Zygote transfer on day 1 versus cleavage stage embryo transfer on day 3: a prospective randomized trial

BACKGROUND: It has been reported that pronuclear morphology is related to embryo quality and viability, and that zygote stage embryos might establish pregnancies after being transferred to the uterus. The objective of this study was to investigate whether transferring zygotes on day 1 would result in similar pregnancy rates compared to transferring cleavage stage embryos on day 3 in a prospective randomized trial. METHODS: Patients undergoing IVF/ICSI treatments were randomized to either day 1 or day 3 transfers by envelope withdrawal technique. Zygotes were classified as 'pattern 0' and 'non-pattern 0' according to the size and alignment of pronuclei, the number and distribution of nucleoli. The two best zygotes or embryos were transferred on day 1 or day 3 respectively. The primary outcome measure was pregnancy rate. RESULTS: Pregnancy rates were higher in day 3 group (55/131, 42%) when compared to day 1 (34/123, 28%, P = 0.024). Similarly, implantation rates were higher in day 3 group (P = 0.03). There were more cycles with cryopreservation in the day 1 group (P < 0.001). Embryo quality on day 3 was similar between pattern 0 and non-pattern 0 zygotes. CONCLUSIONS: Day 3 embryo transfers result in better pregnancy and implantation rates compared to day 1 zygote transfers. The present pronuclei scoring cannot reliably select zygotes for transfer on day 1.     

A prospective, randomized study comparing day 2 and day 3 embryo transfer in human IVF

It is believed that delayed transfer of embryos after IVF allows for a better selection of good quality embryos. Hence, the number of embryos and all other prognostic factors being equal, transfer of day 3 embryos should be associated with higher implantation and pregnancy rates than transfer of day 2 embryos. To investigate this hypothesis, a prospective randomized study was carried out to compare implantation and pregnancy rates between day 2 and day 3 transfers. The relationship between the embryo quality score of day 2 and day 3 embryos and their respective implantation rates was also analysed. In a 2 year period all patients undergoing infertility treatment and in whom at least seven normally fertilized oocytes were obtained were included in the study. A minimization procedure was performed taking into account the patient's age and the method of fertilization (IVF or intracytoplasmic sperm injection). By using a uniform policy of embryo transfer, the number of embryos transferred was similar in both groups. The outcome parameters were embryo quality, implantation and pregnancy rates. No difference was observed in implantation and pregnancy rates between transfers on day 2 versus day 3 (23.8 versus 23.8% and 47.9 versus 46.8% respectively). The incidence of embryos of moderate to poor quality was higher in embryos cultured for 3 days compared with those cultured for 2 days. It is concluded that the outcomes of embryo transfer in terms of implantation and pregnancy rates are comparable for day 2 and day 3 embryos, although the overall embryo quality score decreases when embryos are kept in culture till day 3.     

A prospective randomized trial of blastocyst culture and transfer in in- vitro fertilization

The effectiveness of blastocyst culture and transfer in human in-vitro fertilization (IVF) was evaluated in a prospective randomized trial in patients having a moderate to good response to gonadotrophin stimulation. Embryos were transferred either on day 3 after culture to around the 8-cell stage in Ham's F-10 medium supplemented with fetal cord serum, or on day 5 after culture to the blastocyst stage in the sequential serum-free media G 1.2 and G 2.2. The pregnancy rates after transfer on day 3 or day 5 were equivalent, 66 and 71% respectively; however, significantly more embryos were transferred on day 3 (3.7) than on day 5 (2.2). The number of blastocysts transferred did not affect the implantation rate, and pregnancy rates when either two or three blastocysts were transferred were 68 and 87% respectively. The implantation rate of the blastocysts (50.5% fetal heart beat) was significantly higher compared to the cleavage stage embryos transferred on day 3 (30.1%). The percentage of blastocyst development was not affected by the number of 2-pronuclear embryos, or by maternal age. Irrespective of the number of blastocysts formed, pregnancy rates were similar. Furthermore, the pregnancy rate following blastocyst transfer in patients with 10 or more follicles at the time of human chorionic gonadotrophin administration was not affected by patient age. More than 60% of patients having blastocyst culture and transfer had supernumerary embryos for cryopreservation. The establishment of a pregnancy following thaw and transfer confirmed the viability of cryopreserved blastocysts cultured in the absence of serum or co- culture. The ability to transfer just two blastocysts while maintaining high pregnancy rates will therefore help to eliminate high order multiple gestations and improve the overall efficiency of human IVF.      

Effect of developmental stage of embryo at freezing on pregnancy outcome of frozen-thawed embryo transfer

BACKGROUND: The study aim was to investigate the impact of the developmental stage of embryos on pregnancy outcome of frozen embryo transfer (FET). METHODS: The survival rates of embryos after thawing and pregnancy outcome following FET were compared retrospectively between three cryopreservation strategies utilizing either zygote, day 2 or day 3 embryo freezing. RESULTS: A total of 4006 embryos was analysed in 1657 thaw cycles. The highest (P < 0.0001) survival rate (all cells survived) was observed for zygotes (86.5%), followed by day 2 (61.7%) and day 3 (43.1%) embryos. FET was performed in 1586 (95.7%) of all thaw cycles, resulting in overall clinical pregnancy and implantation rates of 20.7 and 14.2% respectively. The delivery rate per transfer was 16.5%, and live birth rate per transferred embryo 11%. There were no significant differences in clinical pregnancy, implantation, delivery and birth rates between frozen zygote, day 2 and 3 embryo transfers. However, an elevated miscarriage rate was observed in the day 3 group (45%) compared with zygotes (21.3%; P = 0.049) and day 2 embryos (18.3%; P = 0.004). The overall efficacy of FET (birth rate per thawed embryo) was 7.3%. The efficacy was lower in day 3 group (4.2%) than in the zygote (7.1%; P = 0.082) and day 2 (7.6%; P = 0.027) groups. CONCLUSIONS: The developmental stage of embryos at freezing has a profound effect on their post-thaw survival, but seems to have little effect on rates of clinical pregnancy, implantation, delivery and birth after FET. The elevated miscarriage rate for day 3 frozen embryo transfers may be caused by damage during freeze-thaw procedures. The low survival rate and elevated miscarriage rate were both responsible for a reduced overall efficacy for day 3 FET when compared with zygotes and day 2 embryos.     

One year's experience with elective transfer of two good quality embryos in the human in-vitro fertilization and intracytoplasmic sperm injection programmes

High incidences of multiple pregnancies, after transferringa maximum of three embryos, were observed after in-vitro fertilization(IVF) treatment. In a randomized study, it was demonstratedthat, after taking into account embryo quality and other positivelyinterfering parameters, an elective transfer of two good qualityembryos does not significantly influence the pregnancy rate.The intracytoplasmic sperm injection (ICSI) technique was successfullydeveloped in the meantime and high incidences of multiple pregnancieswere also obtained after ICSI. The question arose whether afterICSI there was also room for elective double embryo transferin a well-defined patient group. This report covers 1 year of IVF and ICSI treatment and theresults are presented in relation to the number of embryos transferred.The embryo development is similar for zygotes obtained afterIVF and ICSI; for both techniques 63% of the zygotes developto type A-B embryos and 13% to type C embryos. There is alsono difference in the pregnancy rate after ICSI or IVF. Globally,after IVF, 307 out of the 766 double and triple transfers (40.1%)and 317 out of 774 double and triple transfers (40.9%) afterICSI resulted in a positive HCG. After IVF, 73.9% (227) andafter ICSI 76.3% (242) of the pregnancies were evolutive. Neitherwas there any difference between the two techniques as regardsthe implantation rate per transferred embryo. After IVF, 22.8%of the transferred embryos implanted compared with 21.8% afterICSI. When the elective double embryo transfers were compared,no difference was found between IVF and ICSI. After IVF, 102of the 211 elective double transfers (48.1%) resulted in a pregnancyversus 93 out of 225 (41.3%) after ICSI [not significant (NS)].A high implantation rate per transferred embryo (IVF: 33.2%;ICSI: 26.9%, NS) was obtained in this elective double transfercategory, as was also reported in the randomized study. Thesedata confirm the results obtained in our randomized study andthe effectiveness of the elective double embryo transfer forIVF as well as for ICSI.     

A prospective randomized study: day 2 versus day 5 embryo transfer

BACKGROUND: This randomized controlled study was performed in an unselected IVF/ICSI population to test the hypothesis that blastocyst transfers result in higher clinical pregnancy rates (CPR) per oocyte retrieval when compared with day 2 transfers. METHODS: Blind randomization for transfer on day 2 (group 1) or day 5/6 (group 2) was performed before stimulation. Oocytes and embryos were cultured in sequential media in 5.5% CO(2), 5% O(2), 89.5% N(2) and 90% humidity. A maximum of two embryos was transferred. RESULTS: The two groups were similar for age, IVF indication, number of treatment cycles, rate of ICSI/IVF, number of fertilized oocytes and number of embryos transferred. The CPR/oocyte retrieval was comparable in group 1 (32%) and in group 2 (44%), while the CPR/embryo transfer was significantly higher (P < 0.01) in group 2 (60%) than in group 1 (35%). Similarly, the implantation rate per embryo transferred was significantly higher (P < 0.03) in group 2 (46%) than in group 1 (29%). The cryo-augmented delivery rate/oocyte retrieval was comparable in group 2 (36.3%) and in group 1 (28.6%). CONCLUSION: This randomized study in an unselected population showed a significantly higher CPR/embryo transfer and a tendency toward a higher CPR/oocyte retrieval in patients receiving blastocysts when compared with day 2 transfers.     

Day 3 embryo transfer with combined evaluation at the pronuclear and cleavage stages compares favourably with day 5 blastocyst transfer

BACKGROUND: The respective advantages of day 3 and day 5 embryo transfer are a matter of debate. Previous comparisons did not include pronuclear stage zygote scoring and cumulative success rates (fresh and cryopreserved embryos). METHODS: Patients were randomized prospectively for day 3 or day 5 embryo transfer. Day 3 embryos were selected for transfer and cryopreservation by using combined evaluation at the pronuclear and cleavage stages. RESULTS: There was no difference between day 3 and day 5 fresh embryo transfers as to the rates of pregnancy (58 versus 62%), clinical pregnancy (56 versus 58%), delivery (50 versus 48%), implantation (35 versus 38%) and birth (33 versus 36%) rates. The corresponding values for cryopreserved embryo transfers were also similar. However, day 3 embryo transfer compared favourably with day 5 transfer when the pregnancy (90 versus 66%), clinical pregnancy (85 versus 62%) and delivery (77 versus 52%) rates were calculated per oocyte recovery attempt. CONCLUSIONS: With a selected population of good prognosis patients and our embryo selection criteria, the implantation potential of day 3 and day 5 embryos is equal. Per oocyte recovery attempt, day 3 transfer is more clinically efficient than day 5 transfer, but at least one transfer of cryopreserved embryos is necessary to manifest this superiority.     

A comparison of four different techniques of assisted hatching

BACKGROUND: Assisted hatching (AH) has been proposed as a means to increase the implantation rate in patients with poor prognosis for pregnancy. The procedure appears to be effective when used selectively. Several different methods for AH have been introduced over the years but comparative studies are lacking. The aim of the current study was to compare retrospectively the efficacy of AH performed with four different methods in patients undergoing IVF or ICSI. METHODS: AH was performed prior to day 3 embryo transfer in 794 IVF/ICSI cycles. Indications for AH were females aged >35 years and/or elevated follicular phase FSH levels, previous failed IVF/ICSI cycles, poor embryo quality, and thick zona pellucida (>15 microm). Assignment to one of the four methods of AH was according to the availability of the particular method during the study period. The study was not randomized. RESULTS: Partial zona dissection was used in 239, acid Tyrode in 191, diode laser in 219 and pronase thinning of the zona pellucida in 145. Mean female age, mean number of previous failed IVF/ICSI cycles, number of oocytes retrieved, fertilization and cleavage rates, good quality embryos and zona thickness on day 3 did not differ between groups. Mean number of embryos transferred, implantation rate, clinical pregnancy rate, and abortion rates were likewise similar. CONCLUSIONS: Selective AH using four different methods yields similar implantation and pregnancy rates.     

Characterization of a top quality embryo, a step towards single-embryo transfer.

In most in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) programmes approximately one ongoing pregnancy in three is multiple. The need to characterize embryos with optimal implantation potential is obvious. We retrospectively examined all of 23 double transfers resulting in ongoing twins, occurring between January 1, 1996 and May 19, 1997. Characteristics of these top quality embryos were absence of multinucleated blastomeres, four or five blastomeres on day 2, seven or more cells on day 3, and 2 embryos, 11/31 (35%) were multiple. We applied our top quality criteria to the 221 double transfers: 106 transfers with two top embryos resulted in 65 (63%) ongoing pregnancies with 37 (57%) twins, 65 transfers with one top embryo in 38 (58%) ongoing pregnancies with eight (21%) twins. In the group without top embryos, 12/52 (23%) ongoing singletons occurred, with no twins. The corresponding ongoing implantation rates were 49, 35 and 12%. This analysis suggests that single embryo transfer with an acceptable pregnancy rate might be considered if a top quality embryo is available.     

The predictive value of using a combined Z-score and day 3 embryo morphology score in the assessment of embryo survival on day 5

BACKGROUND: The purpose of this study was to evaluate the ability of using the Z-score alone, or, in combination with the day 3 embryo morphology score, to predict embryo viability at day 5 from a large cohort of embryos derived from patients undergoing treatment with IVF/ICSI. METHODS AND RESULTS: In this retrospective study, a total of 1894 zygotes from 346 treatment cycles (295 couples) was analysed between January 2001 and May 2002. The Z-scoring system was useful in predicting day 5 embryo survival. The mean +/- SD day 5 embryo survival rates were 78.2 +/- 1.7, 49.0 +/- 2.5, 21.4 +/- 3.2 and 11.8 +/- 5.6% for Z-1, Z-2, Z-3 and Z-4 zygotes groups respectively. Embryos derived from Z-1 scores and grade I day 3 embryo scores showed the best day 5 embryo survival and a very high implantation potential. CONCLUSIONS: These data suggest that a combined evaluation of the Z-score and day 3 embryo morphology is highly predictive of embryo outcome after IVF/ICSI. The Z-score could be of great help in the selection of embryos for cultures extended to later stages. The Z-score alone, or preferably in combination with day 3 embryo morphology, is useful in the determination of the most suitable embryos and the number of embryos for transfer, thus achieving the optimal chance of conception while reducing the risk of high order multiple pregnancy.     

Is there any benefit from the culture of a single oocyte to a blastocyst-stage embryo in unstimulated cycles?

BACKGROUND: The aim of the study was to test the influence of 2- and 5-day cultivation of a single oocyte on the pregnancy rate in a non-stimulated cycle. METHODS: A retrospective chart review of 391 consecutive patients undergoing IVF and intracytoplasmic sperm injection in unstimulated cycles was performed. The embryos were kept in MediCult universal IVF medium for day 2 transfers and in BlastAssist System for day 5 transfers. RESULTS: The oocyte recovery rate in the group for 2-day cultivation and in the group for 5-day cultivation was similar, being 79.4 (162/204) and 83.6% (154/187) respectively. The same is true of the fertilization rate (73.8 versus 77.7%). The blastulation rate was 52.8%. The embryo transfer rate per cycle was higher when day 2 embryos were transferred: 64.8% (105/162) compared with 35.7% (55/154) if blastocyst-stage embryos were transferred. The pregnancy rate per transferred embryo was higher when a blastocyst was transferred (40.0%) instead of a day 2 embryo (23.8%). CONCLUSION: The expected pregnancy rate calculated per embryo available on day 2 of cultivation was similar in both groups (23.8 versus 22.2%) and it was not affected by oocyte culture to the blastocyst stage.     

Prevention of twin pregnancy after in-vitro fertilization or intracytoplasmic sperm injection based on strict embryo criteria: a prospective randomized clinical trial.

A prospective randomized study comparing single embryo transfer with double embryo transfer after in-vitro fertilization or intracytoplasmic sperm injection (IVF/ICSI) was carried out. First, top quality embryo characteristics were delineated by retrospectively analysing embryos resulting in ongoing twins after double embryo transfer. A top quality embryo was characterized by the presence of 4 or 5 blastomeres at day 2 and at least 7 blastomeres on day 3 after insemination, the absence of multinucleated blastomeres and <20% cellular fragments on day 2 and day 3 after fertilization. Using these criteria, a prospective study was conducted in women <34 years of age, who started their first IVF/ICSI cycle. Of 194 eligible patients, 110 agreed to participate of whom 53 produced at least two top quality embryos and were prospectively randomized. In all, 26 single embryo transfers resulted in 17 conceptions, 14 clinical and 10 ongoing pregnancies [implantation rate (IR) = 42.3%; ongoing pregnancy rate (OPR) = 38.5%] with one monozygotic twin; 27 double embryo transfers resulted in 20 ongoing conceptions with six (30%) twins (IR = 48.1%; OPR = 74%). We conclude that by using single embryo transfer and strict embryo criteria, an OPR similar to that in normal fertile couples can be achieved after IVF/ICSI, while limiting the dizygotic twin pregnancy rate to its natural incidence of <1% of all ongoing pregnancies.     

Ultrasound-guided embryo transfer maximizes the IVF results on day 3 and day 4 embryo transfer but has no impact on day 5

BACKGROUND: The use of ultrasound-guided embryo transfer has been reported to affect success rates in some centres but not others. In a prospective study, we examined the influence of ultrasound guidance in embryo transfer performed on different days after oocyte retrieval. METHODS: Two different methods of embryo transfer were evaluated in 1069 consecutive transfers. The ultrasound-guided embryo transfer was used in 433 cases, whereas 636 embryo transfers were performed with the tactile assessment ('clinical feel') method. RESULTS: Ultrasound-guided embryo transfer yielded a higher overall pregnancy rate than the 'clinical feel' approach, 47 versus 36% (P < 0.001). This difference was statistically significant where embryos were transferred after 3 or 4 days of culture, 45.9 versus 37.1% (P = 0.001) and 42.3 versus 27% (P = 0.035) respectively but not significant (P = 0.112) on day 5 embryo transfer (56.3 versus 45.7%). Likewise, the implantation rate was significantly different between the two groups on day 3 and 4 embryo transfer, 23.3 versus 15.8% (P < 0.01) and 21.6 versus 15.7% (P < 0.05%) respectively but no statistical difference was noted on day 5 embryo transfer, 26.7 versus 23.6%. CONCLUSION: Ultrasound assistance in embryo transfer on day 3 and 4 significantly improved pregnancy rates in IVF but had no impact on day 5.     

Similar delivery rates in a selected group of patients, for day 2 and day 5 embryos both cultured in sequential medium: a randomized study

BACKGROUND: The existence of a real benefit of blastocyst transfer is still a matter of debate. The aim of this study was to compare, in a prospective randomized trial, the outcome of day 2 and day 5 transfer of human embryos cultured in an 'in-house' sequential medium. METHODS: A total of 193 cycles from 171 patients with less than four previous IVF cycles, <39 years old and with four or more zygotes on day 1, were randomly allocated to day 2 (94 cycles) or day 5 (99 cycles) transfer. Zygotes were kept in fertilization medium until 18 h post-fertilization and then placed in a 'glucose-free' cleavage medium. Embryos allocated for day 5 transfer were placed in a blastocyst medium 66 h post-fertilization. Two or three embryos were replaced according to the morphology. RESULTS: A mean (+/- SEM) number of 2.1 +/- 0.4 and 1.9 +/- 0.3 embryos were replaced on day 2 and day 5 (P < 0.001) respectively. Delivery rates per transfer were 44.1 and 37.1% [P = not significant (NS)], implantation rates were 31.4 and 29.4% (NS) and multiple delivery rates 22 and 36% (NS) for day 2 and day 5 groups respectively. Ten patients (10.1%) had no blastocysts available for transfer. CONCLUSIONS: No clear benefits were observed using blastocyst transfer for patients aged <39 years who had had less than four previous IVF cycle attempts.     

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.     

Embryo morphology or cleavage stage: how to select the best embryos for transfer after in-vitro fertilization

This retrospective study of 1001 in-vitro fertilization (IVF) cycles included a consecutive series of single transfers (n = 341), dual transfers (n = 410) and triple transfers (n = 250) where all the transferred embryos in each cycle were of identical quality score and identical cleavage stage. In our 2 day culture system, transfer of 4- cell embryos resulted in a significantly higher implantation rate and pregnancy rate (23 and 49%) compared with 2-cell embryos (12 and 22%) and 3-cell embryos (7 and 15%). Furthermore, the transfer of 4-cell embryos resulted in a significantly higher pregnancy rate compared with embryos that had cleaved beyond the 4-cell stage (28%). The implantation rate (21%) and pregnancy rate (43%) after transfer of embryos of score 1.0 were significantly higher than after transfer of embryos of score 2.0 (14 and 32% respectively). Transferring embryos of score 2.1 resulted in significantly higher implantation rates (26%) and similar pregnancy rates compared with score 1.0. Transferring embryos of score 2.2-3.0 resulted in a significantly lower implantation rate (5%) and pregnancy rate (15%). A striking finding was that embryos of quality score 2.0 had a significantly lower implantation rate compared with embryos of quality score 1.0 and 2.1 and a significantly lower pregnancy rate compared to embryos of quality score 1.0. We also found a lower implantation rate and pregnancy rate when transferring 3-cell embryos. These findings may indicate periods of increased sensitivity to damage during the cell cycle. In conclusion, these results substantiate the idea of the superiority of 4-cell embryos and demonstrate that minor amounts of fragments in the embryo may not be of any importance. These findings may call for a shift when weighing the two main morphological components (quality score and cleavage stage) in the sense that reaching a 4-cell cleavage stage even with the presence of a minor amount of fragments should be preferred to a 2-cell embryo with no fragments.      

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.     

Fertilization and early embryology: Granulosa cell co-culture enhances human embryo development and pregnancy rate following in-vitro fertilization

A preliminary study and related clinical trial were performedto evaluate the effects of granulosa-lutein cell co-cultureon human embryo development and pregnancy rates for in-vitrofertilization (IVF). In the study, sibling two-pronuclear zygoteswere randomly allocated to culture with (co-culture) or without(control) autologous granulosalutein cells. After 24 h, embryoswere examined for blastomere number and degree of fragmentation.Co-culture had no effect on the average number of blastomeresper embryo at 24 h; however, fragmentation was significantlydecreased in co-cultured embryos (0.7 ± 0.1) comparedwith controls (1.3 ± 0.2; P < 0.05). In the subsequentclinical trial, all two-pronuclear zygotes were co-culturedfor 48 h prior to embryo transfer. The live birth rate per embryotransfer was 43.4% with an implantation rate per embryo of 17.6%.Of the untransferred embryos, 68% developed to the blastocyststage and were cryopreserved. We conclude that the simple systemof autologous granulosa-lutein cell co-culture improves embryodevelopment, implantation and subsequent pregnancy rates forIVF.     

Human preimplantation embryo cryopreservation: selected aspects

This report describes the results of cryopreserving human preimplantation zygotes and cleaved embryos (2-4 cells) in our in-vitro fertilization programme. Cryopreserved zygotes and cleaved embryos resulted in similar post-thaw survival rates (74.8 versus 70.9%). Pregnancy rates per retrieval cycle (RC) and embryos transferred per pregnancy for frozen-thawed zygotes versus frozen-thawed cleaved embryos were 21.8 versus 11.5% (P less than 0.2) and 12.6 versus 17.5 (P less than 0.2), respectively. Pregnancy rates increased significantly for both fresh (P less than 0.0005) and frozen-thawed (P less than 0.05) embryos as the number of embryos replaced per transfer increased from one to three or more. Frozen-thawed embryos resulted in multiple implantation rates per transfer of 25 compared to 6.4% (P less than 0.1) for fresh embryos when two embryos were replaced. Pregnancy rates were reduced for fresh (P less than 0.05) and frozen-thawed (P less than 0.1) embryos obtained from patient retrieval cycle numbers greater than 3. The method of follicular stimulation during the retrieval cycle did not affect frozen-thawed embryo survival rates. There was no difference in pregnancy rates from frozen-thawed embryos replaced during natural or clomiphene citrate transfer cycles. Patients with cryopreserved embryos had cumulative pregnancy rates of 37.1% (66/178) compared to 23.5% (110/468) (P less than 0.01) for patients with no embryos cryopreserved; cryopreservation of preimplantation embryos is a reliable therapeutic procedure that enhances achievement of pregnancy through in-vitro fertilization.     

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).