A single medium supports development of bovine embryos throughout maturation, fertilization and culture

Oocytes and embryos are typically exposed sequentially to varying culture media in standard in-vitro protocols. Expenditures of energy may be required following each medium change to adjust to the changing environment. Therefore, a single base medium was evaluated for its ability to support in-vitro maturation, fertilization and pre-implantation development (IVM/F/C) of bovine oocytes and embryos. Four treatments were examined: a standard maturation [tissue culture medium (TCM) 199 with bovine calf serum (BCS)], fertilization (modified Tyrode's medium with albumin, lactate and pyruvate) and culture (hamster embryo culture medium/TCM with BCS) system (control) and three synthetic oviductal fluid (SOF) treatments; maturation in SOF with bovine serum albumin (SOFBSA), SOF with bovine calf serum (SOFBCS) or the control maturation medium (TCM199 with BCS; SOF199), followed by fertilization and culture in SOF medium. The percentage of total inseminated oocytes successfully developing to the morula and blastocyst stage did not differ (P > 0. 05) between treatments (control, 30.5 +/- 3.5; SOFBSA, 24.6 +/- 3.2; SOFBCS, 22.4 +/- 4.7; SOF199, 27.3 +/- 3.2). Embryos cultured in SOFBCS (92.1 +/- 6.4) had significantly higher cell numbers (P < 0. 05) than those cultured in control (74.8 +/- 4.8) and SOFBSA (71.6 +/- 6.6) but not SOF199 (81.2 +/- 6.8). In conclusion, a single medium can be used successfully throughout maturation, fertilization and pre-implantation embryo development. Moreover, inclusion of serum during maturation in the single medium system resulted in significantly greater cell numbers, possibly reflecting increased quality of the embryos produced.     

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.     

Fertilization and early embryology: Development of in-vitro-fertilized primate embryos into blastocysts in a chemically defined, protein-free culture medium

The formulation of chemically defined culture media that supportprimate embryo development would facilitate studies on primatepreimplantation embryogenesis. The specific aims of this studywere (I) to evaluate the development of macaque embryos in asimple, chemically defined, protein-free medium developed fora rodent embryo model, and (ii) to determine if a two-step progressiveculture system could enhance blastocyst development and zonaescape. In experiment 1, in-vitro-fertilized pronucleate stageembryos (n=81) from nine monkeys were randomly allocated toone of three treatments: (a) hamster embryo culture medium-6(HECM-6; chemically defined, protein- free medium), (b) CMRL-BCSmedium (modified CMRL 106 medium containing 20% bovine callserum; BCS), and (c) a two-step culture procedure (HECM-6 throughto the 8- to 12-cell stage, and CMRL-BCS medium beyond thatstage). Optimal development was attained equally (P0.05) withembryos cultured in CMRL-BCS medium or the two-step procedure(48 and 61% blastocysts respect ively). HECM-6 alone supporteddevelopment to the morula stage (72%) equally as well as CMRL-BCSmedium (80%) or the two-step procedure (69%), but not to theblastocyst stage (22 versus 48 and 61% respectively). Hatchingof the blastocysts was essentially limited to the serum-containingmedia (CMRL-BCS medium, 31% two-step procedure, 44%). In experiment2, in-vitro-fertilized pronucleate-stage embryos (n=87) fromnine monkeys were randomly placed in each of four two-step treatments:(a) HECM-6 through to the 8- to 12-cell stage and CMIRL-BCSmedium beyond that stage, (b) HIECM-6 through to the 8- to 12-cell stage and HECM-6-BCS beyond that stage, (c) HIECM 6 throughto the morula stage and CMIRL-BCS medium beyond that stage,and (d) HECM-6 through to the morula stage and HECM-6-BCS beyondthat stage. Greater (P 0.05) percentages of embryos developedinto blastocysts, expanded blastocysts and hatched blastocystswhen switched at the 8- to 12-cell versus the morula stage inthe second step medium. When transferred into BCS containingmedium at either the 8- to 12-cell or morula stage, embryosunderwent blastulation and expansion equally well in CMIRL-BCSmedium versus HECM-6-BCS. However, when embryos were switchedto the second step medium at the 8- to 12-cell stage, hatchedblastocysts 1690 were obtained more (P0.05) frequently in CMRL-BCSmedium (50.9%) than in HECM-6-BCS (37%). This work is the firstto produce in-vitro-fertilized primate blastocysts culturedfrom the pronucleate stage in chemically defined, protein-freemedium, and demonstrates that while primate embryos can formmorulae in such a medium, their requireents for blastocoel formationand zona escape appear to be more demanding, and may be acquiredas early as the 8-cell stag.     

Development of embryos from natural cycle in-vitro fertilization: impact of medium type and female infertility factors

Single embryos derived from natural cycle in-vitro fertilization(IVF) were graded during the pre-transfer culture period usingmorphological criteria. Most embryos developed well in culturewith 96% showing continuing division and 68% showing good morphologicalappearance, although embryo quality tended to decline with anincreased incidence of fragmentation and uneven cleavage asdivision proceeded. Both the pregnancy rate and the distributionof embryo grades were similar among four different culture mediaused, suggesting that choice of medium had little impact onoutcome. In contrast, there were marked differences in pregnancyrate according to the type of infertility, which was not reflectedin a decrease in embryo quality. However, although embryos frompatients with tubal infertility implanted and formed viablepregnancies irrespective of morphological appearance, only ‘good’quality embryos from patients with non-tubal (or ‘unexplained’)infertility were able to implant. Thus the appearance of theembryo derived from natural cycle IVF in women with unexplainedinfertility may be of clinical relevance.     

Decisions for the fate of frozen embryos: fresh insights into patients' thinking and their rationales for donating or discarding embryos

BACKGROUND: In the final decision for the disposition of unused IVF embryos patients must choose between options involving either donation or destruction, and this decision must be made in a context where there is tension about the status of embryos (i.e. whether viewed as potential children or as a base for further development) and whether embryo donation is adoption or tissue donation. This study explored the emotive experience of making a decision for either the destruction or donation of unused embryos. METHODS: Thirty-three patients (9 women and 12 couples) who discarded embryos and 15 (7 women and 4 couples) who donated embryos were interviewed. Interview data were analysed with particular attention to elements of moral deliberation and use of analogy. RESULTS: Adoption and tissue donation metaphors were both identified, and further, a metaphor of pregnancy termination was identified and found to be highly influential in the decision to donate embryos. Contrary to the majority of current evidence, this study found that participants who discarded embryos emphasized the adoption metaphor while embryo donors emphasized the metaphor of pregnancy termination. For each group the decision was driven by awareness of the option they did not want. CONCLUSIONS: The pregnancy termination metaphor emerged as morally relevant and this holds implications for defining and discussing embryo discard in counselling and consent processes.     

Investigation of respiration of individual bovine embryos produced in vivo and in vitro and correlation with viability following transfer

BACKGROUND: Quantification of oxygen consumption by individual preimplantation embryos has the potential to improve embryo selection. This study investigated whether respiration rates of individual embryos are useful indicators of embryo viability. The effect of the Nanorespirometer on embryo viability was also evaluated. METHODS: The respiration rates of individual day 7 bovine in vivo- (n=44) and in vitro-produced (n=156) embryos were measured using the Nanorespirometer. In vivo-produced embryos were individually transferred to recipients. RESULTS: The respiration rates of in vivo-produced embryos increased with increasing morphological quality and stage of development (P < 0.05). Pregnancy rates on days 35 and 60 were 65 and 60%, respectively. The mean respiration rate did not differ significantly between embryos producing and not producing a pregnancy, but the transfer of embryos with respiration rates <0.78 nl/h, between 0.78 and 1.10 nl/h, and >1.10 nl/h resulted in 48, 100 and 25% pregnancy rate, respectively. The mean respiration rate of in vitro-produced embryos was higher than that of in vivo-produced embryos because of differences in the morphological quality and stage of development. CONCLUSION: The Nanorespirometer does not adversely influence embryo viability, but the sample size was too small to confirm the significance of the correlation observed between respiration rates and viability.     

Comparison between day-2 embryos obtained either from ICSI or resulting from short insemination IVF: influence of maternal age

Short incubation time prevents deleterious effects of cumulus cell degeneration and excess spermatozoa in IVF embryos. We performed a short incubation (3 h) protocol in 328 IVF cycles, in order to compare the developmental potential of regular IVF embryos with those originating from 316 cycles entered our intracytoplasmic sperm injection (ICSI) programme over the same period. Embryo transfers were performed in all patients on day 2. The mean number of embryos transferred was 1.92 for the ICSI group and 1.73 for the IVF group (P < 0.007). This was related only to the wishes of patients. However, the policy of the centre is to transfer a low number of embryos in young patients in order to avoid multiple pregnancies. All spare embryos were permitted to grow to the blastocyst stage for freezing. Shortening incubation time did not decrease fertilization rates. In our overall population, no difference was observed in the implantation rates per embryo for IVF (19%) or for ICSI (20%). An age-related decrease in embryo production was observed for both groups of patients (P < 0.01 for ICSI and P < 0.001 for IVF). The age-related decrease in embryo implantation was only significant for the IVF group (P < 0.03 for patients <30 and >35 years of age). A significant overall decrease in blastocyst formation was observed for spare embryos after ICSI versus IVF (34.2 versus 43.8%; P < 0. 05). The significance of this observation is discussed.     

A prospective randomized study comparing the outcome of in-vitro fertilization and embryo transfer following culture of human embryos individually or in groups before embryo transfer on day 2.

A prospective randomized trial of in-vitro fertilization and embryo transfer was undertaken to investigate the reported beneficial effects of culturing preimplantation human embryos in groups, rather than individually. A total of 159 treatment cycles, in which the women were matched for age, basal gonadotrophin concentrations and number of previous attempts, were included in the study. Of these, 78 cycles were randomized to the 'individual culture' group, and 81 cycles were randomized to the 'group culture' group. The groups did not differ in terms of the median number of oocytes or embryos obtained per cycle. There was no statistically significant difference between the two groups in terms of treatment outcome, as assessed by pregnancies or clinical pregnancies.     

Cryopreservation of human embryos obtained after gamete intra-Fallopian transfer and/or in-vitro fertilization

During a one-year period 636 excess embryos obtained after in-vitrofertilization and gamete intra-Fallopian transfer combined within-vitro fertilization were cryopreserved using two differentprotocols. For early stage embryos including the pronucleatestage, 1,2-propanediol was used as cryoprotectant (procedureA, adapted from Renard) and for later stage embryos dimethylsulphoxidewas used in protocol B, adapted from Trounson and Mohr. Afterthawing 288 embryos, half of them were of sufficient qualityto be replaced. After cryopreservation, procedure A gave thebest survival in embryos having 2 blastomeres; for later stageembryos best survival was obtained using the dimethylsulphoxideprotocol. Survival after cryopreservation was also clearly relatedto the quality of the embryos prior to freezing. Embryos werereplaced during endocrinologically monitored natural cyclesand were transferred in synchrony between endornetrial and embryonicage. After replacement of 126 embryos in 110 patients, 20 pregnanciesoccurred. So far six healthy children have been born, two patientsaborted and 12 pregnancies are ongoing. In this series no statisticaldifference was observed between the implantation rate of embryoscryopreserved by procedure A or B. Six pregnancies occurredin patients from the oocyte and embryo donation programme. Anadequate cryopreservation programme circumvents the difficultproblem of synchronizing the ovarian cycles of donor and acceptorpatients.     

Leukaemia inhibitory factor significantly enhances the blastocyst formation rates of human embryos cultured in serum-free medium

The aim of this study was to investigate the effect of leukaemiainhibitory factor (LIF) on human blastocyst formation ratesin vitro. To do this, it was first necessary to devise a complexserum-free medium (CSFM) in which to test its activity. Blastocystformation rates in CSFM microdrops (18.4%) showed no differenceto those obtained previously for embryos cultured in 1 ml T6medium containing 10% serum (25%). The majority of blastocystswere of optimal blastocyst grade (BG3). The percentage of BG1/BG2blastocysts was decreased in CSFM microdrops (10.2%) comparedto that observed in T6 medium (24.8%). Addition of 1000 IU/mlLIF to CSFM microdrops increased the blastocyst formation ratefrom 18.4 to 43.6% (P < 0.025) and increased the percentageof BG1/BG2 blastocysts (33%; P 0.025) to levels comparablewith those observed in T6 medium. Thus LIF significantly increasedthe quality and number of human blastocysts formed in CSFM medium,increasing the potential for blastocyst transfer in human in-vitrofertilization (IVF)     

In-vitro development and implantation of human embryos following culture on fetal bovine uterine fibroblast cells

Human zygotes resulting from IVF were placed in two differentculture systems to evaluate in-vitro development and to establishpregnancies in patients following embryo replacement. TreatmentA (control) consisted of culturing zygotes in a modified Earle'sBalanced Salt solution while treatment B consisted of culturingzygotes on a monolayer of fetal bovine uterine fibroblasts inthis same culture medium. At the time of embryo replacement,embryos within treatments A and B had 3.7 and 4.3 blastomerespresent, respectively. After 24 h in culture, the cellular fragmentationrate for treatment A embryos was 0.85 which was greater (P <0.05)than the fragmentation rate of 0.44) for embryos within treatmentB. The incidence of implantation for patients whose embryoswere given treatment A was 17.0% which was lower (P <0.05)than 35% for those given treatment B. Implantation rates increasedwith time in culture (43%) for treatment B embryos. Cultureby treatment B of three pronucleate zygotes resulted in 7/9and 4/9 reachIng the blastocyst and expanded blastocyst stages,respectively, whereas only 1/26 three-pronucleate zygotes culturedusing treatment A reached either of these stages.     

Fertilization and early embryology: Improved cleavage rate of human embryos cultured in antibiotic-free medium

Retarded development and blastomere fragmentation of human prelimplantationembryos represent a common phenomenon in in-vitro culture systems.Even though media composition is generally formulated to meetembryo nutritional requirements, the influence of antibioticsupplementation has not been investigated thoroughly. The presentstudy was performed to evaluate the effects of antibiotics onembryo morphology and growth in modified culture media. A totalof 196 zygotes from 18 couples was cultured in three differentmedia: (i) conventional medium (n = 99, control group); (ii)medium modified with half the standard antibiotic concentration(n = 54); and (iii) antibiotic-free medium (n = 43); 49 embryosfrom the control group were selected at the zygote stage andtransferred to the patients on day 2. The remaining 147 zygoteswere cultured to the blastocyst stage for cryopreservatlon;their morphology and cell number were assessed daily at 40,64, 88, and 112 h post-insemination. Overall cleavage rate was95% and embryo scoring revealed 91% grade 1 embryos throughoutthe culture period in the three media. Significantly highercleavage rates were obtained in the antibiotic-free medium ateach observation, including the blastocyst stage, when comparedto the other two groups. In addition, no notable improvementwas observed in the embryos cultured in a reduced concentrationof antibiotics. In conclusion, antibiotic supplementation ofmedia has an adverse effect on the growth rate of preimplantationembryos, even in reduced concentrations, suggesting that antimicrobialdrugs may interfere with the timing of cleavage events eitherby delaying or blocking embryo development.     

Hydrosalpinx fluid does not adversely affect the normal development of human embryos and implantation in vitro

Several retrospectively designed studies have shown an associationbetween the presence of hydrosalpinx and impaired implantationand pregnancy rates among in-vitro fertilization (IVF) patients.In the present study we have evaluated the influence of hydrosalpinxfluid on normal human embryo development and implantation. Surplus,donated frozen embryos (n = 183) from IVF patients were usedto study the effects on blastocyst development of hydrosalpinxfluid at concentrations of 50 and 100% compared with controlsin S2 medium. The fluids were analysed for concentrations ofelectrolytes, osmolarity, protein content, endotoxin levels,bacterial or fungal contamination, pH and haemoglobin content.There was no difference in blastocyst development in culturesunder mineral oil when control cultures (15/42 = 36%) were comparedwith cultures in 50% hydrosalpinx fluid (32/96 = 33%). The onlybiochemical parameter which correlated with capacity for blastocystdevelopment was pH in hydrosalpinx fluid/medium (50/50%) afterequilibration in 5% CO₂ in air. When embryos were cultured in100% hydrosalpinx fluid the blastocyst development was 14% (5/36)in comparison to control 33% (3/9). The original experimentwas repeated in an open culture system without the protectionof mineral oil but still in the presence of 50% hydrosalpinxfluid. The rate of blastocyst development was within the samerange in the open system. In three separate experiments, thecapability of expanded blastocyst to implant on multilayer artificialendometrium was tested. In these experiments, 1/3, 4/5 and 9/9blastocysts implanted. The present study demonstrates that hydrosalpinxfluid does not generally exert any major negative effects onin-vitro development of human embryos or on the implantationprocess in vitro.     

Maternal serum hCG and alpha-fetoprotein levels in pregnancies conceived after IVF or ICSI with fresh and frozen-thawed embryos

BACKGROUND: Studies have shown that levels of serum markers of Down's syndrome were altered in pregnancies conceived after IVF, though the reason for this remains unknown. METHODS: Second-trimester maternal serum levels of hCG and alpha-fetoprotein (AFP) in pregnancies conceived with fresh and frozen-thawed embryos after assisted reproduction were compared with those conceived spontaneously. RESULTS: There were 203 pregnancies with fresh embryo transfers (130 IVF cases, 73 ICSI cases) and 98 pregnancies with frozen-thawed embryo transfers (61 IVF cases, 37 ICSI cases). The controls consisted of 17 145 spontaneous pregnancies. The median hCG multiples of the median (MoM) was significantly increased to 1.24 in 98 pregnancies conceived after frozen embryo transfer. This elevation was observed only in the IVF-frozen embryo transfer subgroup (P < 0.001), but not in the ICSI-frozen embryo transfer subgroup. The median AFP MoM for 203 pregnancies after fresh embryo transfer was 0.90. Among the subgroups, the median AFP MoM was significantly reduced to 0.90 and 0.86 in IVF-embryo transfer (P = 0.04) and ICSI-embryo transfer (P = 0.001) pregnancies respectively, and significantly raised to 1.20 in the IVF-frozen embryo transfer subgroup. CONCLUSIONS: The degree of alterations in maternal serum hCG and AFP levels varied between fresh and frozen-thawed embryos, and also between the mode of fertilization. Pregnancies resulting from ICSI or frozen embryo transfer should be regarded as distinct entities from those of IVF-embryo transfer.     

Microbial contamination of embryo cultures in an ART laboratory: sources and management

BACKGROUND: Although rare, microbial contamination of culture dishes occasionally occurs in our IVF/ICSI programme. Despite stringent culture conditions and the use of medium containing penicillin and streptomycin, an increasing number of infections was observed once they were routinely recorded. In this study, 95 cases of contaminated culture dishes were examined, in an attempt to identify possible causes. METHODS: Relevant data of the IVF/ICSI treatment cycles and the micro-organisms isolated from the infected culture dishes were evaluated retrospectively. RESULTS: Infections were observed only in IVF culture dishes and never after applying intra-cytoplasmic sperm injection. Identification of the contaminating micro-organisms showed that infections were mainly caused by Escherichia coli (n = 56; 58.9%) and Candida species (n = 24; 25.3%). Of the E. coli strains isolated, 41 (73.2%) appeared to be resistant to both antibiotics used in the culture medium and 13 (23.2%) appeared to resist either penicillin or streptomycin. Of all bacterial strains isolated, the resistances were 61.4% to both and 30% to one of the antibiotics used. CONCLUSIONS: Applying the ICSI procedure prevents colonization of the culture dishes by micro-organisms. Infections in IVF culture dishes are mainly caused by bacterial strains insensitive to the antibiotics used or due to yeast colonization by Candida species which frequently reside in the vagina.     

Polyvinyl alcohol and amino acids as substitutes for bovine serum albumin in culture media for mouse preimplantation embryos

The effect of replacing bovine serum albumin (BSA) in a simpledefined medium (KSOM) with polyvinyl alcohol (PVA) and/or aminoacids on the percentages of mouse zygotes that develop to atleast the blastocyst stage and that hatch at least partiallyor completely is reported. Blastocysts could form when BSA wasreplaced with only PVA, but at a moderately reduced rate; however,partial hatching, and hence complete hatching, were severelyimpaired when BSA was replaced with only PVA. The substitutionof BSA with amino acids alone resulted in a high rate of blastocystformation and moderate impairment of hatching. The additionof PVA to BSA-free DSOM supplemented with amino acids had noextra effect. BSA had significant effects when added to BSA-freeKSOM supplemented with amino acids. The BSA caused a significantincrease in the rate of partial hatching, and may even havehad a small effect on the rate of blastocyst formation. Theresults also showed that glucose, at a high concentration of5.56 mM, does not inhibit the development of mouse zygotes tohatched blastocysts when cultured in KSOM supplemented withamino acids.     

Evaluation of variations in pregnancy rates, implantation rates and early abortion rates within an in-vitro fertilization programme

Despite the application at this clinic of a standardized programmefor in-vitro fertilization of human oocytes over the last 27months, great variations in the rates of implantation, clinicalpregnancy and early abortion have been observed during certainperiods. A retrospective evaluation of these results showedthat these variations occurred in periods when various commerciallyavailable batches of Earle‘s medium (the medium was theonly variable changed during the 27 months) were used and thattwo sub-optimal batches of Earle’s medium from one ofthe sources used during one of three periods (period 2) wasmost likely to be responsible for sub-optimal embryo qualityand, consequently, for a halving of the pregnancy rate (30 versus15%) and of the implantation rate (11 versus 5%) and an increasein the early abortion rate (23 versus 50%), It is concludedthat the quality of the culture medium is of major importancefor the success of an IVF programme. The factor(s) in the mediumresponsible for the decrease in embryo quality has not beenidentified.     

Reducing the number of embryos transferred in Sweden-impact on delivery and multiple birth rates

BACKGROUND: Reduction of the number of embryos transferred has been introduced to decrease the multiple birth rates (MBRs) after IVF and the associated risks for the children. The aim of this report is to present the effect of two steps in reduction of the number of embryos transferred, when applied in the majority of the patients, on national data for delivery and MBR after IVF in Sweden. METHODS: This observational study is based on annual reports from all IVF clinics in Sweden to the National Board of Health and Welfare for the time period 1991-2004. RESULTS: The main finding is that despite a successive reduction in the number of embryos transferred, delivery rates were maintained at around 26% while MBR decreased dramatically, from about 35% to around 5%. The same pattern was noticed, independent of age, for all women below 40. In comparison with the USA, lower delivery and MBR were noted for Sweden whereas a higher 'birth per embryo transferred' was found. CONCLUSIONS: Single embryo transfer (SET) results in satisfactory delivery rates and a dramatic decrease in the MBRs, also when applied on a broad scale. The experience from Sweden ought to encourage other countries to introduce SET more widely.     

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.