Effect of chilling on the development of in vitro produced bovine embryos at various cleavage stages

Purpose: Bovine embryos and zygotes are known to be sensitive to “temperature shock” when cooled to temperatures near 0°C. The effect of chilling on in vitro derived embryos at various cleavage stages was investigated. Methods: Cumulus-oocyte-complexes (COCs) were matured in IVM medium with serum. Presumptive zygotes were cultured in serum free in vitro culture (IVC) medium. Embryos were used as chilled or control samples at the 2-cell, 4-cell, 8-cell, morula, and blastocyst stages. Embryos in 0.2 mL PBS in plastic straws were cooled rapidly in ethanol baths at 0°C for 30 min. Embryo viability was assessed by in vitro development. Results: The percentage of control embryos that hatched as blastocysts increased the later stage at which they were selected. Relative proportion of embryos increased from 28% to 48% to 68% when chilled at the 8-cell, morula or blastocyst stages. Conclusions: IVF-produced embryos are differentially susceptible to cooling injury. Cell counts made of those blastocysts formed from chilled embryos indicated subtle effects of chilling.     

Vitrification of mouse embryos at 2-cell, 4-cell and 8-cell stages by cryotop method

Purpose

The objective of this study was to investigate the effects of vitrification on the preimplantation developmental competence of mouse 2-cell, 4-cell and 8-cell stage embryos.

Methods

Mouse 2-cell, 4-cell and 8-cell stage embryos were cryopreserved using the cryotop vitrification method and subsequently warmed on a later date. The embryos were then assessed by their morphology, blastocyst formation and hatching rates. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in hatched blastocysts from the control and experimental groups.

Results

Vitrified embryos at the 2-cell, 4-cell and 8-cell stages appeared morphologically normal after warming. The overall survival rate of vitrified embryos at various stages after warming was 96.7% and there were no significant differences among 2-cell stage (96.0%), 4-cell stage (96.8%) and 8-cell stage (97.1%) embryos (P?>?0.05). The blastocyst formation rate (69.4%) and hatching rate (52.6%) of vitrified 2-cell embryos were significantly lower than that from the control group and vitrified 8-cell embryos (P?<?0.05). In the vitrified 4-cell embryo group, the blastocyst formation rate (90.3%) was similar to the 8-cell group (91.2%), but the hatching rate (60.0%) was significantly lower than that of the non-vitrified control ( 84.1%) and vitrified 8-cell embryo (78.4%) groups (P?<?0.05). When further development to the fully hatched blastocyst stage was compared, hatched blastocysts derived from vitrified 2-cell, 4-cell and 8-cell embryos had significantly lower cell counts both in the ICM and TE, as compared to fresh blastocysts (P?<?0.05). Among the vitrified 2-cell, 4-cell and 8-cell embryo groups, there were no significant differences in the cell counts of ICM and TE (P?>?0.05).

Conclusions

Although cryotop vitrification was suitable for the cryopreservation of mouse embryos from the 2-cell stage, 4-cell stage and 8-cell stage without significant loss of survival, vitrification had an adverse effect on the development of 2-cell embryos. Mouse embryos at the 8-cell stage had the best tolerance for vitrification and would yield the highest level of post-vitrification developmental competence among early cleavage stage embryos. Nevertheless, it is unclear how these findings can be extrapolated to human embryos.     

Factors affecting the success of human blastocyst development and pregnancy following in vitro fertilization and embryo transfer

Objective: To determine the factors affecting blastocyst development and pregnancy after IVF and ET.

Design: Retrospective analysis of data arising from a clinical trial.

Setting: Private in vitro fertilization clinic.

Patient(s): Fifty-six patients aged ≤40 years, undergoing IVF procedures for infertility, recruited specifically for blastocyst transfer.

Intervention(s): All zygotes were cultured to days 5 or 6 after insemination, and one to four of the most advanced blastocysts were transferred to the patient’s uterus.

Main Outcome Measure(s): Development of zygotes to blastocysts in vitro and pregnancy and implantation rates after ET.

Result(s): Fifty-one percent of all zygotes developed to blastocysts. Significant positive correlation between the number of blastocysts formed was observed with the number of oocytes, pronuclear zygotes, and eight-cell embryos formed. There was a negative correlation with male factor infertility. By day 5 or 6, 93% of the patients had at least one blastocysts, and the clinical pregnancy rate per transfer was 43% and the implantation per embryo transferred was 25%. No other clinical factor significantly affected the number of blastocysts formed, pregnancy rate, or implantation rate.

Conclusion(s): The numbers of oocytes, zygotes, and normally developing embryos in culture significantly affects the production of blastocysts in vitro. Male infertility significantly reduces blastocyst production. The number and the quality of the blastocysts transferred significantly influences clinical pregnancy rate.     

First experiences with human blastocyst culture after IVF/ICSI under the conditions of the German embryo protection law

OBJECTIVE: Due to the improvements in human embryo culture in the recent years, it is now possible to transfer embryos five days after oocyte retrieval and IVF or ICSI at the blastocyst stage with favorable implantation rates. In Germany it is illegal to cultivate more than 3 embryos, therefore the selection has to be done at the pronuclear stage. There we report our experiences of human blastocyst culture in a routine IVF/ICSI programme under the conditions of the German Embryo Protection Law. MATERIALS AND METHODS: The data of 100 couples undergoing the IVF-ICSI programme at the University Clinic of Würzburg were analysed prospectively. 14-18 hours after insemination or micro-injection two or three zygotes with the best pronuclear development were selected for further cultivation. Fertilized oocytes were cultured in sequential media and were then transferred into the uterus 5 days after oocyte recovery. The blastocysts were graded from 1-8. RESULTS: In 100 cycles a total of 859 oocytes were collected, of whom 663 were fertilized and reached the pronuclear stage (median fertilization rate 88.9 %). 251 zygotes were selected at the PN stage. 51 % of the selected zygotes achieved the blastocyst stage after 5 days (grade 1-5), 28 % were morulae (grade 6-7) and 21 % of the embryos arrested in their development (grade 8). The clinical pregnancy rate was 26 %. Women who conceived had a significant better development of blastocysts on day 5 (grade 4 versus grade 6, P < 0.01) than those not achieving pregnancy. CONCLUSIONS: In summary, under the current legal conditions in Germany, blastocyst culture cannot improve pregnancy rates as the rate of arrested embryos of over 20 % limits the chances of implantation.     

The Well-of-the-Well system: an efficient approach to improve embryo development

Transfer of human embryos at the blastocyst stage may offer considerable benefits including an increased implantation rate and a decreased risk of multiple pregnancies; however, blastocyst culture requires an efficient and reliable in-vitro embryo culture system. In this study, the effect of the Well-of-the-Well (WOW) system consisting of microwells formed on the bottom of the culture dish was tested in three mammalian species, including humans. The WOW system resulted in significant improvement when comparing the drops for culture of in-vitro-matured and parthenogenetically activated porcine oocytes, and in-vivo-derived mouse zygotes. In human embryos, using a sibling oocyte design, embryos cultured in WOW developed to the blastocyst stage in a significantly higher proportion than did embryos cultured traditionally (55% in WOW and 37% in conventional culture; P < 0.05). In a separate study, also in human, a total of 48 patients with a cumulative 214 unsuccessful previous IVF cycles were selected for the trials. In subsequent intracytoplasmic sperm injection cycles, oocytes/embryos were cultured individually in the WOW system or in microdrops. Transferable quality blastocyst development (48.9% of cultured zygotes) was observed in the WOW system. Ninety-four blastocysts transferred to 45 patients resulted in clinical pregnancy rates of 48.9%, including nine twin pregnancies, seven single pregnancies, five miscarriages and one ectopic pregnancy. The results indicate that the WOW system provides a promising alternative for microdrop culture of mammalian embryos, including human embryos.     

The effect of preimplantation culture conditions on murine embryo implantation and fetal development

Ham's F-10 medium (Gibco, Grand Island, NY) and medium T6 with and without 15% fetal calf serum (FCS) were compared for their ability to support development of murine blastocysts with the capacity to implant and produce normal fetuses when transferred to pseudopregnant females. All media supported equal rates of blastocyst development from 2-cell embryos. In addition, there were no differences in the rates of blastocyst implantation. However, once implanted, blastocysts grown in T6 produced a significantly higher proportion of normal fetuses (58.5% to 65.9%) than blastocysts grown in either Ham's F-10 (2.4%) or T6 with FCS (27.6%). These results demonstrate that the rate of murine blastocyst development from 2-cell embryos in vitro is not a good criterion of healthy embryos. Murine blastocysts transferred in medium with 0% versus 50% FCS implanted and developed into normal fetuses at equal rates.     

Insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP-3 protease activity in the peritoneal fluid of patients with and without endometriosis

OBJECTIVE: To evaluate embryotropic action of hemoglobin (Hb) and ethylenediaminetetraacetic acid (EDTA) on preimplantation embryo development. DESIGN: In vitro model study using mouse embryos. SETTING: University affiliated hospital, Pochon CHA University. ANIMALS: Four-week-old block strain ICR mice naturally mated after superovulation. INTERVENTION(S): One-cell embryos were cultured in serum-free, modified preimplantation-1 medium, to which 1 microg/ml Hb and/or 0.1 mM EDTA were added. MAIN OUTCOME MEASURE(S): Preimplantation development and blastomere number. RESULT(S): More (P<.05) 1-cell embryos developed to the 4-cell (52% vs. 67%-84%), 8-cell (48% vs. 65%-81%), and blastocyst (40% vs. 61%-79%) stages after the addition of hemoglobin (Hb) and/or EDTA than after no addition. Highest proportion of embryos developed to each stage after the combined addition of Hb+EDTA. EDTA specifically stimulated the development before the 8-cell stage, which was as similar as Hb+EDTA. On the contrary, higher ratio of morula to blastocyst transformation was obtained after the addition of Hb or Hb+EDTA than after no addition (0.76 vs. 0.96-0.98). Significant increases in the cell number of blastocysts (46.5-47.2 vs. 53.2 cells), inner cell mass (ICM) cells (16.7-17.5 vs. 21 cells), and the ratio of ICM cells to trophoblasts (0.3-0.37 to 0.39) were found after the combined addition of Hb+EDTA, compared with no addition or with the addition of EDTA or Hb alone. CONCLUSIONS: Hb and EDTA have stage-specific effects on supporting preimplantation embryo development; Hb promotes both the development before the 8-cell stage and the morula to blastocyst transformation, whereas EDTA mainly promotes the development to the 8-cell stage. The combined exposure of embryos to Hb and EDTA improves not only preimplantation development but also the growth and quality of blastocysts.     

Effect of culture medium volume and embryo density on early mouse embryonic development: Tracking the development of the individual embryo

Purpose

To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly.

Methods

(1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining.

Results

The blastocyst development was significantly higher (P < 0.05) when nine embryos were cultured in 50 μl of droplet of culture medium compared with other volumes. The blastocyst development was significantly reduced (P < 0.05) in single embryo culture compared to group embryo culture with or without the WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P < 0.05) in single embryo culture with the WOW system than without.

Conclusions

Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 μl of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced embryonic development with single embryo culture cannot be ameliorated by the WOW system.     

Oestradiol, cyclodextrin-encapsulated 17beta-oestradiol and the oestradiol solubilizer 2-hydroxypropyl-beta-cyclodextrin all impair preimplantation mouse embryo development

The aim of this study was to examine the effects of 2-hydroxypropyl-beta-cyclodextrin (HbetaC) used as a solubilizer for oestradiol, 17beta-oestradiol (ethanol soluble) and HbetaC-encapsulated-17beta-oestradiol on mouse embryo development in vitro. HbetaC had no effect on day 3 development. In contrast, blastocyst development and blastocyst cell number were significantly reduced in the presence of 10(-4) mol/l solubilizer equivalent, but not at lower concentrations. The proportion of compacted embryos was significantly reduced with 10(-4) mol/l 17beta-oestradiol. No blastocysts were formed at 10(-4) mol/l concentration of 17beta-oestradiol, although the rate of blastocyst formation did not differ at lower concentrations. Blastocyst cell number was significantly decreased compared with controls at 10(-5) mol/l 17beta-oestradiol. The dose-response using HbetaC-encapsulated-17beta-oestradiol revealed that at 17beta-oestradiol concentrations of 10(-4) and 10(-5) mol/l, blastocyst development was significantly reduced. Blastocyst cell number was significantly reduced compared with controls for all concentrations of HbetaC-encapsulated-17beta-oestradiol. Exposure of embryos to 17beta-oestradiol (10(-4) mol/l) reduced blastocyst development on days 4 and 5 significantly in cultures initiated at the zygote, 2-cell and 8-cell, but not the morulae, stages of development. Trophectoderm, ICM and blastocyst cell numbers as well as percentage ICM development were reduced significantly, regardless of the stage of development. Therefore, 17beta-oestradiol does compromise embryo development.     

Granulocyte-macrophage colony-stimulating factor stimulates mouse blastocyst inner cell mass development only when media lack human serum albumin

The aim of the current study was to examine the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the development and differentiation of preimplantation mouse embryos from different strains and under different culture conditions. Embryos from F1 hybrid mice were cultured in a modified G1 medium lacking amino acids and EDTA (simple G1), human tubal fluid medium (HTF) or in G1/G2 sequential media, supplemented with GM-CSF (0, 2, 4, 8, and 16 ng/ml). Embryos from CF1 mice were subsequently cultured in G1/G2 with (5 mg/ml) or without HSA, in the absence or presence of GM-CSF (2 ng/ml). GM-CSF had no effect at any concentration on F1 embryo development and blastocyst cell numbers, irrespective of the culture media used. Similarly, GM-CSF had no effect on CF1 blastocyst development. However, a stimulatory effect of GM-CSF was evident on total blastocyst cell number and ICM development when CF1 embryos were cultured in the absence of HSA. When HSA was present in the media the beneficial effect of GM-CSF was negated. There was no difference in the number of apoptotic cells in CF1 blastocysts when G1/G2 were supplemented with GM-CSF with or without HSA. These data indicate that there is no beneficial effect of supplementing either simple (simple G1 or HTF) or more complete (G1/G2) media with GM-CSF when protein is present in the medium. However, when culture conditions are suboptimal and non-physiological, i.e. the absence of protein, GM-CSF stimulates development of both total cell numbers and ICM development of CF1 blastocysts.     

Oxygen concentration and preimplantation development

The recent article by Karagenc et al. once again shows that atmospheric oxygen is detrimental to embryo development in vitro. This study demonstrates that zygotes can readily develop into blastocysts under ambient oxygen, but in spite of their morphologically normal appearance, the viability of many of these embryos is compromised, with the most pronounced effect on ICM development. Numerous other studies have reached the same general conclusions, but in spite of the strong body of evidence derived from animal studies against using atmospheric oxygen, the entire human IVF community does not seem to have been convinced to abandon its use for embryo culture. This commentary argues for adoption of low oxygen concentrations as the standard for human embryo culture, at least for blastocyst production.     

体外受精中3原核胚胎的发育及可利用价值的探讨

目的:探讨体外受精(IVF)中异常的3原核(PN)胚胎的发育及可利用价值。方法:收集IVF治疗周期中废弃的3PN受精卵204个进行体外培养,观察其发育能力,并与同周期的1 138个2PN受精卵进行比较;采用胚胎植入前遗传学筛查(PGS)技术对由3PN发育成的19枚囊胚进行非整倍体分析。结果:3PN组和2PN组的卵裂率无统计学差异(P0.05);但3PN组囊胚形成率显著低于2PN组[9.6%(19/97)vs 37.9%(204/342),P0.01]。整倍体分析显示,10.5%(2/19)的3PN来源的囊胚为正常二倍体核型。结论:3PN受精卵有继续发育能力;囊胚培养和高通量测序可作为有效筛选异常PN受精卵中正常核型胚胎的一种方法。     

Effects of group culture on the development of discarded human embryos and the construction of human embryonic stem cell lines

Purpose

To explore the effect of group culture on the developmental potential of discarded embryos in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles and establish the human embryonic stem cell lines for future research.

Methords

Fresh discarded embryos were collected from the IVF/ICSI-ET program in the reproductive medical center of the first affiliated hospital of Zhengzhou university in this study. All zygotes were individually cultured from Day 1 to Day 3. On Day 3, discard embryos were then cultured in group of 1–4 embryos per droplet (30 μl/droplets) with a constant culture medium until Day 5 or 6. Mechanical method was used to isolate the inner cell mass (ICM) of blastocyst from the embryo. Then we inoculated the ICM on feeder layer. After identification of those cells, the human embryonic stem cell lines (hESCs) were established.

Results

In this study, we collected 1,223 fresh discarded embryos and they were sequential cultured to the blastocysts (18.07 %, 221/1,223), in which good quality blastocysts were 61(4.98 %, 61/1,223). There was no significant difference in the patients. The embryos from 1PN, 2PN, 3PN were sequential cultured to the blastocyst s(39.31 %,92/234;12.87 %,64/497;13.21 %,65/492),in which good quality blastocysts was 13.6 %(32/92),2.61 %(13/64), 3.04 %(15/65).1PN embryo’s blastulation rate and quality embryo formation rate was significantly higher than the 2PN and 3PN embryos’ (P <0.05). Three embryos group cultivation has the highest blastulation rate and quality embryo formation rate (P <0.05). In total, we successfully established 4 hESCs lines.

Conclusion

The group culture of human discard embryos can improve the blastulation rate and blastocyst quality to some extent. Three embryos group cultivate is the better culture number. Human discard embryos are good source for establishment of hESCs.     

Towards a single embryo transfer

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.     

Embryonic development and pregnancies following sequential culture in human tubal fluid and a modified simplex optimized medium containing amino acids

Pregnancies following human blastocyst transfers were established using a protein supplemented modified potassium simplex optimized medium containing amino acids (KSOM(AA)). Zygotes were first cultured for 2 days in protein supplemented human tubal fluid medium. Resulting embryos (day 3) were subsequently cultured in protein supplemented KSOM(AA) until day 5 of development. Pregnancy and implantation rates were noted after culture and transfer of blastocysts following this culture scheme. The impact of conventional insemination, intracytoplasmic sperm injection (ICSI) and maternal age on developmental rates as well as pregnancy rates was also evaluated. Maternal age had a significant impact (P < 0.05) on developmental rates of embryos until day 3 of culture. Overall, rate of blastocyst development for cultured day 3 embryos was 62%. Patients >39 years of age had lower (P < 0.05) rates of blastocyst development than patients in the younger subgroups. ICSI had no impact on developmental rates until day 3 of culture or rate of blastocyst development. Ongoing pregnancy and implantation rates following culture in KSOM(AA) were 51 and 37% respectively. These results indicate that KSOM(AA) supports high rate blastocyst development and resulting pregnancy rates.     

Cryosystem assessment by glucose uptake of murine blastocysts

Glucose uptake was used as a measure of metabolic activity and implantation potential to compare vitrification and slow freezing in a prospective randomized trial using murine blastocysts. Frozen 2-cell embryos (n = 132) thawed and cultured for 48 h to the blastocyst stage were randomly divided into four groups: (i) control - not refrozen; (ii) slow freezing using a programmed rate (PR); (iii) vitrification by super-cooled (VSC) liquid nitrogen; and (iv) vitrification in liquid nitrogen (VLN). Upon re-thawing, embryos were cultured individually for 24 h to determine glucose uptake non-invasively. Morphological assessments included total cell counts and inner cell mass (ICM) detection following immunosurgery. Mean glucose uptake was lower for each treatment (PR and VSC, 4.3 pmol/embryo per h; VLN, 4.9 pmol/embryo per h) versus controls (6.8 pmol/embryo per h). PR and VSC embryos had fewer cells (57.4 +/- 24.2 and 64.1 +/- 31.5) versus controls (85.7 +/- 26.2), and fewer embryos containing a detectable ICM (42.9 and 61.8%) compared with controls (88.2%). The only difference between control and VLN embryos was absolute glucose uptake, although in both treatments glucose uptake was increased from embryos with an ICM compared with those without. Glucose uptake appears to be a sensitive, non-invasive method to validate cryopreservation protocols.     

Cytogenetic analysis of human embryos and embryonic stem cells derived from monopronuclear zygotes

Purpose

By comparing the chromosomal constitution among the arrested cleavage-stage embryos, blastocysts and human embryonic stem cells (hESCs) which are all derived from monopronuclear (1PN) zygotes, it is aimed to determine whether chromosomally normal embryos can be reliably selected by blastocyst culture.

Methods

After 1PN zygotes are sequentially cultured for 5 days, the blastocysts and arrested cleavage-stage embryos were analyzed by fluorescence in situ hybridization (FISH) with probes for chromosomes 18, X and Y; G-banding analysis was adopted to analyze the karyotype of 1PN hESCs.

Results

The diploid rate of blastocysts was 74.6%, which was significantly (P?<?0.001) higher than that of arrested cleavage-stage embryos (31.6%), and the diploid rate of hESCs was 97.0%, which was significantly (P < 0.01) higher than that of blastocysts; the haploid embryos were excluded by blastocyst culture; nevertheless, there still existed such chromosomal abnormalities as mosaic and monosomic in blastocysts and trisomy in hESCs.

Conclusions

Blastocyst culture is an effective method to select against chromosomal abnormalities, especially the haploids in 1PN embryos; however, development to the blastocyst stage is not a reliable marker for mosaicism or aneuploidy.     

Prediction of embryonic developmental competence by time-lapse observation and 'shortest-half' analysis

Selecting an embryo with the highest probability of achieving a pregnancy is a major challenge. Early-cleavage embryos are considered to be of good quality; however, the exact developmental stage that predicts further development has not been defined. The aim of the study was to characterize cleavage rate and distribution of various stages of mouse preimplantation embryos using a time-lapse system. Mated mice were killed 20 h after human chorionic gonadotrophin administration and putative zygotes were recovered and cultured in an incubator-enclosed time-lapse imaging system. The 'shortest half' analysis was used to establish the period in which at least 50% of the embryonic population cleaved within the shortest time. Analysis indicated that through embryonic development, cleavage timing becomes less uniform and the 'shortest half' becomes longer with intervals of 2, 2.5, 3.5 and 5 h for 2-, 4-, 8-cell embryo and blastocyst stages, respectively. The 'shortest half' for the first cleavage was closely synchronized, with 80% of embryos developing to the blastocyst stage. Moreover, slow-cleaving embryos approaching the 2-cell stage expressed inferior developmental potential in comparison to those cleaving within the 'shortest half'. Thus, embryonic cleavage rate seems to be a biological indicator of developmental potential and may be useful for embryo selection.     

Cryopreservation of Embryos,Blastocysts, and Pregnancy Rates of Blastocysts Derived from Frozen-Thawed Embryos and Frozen-Thawed Blastocysts

Purpose: To evaluate the development of cryopreserved embryos when thawed and subsequently cultured to the blastocyst stage in comparison to transferring cryopreserved blastocysts. Methods: In this retrospective clinical study, we have evaluated 170 cycles in patients undergoing IVF treatment for infertility. Cryopreserved embryos were thawed and were subsequently cultured and transferred at the blastocyst stage. Cryopreserved blastocysts (Day 6) were thawed and transferred immediately. Results: Five hundred and sixty embryos and 444 blastocysts have been thawed. In the embryos group, the survival rate was 89% while in the blastocyst group the survival rate was 56%. In the embryos group the blastocyst development rate was 24.5%. The implantation rate in the embryos group was 20.6% per group blastocyst transferred compared to 5.3% in the blastocyst group. Conclusions: The ability of cryopreserved embryos to develop to blastocysts and their implantation potential does not seem to be greatly affected by the cryopreservation procedure.