Implantation: Two instead of three embryo transfer in in-vitro fertilization

The rational of transferring two instead of three embryos wasstudied through 468 in-vitro fertilization (IVF) treatment cyclesin 287 couples. The quality of 1224 embryos was determined accordingto the fragmentation rate and the morphology as good (A) andpoor (B). The influence of the number of embryos transferred(two or three) on the pregnancy rate when the same quality orcombinations of good and poor quality embryos transferred wasexamined. When only good quality embryos were transferred thepregnancy rates in double (AA) and triple (AAA) embryo transferwere 40.5 (17/42) and 42.9% (30/70) respectively (not significant).When only poor quality embryos were transferred, the pregnancyrates in double (BB) and triple (BBB) embryo transfers were11.0% (11/ 100) and 22.9% (16/70) respectively (P < 0.001).On the other hand, when good and poor quality embryos were transferredtogether as AB in double and as AAB and ABB in triple embryotransfer, the pregnancy rates were 36.8 (14/38) and 39.9% (59/148)respectively (not significant). There was no difference in themiscarriage rate between double and triple embryo transfers;16.7 and 18.1% respectively. The multiple pregnancy rate was14.3% for double embryo transfers and 32.4% for triple embryotransfers (P < 0.001). This study demonstrates that if thereis at least one good quality embryo available for transfer,then double instead of triple embryo transfer will not yielda significantly lower pregnancy rate. The influence of the numberof embryos transferred on the pregnancy rate became significantwhen only poor quality embryos were transferred. In conclusion,as long as at least one good quality embryo is available fortransfer, we may consider the transfer of double instead oftriple embryos.     

The use of intra-endometrial embryo transfer for increasing the pregnancy rate

It has been demonstrated previously that pregnancy can be achievedby the direct insertion of embryos into the endometrial stroma(intra-endometrial embryo transfer) of mice. In this study weevaluated whether intra-endometrial transfer resulted in a higherpregnancy rate than conventional embryo transfer. Mouse blastocysts(ICR strain), recovered on day 4 of pregnancy, were transferredinto pseudopregnant day 2, day 3 and day 4 mice of the samestrain; 1-, 2- and 8-cell embryos were also transferred intopseudopregnant day 4 mice. In intra-endometrial embryo transfer,a 27 gauge injection needle was inserted near the utero-tubaljunction into the endometrial stroma and then removed; one blastocystwas transferred into each uterine horn with a glass micropipette.Conventional transfers were performed simultaneously as controls.The pregnancy rates and embryonic viability rates were evaluated9 days after embryo transfer. Furthermore, the rates of livebirth for intra-endometrial and conventional embryo transferswere compared when blastocysts were transferred into pseudopregnantday 4 uteri by both methods. In the transfer to pseudopregnantday 2 recipients, the pregnancy and embryonic viability rateswere significantly higher (P < 0.01) in intra-endometrial[23.4 (11/47) versus 15.9% (15/94)] than in conventional embryotransfer [4.3 (2/46) versus 2.2% (2/92)]. In the transfer topseudopregnant day 3 recipients, both rates were also higher(P < 0.01) in intra-endometrial [90.9 (40/44) versus 87.5%(77/88)] than in conventional transfer [67.4 (31/46) versus64.1% (59/ 92)]. In synchronous transfer to pseudopregnant day4 recipients, there was no difference between methods in thepregnancy rate [conventional, 48.9% (24/49); intraendometrial,50.9% (29/57)] and the embryonic viability rate [conventional,44.9% (44/98); intra-endometrial, 43.0% (49/114)]. In the transferof 1-, 2- and 8-cell embryos into pseudopregnant day 4 mice,pregnancy and embryonic viability rates were very low in bothtransfer methods. Intra-endometrial transfer produced normalliving offspring at a similar rate to conventional transfer.These results reveal that intra-endometrial transfer increasespregnancy and embryonic viability rates in asynchronous embryotransfer in mice, especially when the duration of pseudopregnancyin the recipients was less than the age of the transferred embryos.     

Infertility: Assessments of embryo transfer after in-vitro fertilization: effects of glyceryl trinitrate

The role of embryo transfer and its associated difficultieson the outcome of human in-vitro fertilization (IVF) were examinedusing a standardized procedure and a scoring system (embryotransfer scores 1–5). This system was used to assess anyeffects of the smooth muscle relaxant glyceryl trinitrate (GTN)on embryo transfer. Patients (n = 120) were randomized in adouble-blind manner at their first embryo transfer to receivesublingual GTN or placebo before the transfer. Retrospectiveanalysis showed that higher pregnancy rates were associatedwith uncomplicated transfers (score 1; P < 0.01). The outcomemeasures included pregnancy rate, total time of cervical manipulation(embryo transfer time) and embryo transfer score. All pregnancieshad a transfer score of 1 or 2, but no recorded parameter differentiatedbetween pregnant or non-pregnant cycles, and GTN had no significanteffect on any parameter.     

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.     

A comparative analysis of embryos derived from routine in-vitro fertilization and subzonal microinsemination

Embryos obtained from patients undergoing routine in-vitro fertilization(IVF) and embryo transfer were compared with those undergoingsubzonal microinsemination (SUZI) for male factor infertility.Overall, the proportion of cleaved embryos was significantlyhigher in the IVF group in comparison with the SUZI group at48 h post-insemination [1533 out of 1609 (95.3%) versus 776out of 952 (81.5%)]. The mean ± SD grading score of theIVF-derived embryos of 3.61 ± 0.50 was significantlybetter than that for SUZI of 2.97 ± 0.86 (P < 0.0005)at the same time. The implantation rates following the replacementof IVF or SUZI embryos at 48 h were comparable: 14.3 and 10.0%respectively. However, the IVF embryo implantation rate of 15.1%at 72 h was significantly better than that following the replacementof SUZI embryos at either 48 (10.0%) or 72 h (8.0%). The replacementof SUZI-derived embryos at 48 h resulted in significantly higherpregnancy (25.0%) and implantation rates (10.0%) than at 72h, with rates of 10.8 and 8.0% respectively. Similarly, theoverall embryo quality deteriorated following in-vitro culturefor up to 72 h. The clinical pregnancy loss rate (33.0%) washighest following the replacement of SUZI embryos at 72 h, althoughthe data were limited. It is suggested that these data indicatethat a combination of in-vitro manipulation, the injection ofmultiple spermatozoa into the subzonal space and probably thegenomic capacity of spermatozoa derived from poor-quality semenmay contribute to the poorer outcome of embryo development followingSUZI. Prolonged in-vitro culture beyond 48 h appears to be deleteriousto the development of SUZI cleaved embryos and the subsequentoutcome of treatment, and hence should be avoided.     

Fertilization and early embryology: The applicability of the cumulative embryo score system for embryo selection and quality control in an in-vitro fertilization/embryo transfer programme

The cumulative embryo score system involves three aspects ofrelevance in pregnancy achievement during in-vitro fertilization(IVF) and embryo transfer: cleavage rates, morphological qualitiesand the number of embryos transferred. The scores of 602 IVF/embryotransfer trials were calculated and analysed to determine thesystem's relationship to pregnancy rate, pregnancy outcome andthe incidence of twin and triplet pregnancies. The system wasalso applied to cycles where endotoxins were either presentin or absent from culture medium, in order to evaluate its validityin quality control analyses. Pregnancy rates were found to increasefrom 4%, with scores between 1 and 10, to 35% in the 41–50group. The score of 20 was the criterion for separating patientsinto poor and good pregnancy prognosis groups (P = 0.00001).Biochemical abortions occurred more frequently with scores <20(P = 0.00978), but a similar relationship was not found in clinicalabortion rates (P = 0.62206). Birth rates below and above ascore of 20 (2.8 and 19.2%, respectively) differed significantly(P = 0.0005). The scores of twins overlapped extensively withthose of singleton births, but those of all triplets were >40. The system did not reflect a correlation between embryoquality and the presence of endotoxins in culture medium.     

Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF

BACKGROUND: A biochemical marker for embryo development wouldincrease the chance of a successful pregnancy with IVF by optimizingoocyte and embryo selection, and allow fewer embryos to be transferred.In this study, we correlated cumulus granulosa cell gene expressionof hyaluronic acid synthase 2 (HAS2), cyclooxygenase 2 (COX2;PTGS2) and gremlin (GREM1) with subsequent embryo developmentin search of a parameter for embryo selection. METHODS: Cumuluscell gene expression was determined prospectively on eight consecutivepatients undergoing IVF with ICSI. Immediately following oocyteretrieval, the cumulus was stripped from the oocyte, and cumulusgene expression for PTGS2, HAS2 and GREM1 was assessed usinga one-step real-time quantitative RT–PCR assay. Oocytequality, fertilization and embryo morphology were correlatedto relative gene expression. RESULTS: Gene expression data wereavailable on cumulus cells from 108 oocytes that developed into70 embryos (64.8% fertilization rate). Cumulus PTGS2, HAS2 andGREM1 expression was higher from oocytes that developed intohigher quality embryos (grades 3, 4 and 5) compared with lowerquality embryos (grades 1 and 2) (P<0.05, P<0.001 andP<0.001, respectively). HAS2 and GREM1 expression was alsohigher from the cumulus surrounding oocytes that gave rise tohigher grade embryos (P<0.001). The expression of PTGS2 andHAS2 was 6-fold higher, and that of GREM1 was 15-fold higherin cumulus yielding higher grade embryos versus lower gradeembryos. CONCLUSION: PTGS2, HAS2 and GREM1 gene expression correlatesto morphological and physiological characteristics and providesa novel approach to predict human embryo development. Ultimately,with better predictors of follicular and embryonic health, higherquality embryos can be selected and transferred, reducing higherorder pregnancy rates.     

Fertilization and early embryology: Timing of development is a critical parameter for predicting successful embryogenesis

Development of embryos from the 1-cell stage into blastocystsin vitro is generally slower than the time-course for developmentin vivo. It was the objective of this work to determine whetherembryos that reach the 8-cell stage within a normal time-framehave a developmental advantage (both in vitro and post-embryotransfer) over slower embryos. Hamster 1-cell embryos were collected10 h post-egg activation (PEA) and cultured for 48 h (58 h PEA= t⁵⁰ for 8-cell embryo development in vivo) in hamster embryoculture medium-6. Embryos were sorted according to stage reached,culture was continued in fresh medium and stage of developmentwas observed at 78, 82 and 86 h PEA. At 58 h PEA, embryos were<4-cell (4%), 4-cell (19%), 5- to 7-cell (16%) or 8-cell(61%). The 58 h 8-cell embryos had a significantly greater abilityto develop to the blastocyst stage than 58 h 4-cell embryosat 78, 82 and 86 h PEA (74 versus 13%, 69 versus 25% and 65versus 37% respectively). The percentages of 14-day-old fetusescollected after embryo transfer indicated that morulae and blastocystsderived from 58 h 4-cell embryos were, on average, less viable(26% fetuses) than morulae and blastocysts from 58 h 8-cellembryos (51% fetuses). Thus morulae and blastocysts developingin vitro from faster or slower cleaving embryos can be qualitativelyas well as quantitatively different. These data indicate thatthe timing of development in vitro, specifically the timingof completion of the third cell cycle, is a critically importantparameter for predicting successful embryogenesis in the hamster.     

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.     

Endocrinology: High incidence of embryo transfer cancellations in patients with polycystic ovarian syndrome

This study was aimed at assessing the outcome of in-vitro fertilization(IVF) and embryo transfer in patients with polycystic ovariansyndrome (PCOS). The results of IVF and embryo transfer in PCOSpatients (PCOS group, 78 cycles of 26 patients) were comparedwith those of a control group (423 cycles in 202 patients withoutmale factor; age and ovarian stimulation protocol were matched).Although the pregnancy rate per transfer was not different inthe two groups of patients (25 versus 34%, PCOS versus controlgroup), the PCOS group had a significantly lower pregnancy rateper follicle aspiration (19 versus 31%, P < 0.05). A notableresult was a significantly higher incidence of embryo transfercancellations in the PCOS group (22 versus 8%, P < 0.01),which resulted from unpredictable failure of either oocyte recoveryor fertilization. The incidence of unexplained complete failureof fertilization was significantly higher in the PCOS group(18 versus 5%, P < 0.01). These results may reflect a reducedquality of the oocytes in the PCOS group, and there was a subgroupof PCOS patients who repeatedly produced poor results of treatment.Although the ovarian stimulation regimen best suited to PCOSpatients remains to be determined, special care should be takenduring ovarian stimulation, especially when the PCOS patientshad experienced unexplained failure of oocyte recovery or fertilizationin the previous treatment cycle(s).     

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).     

Fertilization and early embryology: Prolonged sperm-oocyte exposure and high sperm concentration affect human embryo viability and pregnancy rate

A reduced time interval of oocyte exposure to spermatozoa wasinvestigated to assess whether it could enhance oocyte developmentand improve embryo viability, especially in cases of male factorinfertility. A total of 167 patients were included in a prospectiverandomized study. They were randomly allocated to two majorstudy groups, A (n = 85) and B (control group; n = 82). Theoocytes from group A patients were exposed to spermatozoa foronly 1 h; those from group B were exposed for 16 h. The twostudy groups were then subdivided according to semen qualityfor further analysis of the results. Significantly higher percentageswere obtained in group A than in group B in terms of the fertilizationrate (74 versus 68%, P < 0.025), cleavage rate (53 versus41%, P < 0.005), pregnancy rate (27 versus 12%, P < 0.05)and implantation rate (11 versus 6%, P < 0.05). In addition,an increased fertilization rate was achieved in oocytes exposedto male factor spermatozoa for only 1 h compared with the conventionalincubation period (78 versus 65%, P < 0.01). Advanced cellularstages (55 versus 41%, P < 0.02) and higher implantationrates (13 versus 4%, P < 0.05) were attained in the subgroupwhose oocytes were exposed to normal spermatozoa for 1 h comparedwith the male factor spermatozoa with the standard culture interval.The higher fertilization rates, enhanced embryo developmentand viability achieved in group A indicate that prolonged exposureof oocytes to high concentrations of spermatozoa is detrimental,decreasing sperm-oocyte interaction and subsequent embryo implantation,particularly in male factor patients.     

Delaying transfer to the third day post-insemination, to select non-arrested embryos, increases development to the fetal heart stage

The purpose of this study was to determine whether delayingembryo transfer by 24 h, until day 3 post-insemination, allowedimproved selection of non-arrested embryos for transfer. Wehave retrospectively analysed pregnancy rates in a large seriesof patients who had embryo transfer either on day 2 or on day3 post-insemination over a 27 month period. From January 1990to March 1992, 567 patients received embryo transfer on day2, and 661 patients had transfer on day 3 post-insemination,but these transfers were not contemporary. Pregnancy rates wereslightly higher in patients who had embryo transfer on day 3(37%) than in those patients who had their embryos transferredon day 2 (35%), but this difference was not significant. Theimplantation rate, as measured by the proportion of embryosdeveloping to the fetal heart stage, was significantly higherfollowing transfer on day 3 (23%) than after transfer on day2 (19%) (P < 0.05), suggesting that selection of viable embryosis improved on day 3. Furthermore, of the embryos which gaverise to a fetal sac, significantly fewer miscarried before thefetal heart stage (P < 0.05) following transfer on day 3(6%) than after transfer on day 2 (12%). Delaying transfer untilday 3 provides a further 24 h to observe embryo development.During this period 16% of embryos arrested or became developmentallyretarded; thus waiting until day 3 allowed these embryos tobe identified and avoided for consideration for transfer. Embryotransfer may be safely delayed until day 3, and this may helpin selecting embryos most likely to implant and develop aftertransfer.     

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.      

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.     

Elective single embryo transfer: the value of cryopreservation

In-vitro fertilization is associated with a high rate of multiple pregnancies, a consequence of the number of embryos transferred. There is a challenge in avoiding even twin pregnancies in assisted reproduction, and this can be accomplished with elective single embryo transfer and a good cryopreservation programme. In our follow-up study, we analysed all our elective single embryo transfers during 1998-1999. In all these cycles at least one embryo was frozen. A total of 127 elective single embryo transfers were performed with a clinical pregnancy rate of 38.6%. The highest implantation rate was obtained with four-cell embryos with <10% fragmentation (39.8%). Thirty-four patients have delivered (26.8%), one of these being a monozygotic pregnancy. In total 129 frozen-thawed cycles have been achieved in 83 patients. One frozen-thawed embryo has been transferred in 46 cycles with a clinical pregnancy rate of 17.4%, and two embryos have been transferred in 83 cycles, with a clinical pregnancy rate of 37.3%. Up until now, 66 of 125 patients in our single embryo transfer programme have delivered or have on-going pregnancies, and 77 still have embryos frozen. The cumulative delivery rate per oocyte retrieval is 52.8% and the twin rate 7.6%. We conclude that elective single embryo transfer with a good cryopreservation programme results in very acceptable pregnancy rates with a low risk of twins. This is a cost-effective practice that substantially reduces all risks associated with multiple pregnancies and lowers the cost per delivery.     

Embryonic morphology and rate of implantation of human embryos following co-culture on bovine oviductal epithelial cells

A study was undertaken to evaluate embryonic development andestablish pregnancies with human embryos after in-vitro culturein two different systems. Treatment A consisted of culturingzygotes in serum-supplemented human tubal fluid culture medium(HTF). Treatment B consisted of culturing zygotes on a monolayerof bovine oviductal epithelial cells with HTF. At the time ofembryo replacement, embryos in treatment B had 4.11 blastomerespresent, which was greater (P < 0.05) than the 3.81 presentfor embryos in treatment A. In addition, the cellular fragmentationrate for treatment A embryos was 1.10, which was greater (P< 0.05) than the fragmentation rate of 0.38 for embryos withintreatment B. The incidence of ongoing pregnancy was higher afterreplacement of co-cultured embryos (treatment B) (43%) thanreplacement of conventionally cultured embryos (treatment A)(29%). The implantation rate per embryo increased (P < 0.05)from 11.5 to 18.4% after co-culture. In treatment B the proportionof ‘spare’ embryos developing to expanded blastocystswas 58.5%, which was greater (P < 0.05) than the blastocystdevelopment rate of 29.3% observed for embryos within treatmentA.     

Fertilization and early embryolgoy: A comparison of the effects of different biopsy strategies on the post-thaw survival of 8-cell-stage mouse embryos: implications for preimplantation diagnosis

The aim of this study was to investigate the effects of twodifferent biopsy strategies, zona slitting and zona piercing,on the post-thaw survival and subsequent in-vitro developmentof 8-cell mouse embryos. From control experiments it was determinedthat neither biopsy by zona slitting nor zona piercing adverselyaffected embryo development in vitro, as similar rates of blastocystformation and hatching were found between biopsied and zona-intactembryos; there was, however, a trend towards a lower rate ofblastocyst hatching in embryos biopsied by zona piercing (78.3%compared with 91.9% of zona-intact embryos). When biopsy wasfollowed by cryopreservation a different picture was seen: similarrates of freeze—thaw survival were found for zona-slit,zona-pierced and zona-intact embryos (84.2, 88.5 and 87.2% respectively),but this was superseded by a significant (P < 0.05) reductionin the blastocyst formation rates (61.4% zona-slit and 63.9%zona-pierced versus 78.7% zona-intact) and hatching rates (51%zona-slit and 52.5% zona-pierced versus 72.3% zona-intact) ofthe biopsied embryos. When the effects of zona slitting andpiercing were considered in isolation, i.e. without performingbiopsy, it was found that the larger holes produced by zonaslitting rendered embryos more susceptible to freeze-thaw damage.Significant (P < 0.05) reductions occurred in the blastocystformation rates (63.7% zona-slit versus 78.7% zona-intact embryos)and hatching rates (52.3% zona-slit versus 72.3% zona-intactembryos) of the zona-slit embryos.     

Comparison of a fixed and dynamic protocol for embryo replacement in an IVF/ET programme

Implantation after embryo transfer is considered a major obstadein terms of pregnancy rates after in-vitro fertilization. Aflexible approach to the date of replacement, based on the factthat the most suitable embryonic structure for proper implantationis the four- to eight-cell embryo, has been studied. One-hundred-and-twentypatients with various aetiologies of infertility were stimulatedwith HMG or combined HMG and FSH, then treated by three differentmethods of embryo replacement. In group I embryos were replacedin mothers 48 h after ovum retrieval; in group II replacementswere carried out 72 h after retrieval; and in group III replacementswere related to embryonic cleavage development. Mean levelsof oestradiol when HCG was given averaged 1301 ± 121pg/ml, 1016 ± 96 pg/ml and 1182 ± 101 pg/ml inthe three groups, respectively. There was no significant differencein the average number of embryos transferred among the variousgroups. The pregnancy rates per transfer were 21.8, 24.2 and38.7%, respectively (P < 0.001). Although more investigationis required, a dynamic approach to embryo replacement mightsignificantly improve pregnancy rates, because of improved interactionsbetween the embryos and the uterus.