Pregnancies following blastocyst stage transfer in PGD cycles at risk for beta-thalassaemic haemoglobinopathies.

BACKGROUND: Preimplantation genetic diagnosis (PGD) usually involves blastomere biopsy 3 days post-insemination (p.i.), followed by genetic analysis and transfer of unaffected embryos later on day 3 or 4. We evaluate a strategy involving embryo biopsy on day 3 p.i., genetic analysis on day 4 and, following culture in blastocyst sequential media, transfer of unaffected embryos on day 5 p.i. METHODS: PGD cycles were initiated in 15 couples at risk of transmitting beta-thalassaemia major. Oocyte retrieval and ICSI were performed according to standard protocols. Embryo culture used blastocyst sequential media. Embryos were biopsied on day 3 p.i. using acid Tyrode's for zona drilling, and the single blastomeres were genotyped by a protocol involving nested polymerase chain reaction and denaturing gradient gel electrophoresis analysis. RESULTS: Forty of 109 (37%) embryos biopsied on day 3 p.i. developed to blastocysts by day 5 p.i., with at least one blastocyst available for transfer in 12 cycles (80%). Genotype analysis characterized 51/109 (47%) embryos unaffected for beta-thalassaemia major, of which 28 were blastocysts. Transfer of 37 day 5 p.i. embryos (blastocysts and non blastocysts) initiated eight clinical pregnancies. Implantation rate per embryo transferred was 12/37 (32%). CONCLUSIONS: Embryo biopsy on day 3, followed by delayed transfer until day 5 p.i. offers a novel and effective strategy to overcome the time limit encountered when performing PGD, without compromising embryo implantation.     

Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation genetic diagnosis of beta-thalassaemia: a pilot study

BACKGROUND: Trophectoderm biopsy at the blastocyst stage is an emerging approach in preimplantation genetic diagnosis (PGD). This study aimed to compare genotyping success and implantation rates in PGD cycles for beta-thalassaemia following biopsy at the cleavage versus the blastocyst stage, with transfer of blastocysts. METHODS: This pilot study included 20 cycles: Group A: 10 cycles, day 3 blastomere biopsy, day 5 transfer; Group B: 10 cycles, day 5 trophectoderm biopsy, day 6 transfer. Standard-assisted reproduction and laser biopsy procedures were used. Biopsied cells were genotyped using real-time PCR multiplexed with fluorescent microsatellite analysis. RESULTS: In Group A, 131 fertilized eggs developed to 101 embryos suitable for single blastomere biopsy; 76/101 blastomeres were diagnosed (75.2%), 30 unaffected blastocysts were transferred resulting in six pregnancies (eight fetal hearts, 26.7% implantation rate). In Group B, 128 fertilized eggs developed to 53 blastocysts for trophectoderm biopsy (four to five cells), with 50/53 blastocysts diagnosed (94.3%), 21 unaffected blastocysts transferred and 6 pregnancies initiated (10 fetal hearts, 47.6% implantation rate). Overall, nine pregnancies reached >10 weeks gestation and were confirmed unaffected by prenatal diagnosis, with 12 healthy babies born. CONCLUSIONS: This pilot study suggests that trophectoderm biopsy and blastocyst transfer may be more advantageous than cleavage stage biopsy with respect to outcome of PGD for monogenic diseases.     

The Graduated Embryo Score (GES) predicts blastocyst formation and pregnancy rate from cleavage-stage embryos

BACKGROUND: Embryo morphology and cleavage rates alone do not consistently identify embryos with high implantation potential following IVF. Blastocyst transfer has been reported to improve success rates by identifying potentially superior quality embryos. Algorithms for predicting IVF outcomes based on the presence of early developmental milestones have been proposed. Here we introduce the Graduated Embryo Score (GES). METHODS: Nucleolar alignment along the pronuclear axis, regular cleavage and degree of fragmentation at the first cell division, and cell number and morphology on day 3 were weighted to create a possible GES of 100 for each of 1245 fertilized embryos derived from 109 patients aged <40 years. The GES was correlated with IVF outcome. RESULTS: Of 983 embryos for extended culture, 349 (36%) developed to blastocyst and 180 (18%) were good quality (grade I-II). When ranked by cell number and morphology alone, 34% of embryos with > or =7 cells and <20% fragmentation formed good quality blastocysts. Using GES, embryos scoring 90-100 had 64% blastocyst formation compared with 31% scoring 70-85 and with 11% scoring 30-65. Embryos scoring 70-100 had 44% blastocyst development compared with 9% scoring 0-65. Fifty-six patients (51%) conceived on-going gestations from 294 transferred embryos. In patients with at least one transferred embryo scoring > or =70, the pregnancy rate was 59% compared with 34% if all embryos scored <70. The overall implantation rate was 28%. Among embryos scoring 70-100, an implantation rate of 39% was seen, compared with 24% among embryos scoring 0-65. CONCLUSIONS: Predicting which cleaved embryos will form blastocysts could permit the high success rates associated with blastocyst transfer to be achieved from day 3 embryo transfer.     

Cleavage anomalies in early human embryos and survival after prolonged culture in-vitro

This study examines the relationship between common morphological anomalies of cleaving embryos and their ability to form apparently normal blastocysts in vitro. The impact of cleavage rate, fragmentation, and multinucleation on compaction, cavitation, along with inner cell mass and trophectoderm formation has been assessed. The study population consisted of 102 patients who elected or were selected to have a day 5 embryo transfer. Clinical pregnancy and implantation rates were 66.7 and 49% respectively. Slow and fast cleavage had a significant negative association with normal blastocyst formation. Only 13.8% (67/484) of embryos with <7 cells and 27.5% (25/91) of those with >9 cells on day 3 formed blastocysts with apparently normal morphology, compared to 41.9% (252/602) with 7-9 cells on day 3 (P < 0.001). Fragmentation had a negative impact on normal blastocyst formation. Embryos with >15% fragmentation formed normal blastocysts at a significantly lower rate (46/279; 16.5%) than embryos with 0-15% fragmentation (311/935; 33.3%) (P < 0. 001). Furthermore, the pattern of fragmentation was associated with blastocyst formation. Type IV fragmentation led to a significant reduction in blastocyst formation (25/170 or 14.7%), compared to types I, II and III which performed much better (38.6, 32.9 and 32.4% respectively). Only 15.9% (22/138) of embryos with one or more multinucleate cells on day 2 and/or 3 formed normal blastocysts compared with 31.9% (335/1051) (P < 0.001) of those without multinucleation. Collectively, the data suggest that cleavage anomalies, some of which do not preclude development after short-term culture, may reduce the developmental competence of embryos after prolonged culture.     

Embryos with high implantation potential after intracytoplasmic sperm injection can be recognized by a simple, non-invasive examination of pronuclear morphology

Embryos are conventionally selected for transfer based on the evaluation of the cleavage speed and extent of blastomere fragmentation. Here we examined whether the predictive value of these criteria, as indicators of the chance of embryo implantation, can be further potentiated by adding previously described criteria reflecting the regularity of pronuclear development. In a group of embryos selected for transfer in 380 fresh embryo transfer cycles according to the conventional criteria, the transfer of only those embryos that developed from zygotes judged normal at the pronuclear stage (pattern 0) gave significantly higher pregnancy (44.8%) and implantation (30.2%) rates compared with the pregnancy (22.1%; P < 0. 05) and implantation rates (11.2%; P < 0.001) for the transfers of only those embryos that developed from zygotes judged abnormal (non-pattern 0). The transfer of only one pattern 0 embryo was sufficient for the optimal chance of pregnancy (no differences in pregnancy rates after transfer of one, two or three pattern 0 embryos), whereas the transfer of two pattern 0 embryos mostly resulted in a twin pregnancy. The inclusion of the criteria based on pronuclear morphology can thus lead to the application of a single embryo transfer policy and optimize the selection of embryos for transfer and cryopreservation.     

Morphological evaluation of human embryos and derivation of an embryo quality scoring system specific for day 3 embryos: a preliminary study

A scoring system specific for day 3 embryos has not been extensively explored. Most IVF laboratories continue to grade embryos solely on the basis of cell number and percentage fragmentation as was traditionally done for day 2 embryos. Additional morphological features, some unique to day 3 embryos, may be useful in selecting embryos most likely to blastulate and implant. The objective of this study was to derive an embryo scoring system for day 3 transfers which is predictive of positive pregnancy outcomes. A total of 316 transferred embryos from 93 patients was recorded on videotape and evaluated. The following parameters were used to grade the embryos: cell number, fragmentation pattern (FP), cytoplasmic pitting, compaction, equal sized blastomeres, blastomere expansion and absence of vacuoles. The clinical pregnancy rate was 41.9%, with an implantation rate of 18% per embryo transferred. The mean number of embryos transferred per patient was 3.4. Three formulae were derived to score embryo quality in each transfer based on the average score of individual embryos transferred. In the first scoring system, cell number alone was used to predict pregnancy outcome. The second scoring system was based on blastomere number and the observed FP. The third scoring system utilized both blastomere number and FP but also combined this with five morphological criteria to yield a final day 3 embryo quality (D3EQ) score. We found the D3EQ score to be prognostic of pregnancy outcome. This study suggests that although cell number and FP are certainly predictors of positive pregnancy outcomes, additional parameters specific to day 3 embryos should be used to stratify a cohort of embryos further.     

Parameters guiding selection of best embryos for transfer after cryopreservation: a reappraisal

BACKGROUND: The purpose of this study was to evaluate the respective influences of blastomere survival and resumption of mitosis on the outcome of frozen-thawed embryos. METHODS: A retrospective analysis was performed in our centre on 363 thawing cycles, involving 4-cell day 2 grade 1 embryos with <10% fragmentation. RESULTS: A higher implantation rate per transferred embryo was observed when all transferred embryos were characterized by fully intact blastomeres (100% blastomere survival) as compared with damaged embryos (50 or 75% blastomere survival) (22.0 versus 7.2%; P < 0.0001). Moreover, the implantation rate per transferred embryo was significantly higher for cleaved embryos compared with uncleaved embryos (19.7 versus 3%; P < 0.0001). Transfer of fully intact, cleaved embryos resulted in the highest implantation rates compared with transfer of damaged and uncleaved embryos (27.4 versus 0%; P < 0.0001). Intermediate implantation rates were observed when only one of the two criteria was fulfilled (13 versus 11% respectively; P > 0.05). Multivariate analysis showed that the clinical pregnancy rate was influenced by both criteria (odds ratio = 3.4 for transfer of embryos with six or more cells versus embryos with less than six cells. CONCLUSION: The results of our study suggest that the most important factor to predict further embryo development is the total number of blastomeres in transferred embryos, however they are obtained (good survival and/or resumption of mitosis).     

The effect of pronuclear morphology on embryo quality parameters and blastocyst transfer outcome.

BACKGROUND: Embryo quality may be accurately assessed as early as the pronuclear zygote phase, as shown in recent studies. However, it is not known whether good quality zygotes are destined to become good quality cleavage stage embryos and blastocysts. METHODS: In this retrospective study, 86 intracytoplasmic sperm injection-embryo transfer cycles were studied where each available embryo was scored from the zygote until the blastocyst stage. Embryonic normality parameters such as pronuclear pattern, early cleavage, cleavage stage embryo grade, the presence of embryos with > or =8 cells on day 3 and blastocyst quality were recorded. Embryo transfer was undertaken at the blastocyst stage and the outcome was studied according to the pronuclear pattern exhibited by the zygotes. RESULTS: Embryos that showed an ideal pronuclear pattern (0 PN pattern) cleaved earlier and faster and resulted in better quality cleavage stage embryos and blastocysts. The incidence of blastocyst formation was 72% in zygotes showing a 0 PN pattern, compared with 12.7% in zygotes with double pronuclear abnormality. Higher implantation and pregnancy rates were obtained when at least one blastocyst derived from a 0 PN pattern zygote was included in the set of embryos to be transferred. CONCLUSIONS: Our results indicate that the pronuclear pattern of the zygote is closely related to blastocyst formation and quality. Blastocysts derived from 0 PN zygotes have a higher potential for implantation.     

Improved development of human embryos in vitro by a human oviductal cell co-culture system.

This study was undertaken to determine the effect of co-culture with human oviductal cells on human embryos. Spare embryos from gamete intra-Fallopian transfer (GIFT), pronuclear stage transfer (PROST) and in-vitro fertilization/embryo transfer (IVF/ET) programmes were either cultured in serum-supplemented Earle's balanced salt solution alone, or co-cultured in the same solution with oviductal cells from the pronuclear stage (day 1 post-insemination) or two- to four-cell stage (day 2 post-insemination). The co-cultured embryos appeared to have a higher developmental potential (higher rate of blastocyst formation and lower fragmentation rate), although there was no statistical difference in their rate of development, degree of fragmentation and stages attained, when compared with conventionally cultured embryos. The percentage of hatching blastocysts was significantly higher (P less than 0.05, Fisher's exact test) for embryos co-cultured from day 1 post-insemination (38%) than for embryos which had not been co-cultured (7%). The blastocyst hatching rate for embryos co-cultured from day 2 post-insemination was 15%. It was therefore concluded that co-culture of human embryos with oviductal cells could improve the development of the embryos in vitro. The degree of improvement was more pronounced when the co-culture started at an earlier stage.     

Extended embryo culture in human assisted reproduction treatments

In order to evaluate the niche of extended embryo culture in an IVF programme, retrospective analysis of non-selected IVF patients, who underwent ovarian stimulation from April 1998 to June 1999 in a single private practice assisted reproductive technology centre, was performed. Embryos were cultured for 48 h in S1/G1.2 medium followed by 48 to 72 h of culture in S2/G2.2 to day 5 or day 6. Only fertilized oocytes exhibiting two pronuclei from donor and non-donor IVF and intracytoplasmic sperm injection (ICSI) cases were examined to determine the relationship between embryo cell number on day 3 and subsequent rate of blastocyst formation. Results indicated that a proportional relationship existed between the number of blastomeres present in day 3 embryos and the rate of blastocyst formation. Fifty-four per cent of embryos that had six cells on day 3 formed blastocysts, while 76% of those embryos with eight cells formed blastocysts. Blastocyst development did not increase further when embryos had more than eight cells on day 3, indicating that embryos with greater cell numbers on day 3 are not always predictive of a greater likelihood of blastocyst formation. Fertilized oocytes exhibiting two pronuclei from donors produced significantly more blastocysts (67%) than those from IVF patients (52%; P < 0.01), and had a significantly higher implantation rate (54%) compared with IVF patients (30%; P < 0.01). Furthermore, blastocyst cryopreservation resulted in significantly higher implantation rates than cryopreserved cleavage stage embryos (P < 0.001).     

Development of serum-free media for the culture and transfer of human blastocysts

The culture and transfer of the blastocyst stage embryo has several advantages for assisted reproduction in the human. However, due to inadequacies of present culture conditions in human in-vitro fertilization (IVF), embryos are routinely transferred to the uterus on either day 2 or day 3 of development around the 4- to 8-cell stage, with resultant implantation rates of only 10-25%. In other mammalian species the transfer of cleavage stage embryos, which normally reside in the oviduct, results in a significantly lower implantation rate compared with the transfer of blastocysts. Extended culture of human embryos in vitro will help to identify those embryos with little, if any, developmental potential. It is therefore plausible that the blastocyst has an intrinsically higher viability than the cleavage stage embryo. It has now been shown in human IVF that sequential serum-free media can support > 50% blastocyst development, with an implantation rate per blastocysts of 50%, double that obtained for cleavage stage embryos. As the implantation rate of the blastocyst is higher than the cleavage stage embryo, fewer blastocysts are required for transfer. The development of completely defined embryo culture media may prove feasible by the replacement of protein with the glycosaminoglycan hyaluronate. Hyaluronate, which is protein-free, is more suitable than albumin in supporting implantation in the mouse, and can eliminate the biological variation inherent when using protein and the potential for contamination when using blood products such as albumin.     

The rate of development and time of transfer play different roles in influencing the viability of human blastocysts

Improved embryo culture protocols now render more feasible the possibility of obtaining human blastocysts after in-vitro fertilization. In this study we present: (i) results of blastocyst development from supernumerary embryos after co-culture on green monkey kidney epithelial cells and (ii) pregnancy rates after transfer of frozen blastocysts. In addition, we have examined the influence of the day of blastocyst freezing and the day of transfer after the luteinizing hormone (LH) peak on pregnancy and implantation rates. Of 423 supernumerary embryos, 200 developed to the blastocyst stage (47.3%). By days 5 and 6, 67% of the blastocysts had reached the blastocyst stage, and were frozen, compared to 28.5% by day 7. When we compared the cases where only blastocysts frozen on days 5 and 6 were transferred compared to those frozen and transferred on or after day 7 the pregnancy rates were 7/18 (38.9%) and 1/16 (6.2%) respectively. In contrast, when we examined the influence of the day of transfer we found that pregnancies were established from day 5 up to day 9 post LH peak. Based on these results, we suggest that every attempt should be made to increase the development rate of supernumerary embryos to the blastocyst stage, as it appears that the quality of blastocysts transferred, as shown in this study by rate of development, plays a more crucial role than the timing of transfer.      

Cryopreservation of biopsied embryos at the blastocyst stage

BACKGROUND: The availability of an efficient cryopreservation program is especially important in the case of embryos that have undergone blastomere biopsy for PGD. Unfortunately, the freezing/thawing of biopsied embryos has given disappointing results when performed at the cleavage stage. In this study, embryos diagnosed as normal after PGD were grown to the blastocyst stage, frozen and thawed for successive frozen embryo transfer. METHODS: A total of 34 patients performed a thawing cycle in which 47 blastocysts were thawed. The cryopreservation solutions were based on HEPES-buffered medium supplemented with human serum albumin (HSA), sucrose and 1,2-propanediol. The same protocol was applied to embryos from 88 IVF/ICSI patients, which underwent 92 thawing cycles with 150 thawed blastocysts. RESULTS: The survival rate was similar in the two groups (53% after PGD and 58% in IVF/ICSI cycles), as well as the cumulative pregnancy rate per patient (59% after PGD versus 47% in IVF/ICSI cycles), despite a higher maternal age and a lower proportion of embryos available for transfer or cryopreservation in the PGD group. CONCLUSIONS: Neither the survival rate nor the subsequent development and chances of implantation, differed between embryos frozen at the blastocyst stage following biopsy and those frozen intact.     

Blastocyst formation and cell numbers in human frozen-thawed embryos following extended culture

BACKGROUND: Blastomere loss following human embryo cryopreservation has been shown to be associated with reduced implantation potential. In order to elucidate the underlying mechanism, the present study was designed to investigate the consequences of blastomere loss on subsequent preimplantation development in vitro. METHODS: Cryopreserved embryos destined for disposal were thawed and cultured for 96 h prior to determination of total cell numbers in resultant blastocysts. RESULTS: The proportion of embryos which formed blastocysts in vitro was significantly reduced when blastomere loss was evident in thawed embryos (25 versus 41% in fully intact thawed embryos). In addition, blastocysts from partially intact thawed embryos exhibited a significant reduction in total cell number (45.0 versus 58.4 in blastocysts from fully intact thawed embryos). Development to the blastocyst stage was significantly less frequent in fully intact thawed embryos which had been generated using ICSI (19%) compared with standard IVF (47%), although cell numbers in the resultant blastocysts were similar (57.1 and 58.6 respectively). CONCLUSIONS: Blastomere loss following cryopreservation impairs preimplantation development and results in reduced cell numbers at peri-implantation stages. ICSI is associated with reduced potential for post-thaw development of cryopreserved embryos in vitro.     

Influence of group culture and culture volume on the formation of human blastocysts: a prospective randomized study.

The optimal culture conditions for embryos to reach the blastocyst stage are under investigation. One factor is the putative influence of autocrine or paracrine factors produced by the embryo itself. Studies in mice blastocysts showed a beneficial influence of micro-cultures and communal growth on pregnancy and implantation rates, attributed to these growth or survival factors. In humans, studies have only involved embryos up to day 2 or 3 without consistent results. Therefore a prospective, randomized study was designed to assess whether group culture or incubation volume would influence day 3 human embryos to develop into blastocysts. Embryos were initially cultured in groups until day 3, after which patients were randomly allocated to four groups. Group 1: group culture in a small volume; group 2: single culture in a small volume; group 3: single culture in a large volume; group 4: group culture in a large volume. No significant differences in blastocyst formation could be found between the four groups (35, 45, 36 and 36% respectively). Therefore any of the four modes can be used, whichever is most convenient. The only factor which made a statistically significant contribution was the number of embryos on day 3. An increase in the number of embryos by one embryo decreased the odds of obtaining a blastocyst by 6%. The overall pregnancy rate per transfer was 40%, and the implantation rate was 28%. Single culture in a small volume allows a direct and individual assessment of embryo morphology, and as such it seems preferable.     

Use of co-culture of human embryos on Vero cells to improve clinical implantation rate

Co-culture of human embryos (n = 384 cycles) to the blastocyst stage using Vero cell monolayers was carried out between August 1995 and December 1997. A total of 2868 zygotes were co-cultured and 1027 embryos reached the blastocyst stage (blastocyst formation rate 35.8%). The blastocysts were frozen in 43.7% of patients. A mean of 1.8 blastocysts was transferred per patient and 95 pregnancies were obtained (pregnancy rate/cycle 24.7%). The blastocyst implantation rate was 23.6%. Miscarriage occurred in 15 patients (15.7%) and ectopic pregnancy in three (3.1%) patients. The multiple pregnancy rate was 32.6%. No differences were observed in the blastocyst rate between poor, normal or high response patients. Blastocyst formation was significantly lower when frozen donor spermatozoa were used. Significantly higher pregnancy rates per transfer and blastocyst implantation rates were attained when embryos were transferred on days 5 or 6 compared with day 7. No advantage was observed when co-culture was used in first cycle IVF patients, in comparison with conventional day 2 replacements. The use of blastocysts for preimplantation genetic diagnosis (PGD) increases the diagnostic reliability and widens diagnostic possibilities. A total of 215 cycles with frozen-thawed co-cultured blastocysts were carried out, with a pregnancy rate of 22.7% per replacement.     

Pregnancy achieved by transferring blastocysts into endometrial stroma in mice.

To prevent the extra-uterine discharge of transferred embryos, we directly inserted mouse embryos into the endometrial stroma (intra-endometrial embryo transfer). A 27G injection needle was inserted near the utero-tubal junction into the endometrial stroma. After removal of the needle, a glass micropipette was inserted and one embryo was transferred with a very small amount of culture medium. To determine the feasibility of this method, the uterine lumen was flushed with phosphate-buffered saline from the tubal ends immediately after transferring blastocysts into pseudopregnant mice on day 2 and day 4. The rates of recovery of embryos from the uterine lumen were 5.0% (1/20; day 4) and 15.0% (3/20; day 2). These results suggest that a high rate of intra-endometrial embryo transfer is possible. The embryonic viability rates (number of viable grown fetuses/number of blastocysts transferred) of this method were 50.0% (28/56; day 4) and 25.0% (5/20; day 2). Living offspring were delivered from both recipients which had received embryos on day 2 and day 4 of pseudopregnancy. In human in-vitro fertilization and embryo transfer, attempts have also been made to immobilize the embryos, and this method might be clinically applicable. Moreover, this method will be a good in-vivo model for studies on the mechanism of implantation.     

Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing

BACKGROUND: The present study was undertaken to examine the usefulness of both vitrification and assisted hatching (AH) on blastocysts that originate from embryos showing different qualities during their cleavage stage. METHODS: A total of 281 blastocysts were vitrified (93 vitrification-warming cycles) in a mixture of ethylene glycol-dimethylsulphoxide-Ficoll and sucrose using the Hemi-Straw (HS) carrier system. After warming, AH using the partial dissection technique was performed in 36 cycles. RESULTS: After warming and culture for 24 h, a total of 168 blastocysts (60%) was suitable for embryo transfers and a total of 25 ongoing pregnancies (27%) was obtained. Forty-nine transfers of 96 no-AH blastocysts and 36 transfers of 72 AH blastocysts resulted in an implantation rate of 13 and 22% respectively (P < 0.05). The percentage of transfers with at least one hatching blastocyst was significantly higher after application of AH (69 versus 33%) (P < 0.001). In all, 73 and 38% of blastocysts showing respectively optimal and non-optimal embryo development during the early stage were available for transfer (P < 0.001). Consequently, implantation rates of 19 and 6% were obtained after transfers of blastocysts showing respectively optimal and poor embryo development. CONCLUSIONS: Artificial opening of the zona pellucida after warming of vitrified blastocysts significantly improved the rate of transfers with hatched blastocysts and the implantation and pregnancy rates. The percentage of blastocysts that survived the HS vitrification procedure and were available for embryo transfer is related to their previous developmental quality.     

Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts

BACKGROUND: Manual puncture of the trophectoderm of human blastocysts with a needle before vitrification increases their survival rate, but the embryos take a long time to re-expand. This study examined whether causing human blastocysts to collapse by manual pipetting before vitrification would allow more rapid re-expansion and improve pregnancy rates. METHODS: After embryo transfer in IVF cycles, surplus embryos that developed to the expanded blastocyst stage were placed in cryoprotectant and then artificially shrunk by mechanical pipetting with a fine hand-drawn glass pipette slightly smaller in diameter than the blastocyst. The shrunken embryos were placed in a small volume of vitrification solution and plunged into liquid nitrogen on a cryotop. The blastocysts were thawed by warming and then dilution in 1 mol/l sucrose. RESULTS: Of 49 expanded vitrified blastocysts, 48 (98%) re-expanded within 3 h after warming. Following transfer (48 blastocysts in 28 cycles), 14 women (50%) became clinically pregnant, and the implantation rate was 33% (16/48). Eight healthy babies have been born in six deliveries, and the other eight pregnancies are ongoing. To date, there have been no spontaneous abortions. CONCLUSIONS: The results suggest that artificial shrinkage with pipetting is a simple and effective technique to assist successful cryopreservation of expanded blastocysts by vitrification.     

胚胎干细胞与四倍体互补产生转基因小鼠

目的 将四倍体胚胎补偿技术与转基因相结合，构建基因打靶载体转化的胚胎干细胞(ESCs)，获得小概率的同源重组事件，进而直接得到所要的突变品系。 方法 通过电穿孔的方法将增强型绿色荧光蛋白（EGFP）表达质粒转入ESCs，用G418筛选获得EGFP-ESCs。另外，通过电融合获得四倍体囊胚细胞，显微注射法将19～21个EGFP-ESCs注入每个四倍体囊胚腔，分别移植到假孕2.5d雌鼠子宫或假孕0.5d的输卵管内。 结果 获得稳定表达的EGFP-ESCs，染色体数目正常 (2n＝40条)；四倍体细胞融合率为95.07%，囊胚发育率为95%；共获得410个重构囊胚；移植后未得到出生小鼠，但共观察到了151个着床位点，子宫移植的着床率为29.41%，输卵管移植的着床率为64.37%，获得的胚胎中EGFP蛋白有散在的表达。 结论 稳定表达的EGFP-ESCs参与到了转基因胎鼠的发育中；本实验中输卵管移植的着床率高于子宫移植的着床率。