Human embryo cryopreservation, extrinsic and intrinsic parameters of success

Freezing and thawing (F-T) was applied to 490 early human embryosusing propanediol as cryoprotectant. The survival rate of embryosfrozen with propanediol alone did not exceed 31% (26/83). Thecombination of propanediol and sucrose, however, significantlyincreased the percentage of surviving (248/407 = 61%) and intact(188/407 = 46%) embryos and seemed to enhance embryo viabilityas suggested by the implantation rate (14.5 versus 8%) without,however, any statistical significance. Embryo survival, butnot viability, was correlated with morphological features, whereasneither the age of embryos (1, 2 or 3 days post-insemination)nor the segmentation stage (regular or intermediate) were involvedin F—T ability. Thirty-eight F—T embryos implantedwhen replaced in uterro, representing 8% of all F—T embryosand 14% of the F—T replaced embryos. The pregnancy rateper transfer reached 19% (35/185) and was identical to the pregnancyrate per transfer of fresh embryos (253/1149 = 22%). In oocytedonation, too, embryo freezing did not impair the pregnancyrate (25%). In spontaneous cycles, synchronous transfer gavebetter results than asynchronous transfers (20 versus 10%),but spontaneous cycles had no significant advantage (16% pregnaocy/transfer)as compared to stimulated (26%) and artificial (27%) cycles.     

Fertilization and early embryology: Embryo quality and pregnancy potential of fresh compared with frozen embryos--is freezing detrimental to high quality embryos?

To determine the effect of cryopreservation on embryo qualityand the pregnancy potential of embryos, donated oocytes fromthe same donor (n = 24) were randomly allocated, with subsequenttransfer to two or more different ovum recipients resultingin at least one fresh and one frozen embryo transfer cycle fromthe same cohort of oocytes. Endometrial receptivity was controlledin all ovum recipients, and male factor patients were excluded.The number of embryos transferred, mean embryo grade transferred,number of high quality embryos (grade 2.5, grade 1 being best)transferred and embryo implantation and live birth rates arereported. Significantly more embryos (4.4 ± 1.2 versus3.3 ± 1.2, P < 0.00003) of higher quality (1.9 ±0.5 versus 2.1 ± 0.5, P < 0.013) and of a more advancedcell stage (3.0 ± 0.6 versus 2.6 ± 0.7, P <0.019) were transferred fresh than after cryopreservation respectively.Implantation rates/embryo [19/151 (12.6%) and 9/111 (8.1%)]and live birth rates/transfer [11/42 (26.2%) and 6/45 (13.3%)],from fresh and frozen transfers respectively, were not significantlydifferent despite the larger number of high quality embryostransferred fresh. Embryo cryopreservation adversely affectsembryo quality, but does not have detrimental effects on theimplantation or pregnancy potential of high quality embryos.Because of the loss of embryos during freeze — thawingduring frozen embryo cycles, every effort should be made toattempt a fresh transfer.     

Fertilization and early embryology: Preliminary experience with human oocyte cryopreservation using 1,2-propanediol and sucrose

Feasibility of cryopreservalion of mature human oocytes using1,2-propanediol and sucrose was studied initially utilizrng1 and 2 day old unfertilized oocytes. Of these 285 aged oocytes55% survived thawing, and 41% of 128 oocytes inseminated bysingle sperm intracytoplasmlc injection (ICSI) fertilized normally.Limited embryonic development occurred in 51% of these embryos(n = 27) observed for the next 4 days. Cryosurvlval of freshdonated oocytes (n = 81) was poorer (n = 20; 24.7%), while fertilization(n = 13; 65%) and embryo development (100%) was good prior uterinetransfer on day 3. Eight oocyte recipient cycles were undertaken,in which cryopreserved donated oocytes were thawed and inseminatedby ICSI. Five of these cycles reached embryo transfer, and threepregnancies were initiated though none went successfully toterm. Oocyte cryopreservatlon will ultimately facilitate oocytedonation procedures; however, cryosurvival of fresh frozen oocytesmust be improved to at least the degree observed with aged unfertilizedoocytes.     

Comparison of clinical outcome after cryopreservation of embryos obtained from intracytoplasmic sperm injection and in-vitro fertilization

The impact of intracytoplasmic sperm injection (ICSI) on cryopreservedzygotes and embryos was evaluated by comparing embryo survivaland implantation between embryos derived from ICSI and thosederived from standard insemination procedures. The study includedpatients whose excess zygotes and embryos were cryopreservedbetween September 1993 and December 1994 and who subsequentlyunderwent a frozen embryo transfer. Embryo survival, clinicalpregnancy rates per transfer and pregnancy outcome were compared.Three hundred and thirty eight cryopreservation cycles, duringwhich 1471 embryos were cryopreserved, were included in thisstudy. Of those, 961 were derived from oocytes fertilized byinsemination in vitro and 510 were derived from oocytes fertilizedby ICSI. A total of 690 of the embryos (451 in the inseminationgroup and 239 in the ICSI group) have since undergone a thawcycle. The embryo survival rates were similar between the twogroups (70.5 and 73.2%, insemination and ICSI respectively)and were not significantly affected by the stage at cryopreservation.There was no significant difference in pregnancy rates per transfer(31.8 and 32.3%), the preclinical pregnancy loss rate (16.7and 23.8%), or the clinical miscarriage rate (16.7 and 23.8%)between the insemination and the ICSI groups respectively. Itis concluded that ICSI does not have an adverse impact on thesurvival and successful implantation of cryopreserved and thawedembryos.     

Transfers of frozen-thawed human embryos in cycles stimulated by HMG

A total of 130 transfers of frozen-thawed (F-T) human embryos was carried out after moderate ovarian stimulation with human menopausal gonadotrophin (HMG). Embryos were replaced 3 days after the spontaneous luteinizing hormone (LH) surge or 4 days if ovulation was induced by human chorionic gonadotrophin (HCG). Embryos were thawed a few hours prior to transfer. One-hundred-and-twenty-three transfers were effective and 23 pregnancies were achieved. The rate of ongoing pregnancies per transfer was 17.9% (22/123). The survival rate of embryos originating from cycles stimulated by a combination of an LHRH analogue and HMG in a long protocol (LA-HMG protocol) was significantly lower when compared with the rate of embryos retrieved from clomiphene citrate-HMG (CC-HMG protocol) stimulated cycles (52 versus 67%, P less than 0.05). When fresh embryos originated from cycles stimulated with an LHRH analogue and HMG in a short protocol (SA-HMG protocol), the survival rate was not affected (59 versus 67%, NS). Although the difference was not significant, the ongoing pregnancy rate per transfer according to the three protocols from which the embryos originated seemed to be better with the SA-HMG protocol: 16% with the CC-HMG protocol, 14.5% with the LA-HMG protocol versus 27.6% with the SA-HMG protocol. The success rate was independent of the number of F-T transferred embryos if at least one embryo with 100% intact blastomeres was replaced.     

High serum oestradiol concentrations in fresh IVF cycles do not impair implantation and pregnancy rates in subsequent frozen-thawed embryo transfer cycles

High oestradiol concentrations may be detrimental to the success of in-vitro fertilization (IVF) treatment. A total of 1122 women aged <40 years who were undergoing their first IVF cycle were evaluated retrospectively. Serum oestradiol concentrations on the day of human chorionic gonadotrophin (HCG) administration were categorized into three groups: group A <10 000 pmol/l; group B 10 000-20 000 pmol/l and group C >20 000 pmol/l. In fresh cycles, group A had significantly lower pregnancy rates per transfer (16.2 versus 23.7% respectively, P = 0.005, chi(2)) and implantation rates (8.7 versus 11.7% respectively, P = 0.037, chi(2)), when compared with group B. The pregnancy rate per transfer in group C was significantly lower than that in group B (12.1 versus 23.7%, P = 0.049, chi(2)) and group C had the lowest implantation rate (6.4%). In frozen-thawed embryo transfer cycles, implantation rates in groups A, B and C were similar (7.5, 8.1 and 9.6% respectively) and the pregnancy rates were also comparable in all groups. In conclusion, high serum oestradiol concentrations in fresh IVF cycles may adversely affect implantation and pregnancy rates. Embryo quality seemed unaffected as excess embryos from different groups had similar implantation and pregnancy rates in frozen-thawed embryo transfer cycles. The reduced implantation was probably due to an adverse endometrial environment resulting from high serum oestradiol concentrations.     

Clinical experience with ultrarapid cryopreservation of human embryos resulting from intracytoplasmic sperm injection: brief communication.

A total of 41 patients requested thawing of supernumerary embryos in an intracytoplasmic sperm injection (ICSI) programme. Mean patient age was 30.8 +/- 3.8 years. Embryo freezing by the ultrarapid method was performed at room temperature in 3 mol/l DMSO and 0.25 mol/l sucrose. Total freezing time was 2.5 min including filling of the straw. In the thawing process, the embryos were removed from liquid N(2), left at room temperature for 30 s, immersed for 40 s at 30 degrees C, and then successively transferred at room temperature for 10 min to each of three sucrose solutions of decreasing concentration. The embryos were kept in culture and only those that presented cleavage after 24 h were transferred. Embryos from 42 cycles were thawed and a total of 24 transfers was performed. The mean number of thawed embryos was 5.0 +/- 3.2 per cycle and the mean number of transferred embryos was 2.83 +/- 1.3. The clinical pregnancy rate per cycle obtained after the thawing process was 16. 6%. The clinical pregnancy rate per transfer was 29.2% and the implantation rate was 13.2%. The abortion rate was 14.3%. Six deliveries have been performed, with the birth of seven infants.     

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.     

Implantation: Embryo score to predict implantation after in-vitro fertilization: based on 957 single embryo transfers

The purpose of this study was to devise an embryo score to predictthe likelihood of successful implantation after in-vitro fertilization(IVF). Unlike most studies dealing with the influence of embryostage and morphology on pregnancy, our study was based on singlerather than multiple embryo transfers. A total of 957 singleembryo transfers were carried out. No delivery was obtainedafter any of the 99 transfers using 1-cell embryos or embryosobtained after delayed fertilization. In the remaining 858 transfers,the embryos had cleaved. Higher pregnancy rates were obtainedwith embryos displaying no irregular cells (11.7 versus 6.9%;P < 0.01) and embryos displaying no fragmentation (11.5 versus8.1%; P < 0.05). The 4-cell embryos implanted 2-fold moreoften than embryos with more or less cells (15.6 versus 7.4%;P < 0.01). Based on these observations, we devised a 4-pointembryo score in which embryos are assigned 1 point each if they(i) are cleaved, (ii) present no fragmentation, (iii) displayno irregularities, and (iv) have four cells. Both pregnancyrate and take home baby rate were significantly correlated withembryo score. Each point of this score corresponds to a 4% increasein pregnancy rate. Interestingly, pregnancy rate was significantlylower in women aged >38 years (8.2 versus 11.4%; P < 0.05),even though embryo quality was similar regardless of age. Singleembryo transfer allowed us to define a simple and useful embryoscore to choose the best embryo for transfer to optimize IVFand embryo transfer outcome. The use of this embryo score coulddecrease multiple pregnancies after multiple embryo transfers.     

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.     

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.     

Fertilization and early embryology: Granulosa cells improve human embryo development in vitro

A total of 17 couples with repetitive implantation failure aftertransfer of fresh or frozen—thawed embryos had half oftheir zygotes cultured in standard conditions and frozen atday 2 after insemination, and the other half cocultured withautologous granulosa cells and transferred at the morula orblastocyst stage at day 5 or 6 after oocyte retrieval. At theend of the culture period, supernatants of cocultures were recoveredfor steroid assays. Monolayers were stained for granulosa cellgrowth and morphological assessment. We observed that granulosacells improve embryo development in vitro since 32 out of 60(53%) reached the morula stage and 18 (30%) the blastocyst stage,leading to a total of 83% embryos available for transfer (comparedwith 3% without coculture). The ongoing pregnancy rate of thesepatients who were selected because they had at least three previousimplantation failures, is only 5.9%, however, which is similarto the control group without coculture (6.3%). To conclude,granulosa cells improve embryo development but not the pregnancyrate after transfer of cocultured embryos in patients with multipleprevious implantation failures.     

Preimplantation adoption: establishing pregnancy using donated oocytes and spermatozoa

The experience of transferring embryos produced through in-vitrofertilization (IVF) utilizing donated oocytes and spermatozoais described. Recipients (n = 28; aged 38–59 years) receivedoral micronized oestradiol and i.m. progesterone and were synchronizedto donors undergoing ovarian stimulation. Reasons for selectingtherapy included advanced reproductive age (>42 years; n= 21) or hyper-gonadotrophic hypogonadism (n = 7), combinedwith severe male factor infertility in 23 couples. Five womenwere single and without partners. Oocytes were fertilized bycryopreserved spermatozoa designated for use by the recipient.Up to five embryos were transferred trans-cervically. Supernumeraryembryos were cryopreserved. A total of 36 aspirations produced15.6 ± 7.3 oocytes per retrieval. In 10/36 cycles (27.8%),embryos were available for cryopreservation. Using fresh embryos,the overall pregnancy rate was 38.9% (14/36), clinical pregnancyrate 33.3% (12/36), and ongoing/delivered pregnancy rate 30.6%(11/36). Three ongoing pregnancies were later established bytransferring cryopreserved embryos. Adjusting for these events,the per aspiration overall pregnancy rate per retrieval was47.2%, clinical pregnancy rate 41.7%, and ongoing/deliveredpregnancy rate 38.9%. Implantation rates per individual embryotransferred were 16.6% following fresh embryo transfer. A viablepregnancy was achieved by 14 of 28 women (50% cumulative pregnancyrate). We conclude that using donor oocytes and donor spermatozoais efficacious and allows couples of whom both members sufferfrom severe gamete abnormalities and single functionally agonadalwomen an effective means of achieving pregnancy.     

The effect of intercourse on pregnancy rates during assisted human reproduction

Intercourse during an IVF cycle has the potential to improve pregnancy rates since exposure to semen is reported to promote embryo development and implantation in animals. Conversely, coitus-induced uterine contractions or introduction of infection may have a detrimental effect. A multicentre prospective randomized control trial was conducted to determine if intercourse during the peri-transfer period of an IVF cycle has any influence on pregnancy success. Participants undergoing thawed embryo transfer (Australian centre) or fresh embryo transfers (Spanish centres) were randomized either to abstain or to engage in vaginal intercourse around the time of embryo transfer. The transfer of 1343 embryos during 478 cycles of IVF resulted in 107 pregnancies (22.4%), with 125 viable embryos remaining by 6-8 weeks gestation. There was no significant difference between the intercourse and abstain groups in relation to the pregnancy rate (23.6 and 21.2% respectively), but the proportion of transferred embryos that were viable at 6-8 weeks was significantly higher in women exposed to semen compared to those who abstained (11.01 versus 7.69 viable embryos per 100 transferred embryos, P = 0.036, odds ratio 1.48, 95% confidence interval 1.01-2.19). Hence exposure to semen around the time of embryo transfer increases the likelihood of successful early embryo implantation and development.     

Comparative results on survival of human and animal eggs using different cryoprotectants and freeze-thawing regimens. II. Human

A total of 269 human pronuclear embryos and 84 oocytes weresubjected to four different protocols of cryoprotectant equilibration,washing out and freeze-thawing. The morphological survival,rate of development, fertilization in vitro and overall survivalrate were estimated in the groups of fresh, aged oocytes, diploidand multipronuclear embryos used. With some restrictions, theconclusion can be drawn that slow, low intermediary temperature1, 2-propanediol (1, 2-PROH) and 1, 2-PROH/dimethyl sulphoxide(DMSO) systems are superior to the rapid, high intermediarytemperature 1, 2-PROH and the traditionally used DMSO systems.The best success rates were reached with the combination ofcryoprotectants in the low intermediary temperature group. Thirty-threepatients had 37 transfers of cryopreserved pronuclear embryos(n= 103), resulting in eight pregnancies (24.2% per transfer)thus doubling the pregnancy rate in the stimulation cycles forthe same period (21% per transfer). Thawing at a rate of 50?C/minis not incompatible with the survival of slowly frozen humanoocytes and pronuclear embryos cooled to an intermediary temperatureof –70?C using the DMSO system.     

Day 2 elective single embryo transfer in clinical practice: better outcome in ICSI cycles

BACKGROUND: Single embryo transfer (SET) after IVF/ICSI has been shown to result in an acceptable pregnancy rate in selected subjects. In our unit, SET is routinely carried out among women under the age of 36 in the first or second treatment cycle when a top-quality embryo is available. In order to define further the selection criteria for SET, we have analysed the outcome of elective SET (eSET), including the cumulative pregnancy rate after frozen embryo transfers, performed in the years 2000-2002 in the Oulu Fertility Center. METHODS: During the study period, a total of 1271 transfers were performed, and in 468 cycles SET (39% of all transfers) was carried out. Of the SET cycles, in 308 cases a top-quality embryo was transferred on day 2 and extra embryos were frozen. Of these eSET cycles, ICSI was carried out in 87 cycles (28%). RESULTS: The overall clinical pregnancy rate per transfer was 34.7% in the eSET cycles. In the eSET ICSI cycles, the clinical pregnancy rate was significantly higher than in the corresponding IVF cycles (50.6 versus 28.5%, P < 0.001). The cumulative pregnancy rate per patient after fresh and frozen embryo transfers was also significantly higher after ICSI (71.2 versus 53.4%, P < 0.01). CONCLUSIONS: A high cumulative pregnancy rate per oocyte retrieval can be achieved after eSET in daily clinical practice. The implantation rate of fresh top-quality embryos in the ICSI cycles was significantly higher than in the IVF cycles, possibly due to more successful selection of the embryo for embryo transfer on day 2 after ICSI. In addition, our data suggest that embryo quality is a more important determinant of outcome than the age of the woman.     

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.     

A randomized clinical trial comparing recombinant hyaluronan/recombinant albumin versus human tubal fluid for cleavage stage embryo transfer in patients with multiple IVF-embryo transfer failure

BACKGROUND: We aimed to examine the efficacy of using an embryo transfer medium enriched with hyaluronan (HA) to improve implantation in a selected group of patients aged <43 years with repeated (>4) implantation failures after IVF-embryo transfer. METHODS: About 101 patients, meeting our selection criteria, were randomly allocated to undergo embryo transfer either using our routine embryo transfer medium without HA (control group) or a HA enriched commercial embryo transfer medium (study group). The primary outcome was clinical pregnancy rate. RESULTS: After a similar treatment protocol, the ovarian hormonal response, the mean number of ova retrieved and injected per patient, fertilization and cleavage rates and mean embryo quality were comparable between the study and control groups. Although a similar number of embryos was transferred in both groups (3.1 +/- 0.7 versus 2.9 +/- 0.6, mean +/- SD), a significantly higher implantation rate (16.3% versus 4.8%, P = 0.002) and clinical pregnancy rate (35.2% versus 10.0%, P = 0.004) and delivered or ongoing pregnancy rate (31.3% versus 4.0%, P = 0.0005) were observed in the study group. When mean implantation rate per patient was calculated, the difference between the study (0.148 +/- 0.23) and control (0.04 +/- 0.13) group was significant (P = 0.003). CONCLUSIONS: In this selected group of patients after multiple IVF-embryo transfer failures, the use of HA enriched embryo transfer medium is beneficial.     

Fertilization and early embryology: Cryopreservation of immature mouse oocytes

Mouse oocytes enclosed in cumulus cells were isolated from antralfollicles at the germinal vesicle (GV) stage. They were storedin straws at – 196°C by a conventional mouse embryofreezing method using dimethylsulphoxide (1.5 M) as the cryoprotectant.Overall survival assessed after removal of the cumulus cellswas 93% (299/320). A significantly greater proportion of freshoocytes remained arrested at the GV stage during culture (11versus 1%), but the rate of maturation to metaphase II was notsignificantly different between frozen and fresh oocytes (83versus 74%). The rate of fertilization in vitro was similarfor frozen and fresh oocytes matured in vitro (70 versus 81%)but significantly less than with mature ovulated oocytes (96%).Fertilization of frozen and fresh oocytes arrested after germinalvesicle breakdown was similar (77 versus 95%. No evidence ofparthenogenetic activation was found in the different groupsafter overnight incubation of metaphase II oocytes. Implantationwas similar for embryos derived from fresh and frozen GV-stageoocytes matured in vitro and mature ovulated oocytes, but theloss of embryos after implantation was significantly higherin the in-vitro matured groups (frozen, 40% and fresh, 46% versus24%). The overall survival of oocytes frozen at the GV stagewas 27%. This compares favourably with the estimated overallsurvival of mature oocytes cryopreserved by a similar procedure.We conclude that the increased post-implantation loss is dueto suboptimal conditions for maturation in vitro rather thanfreezing injury.     

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).