ICM) and trophectoderm cells, according to Gardner’s criteria. A good-morphology blastocyst was defined as having a well-expanded blastocoel (B3–B6) on day 5 or (B4–B6) on days 6–7, a well-defined inner ICM (A or B) and a single layer of trophectoderm cells surrounding the cavity (A or B) [Gardner et al. Citation2000]. There were no changes to our laboratory protocols during the study period.
Embryo transfer
Two or three embryos on day 3, or one or two blastocysts on day 5, were transferred to the uterus with a Wallace catheter (Wallace, Brisbane, Australia) under abdominal ultrasound guidance. If more embryos were available, they were vitrified.
Definitions of analyzed parameters
The ‘blastocyst formation rate’ was defined as the percentage of blastocysts formed from the total number of cleaved embryos. The ‘good-morphology blastocyst rate’ was the percentage of good-morphology blastocysts obtained from the total number of blastocysts. Observation of a gestational sac on days 30–35 after embryo transfer was defined as a clinically positive pregnancy. Developmental arrest of the embryo, or spontaneous abortion before 20 weeks of gestation, was defined as a miscarriage.
Statistical analysis
Comparisons of continuous variables were carried out using the Student’s t-test when data distribution was normal. For non-continuous variables, statistical comparisons were carried out using a Chi-square test or Fisher’s exact test, as appropriate. Pearson correlation analysis was also applied to some variables. Statistical analyses were performed with the statistical package SPSS 13.0 (SPSS Inc., Armonk, NY, USA). Significance was defined as having a p value < 0.05.
| Abbreviations | ||
| IVF | = | in vitro fertilization |
| ICSI | = | intracytoplasmic sperm injection |
| ART | = | assisted reproductive technology |
| ICM | = | inner cell mass |
Declaration of interest
None of the authors have any conflicts of interest.
Author contributions
Conception and design of the experiments: HY, HJ; Performance of the experiments: HY, HJ, RH, CW, JZ, KL; Analysis of the data: HY; Contribution of reagents, materials, and analysis tools: RH; Writing of the manuscript: HY, HJ.
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