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.     

Embryo transfer under ultrasound guidance improves pregnancy rates after in-vitro fertilization

Between October 1998 and January 1999, we examined the influence of ultrasound guidance in embryo transfer on pregnancy rate in 362 patients from our in-vitro fertilization (IVF)-embryo transfer programme. These patients were prospectively randomized into two groups: 182 had ultrasound-guided embryo replacement, and 180 had clinical touch embryo transfer. There were no statistically significant differences between the two groups with respect to age, cause of infertility and in the characteristics of the IVF cycle. The pregnancy rate was significantly higher among the ultrasound-guided embryo transfer group (50%) compared with the clinical touch group (33.7%) (P < 0.002). Furthermore, there was also a significant increase in the implantation rate: 25.3% in the ultrasound group compared with 18.1% in the clinical touch group (P < 0.05). In conclusion, ultrasound assistance in embryo transfer significantly improved pregnancy and implantation rates in IVF.     

Ploidy in human cleavage stage embryos after fertilization in vitro

Chromosome preparations were made from 15 cleaved human embryosin the 2- to 12-cell stage after in-vitro fertilization. Allshowed two pronuclei before the first cleavage. Twelve had atleast one diploid metaphase, while three had interphase nucleionly. There was no evidence of partheno-genetic haploid cleavage.As expected, the frequency of metaphases increased with durationof colchicine treatment: 25 and 55% of the cells reached metaphaseafter 6–13 and 16–24 hours' treatment, respectively.Cleaved embryos with ‘ideal’ blastomere numbers(2, 4 or 8) showed a considerably higher metaphase frequencythan others. For a more detailed chromosome analysis the techniquewill have to be further improved. The asynchronous cleavageof blastomeres makes optimal treatment by mitogens difficult.     

Cost-effectiveness analysis of in-vitro fertilization: estimated costs per successful pregnancy after transfer of one or two embryos

Standard protocols for in-vitro fertilization (IVF) include transfer of two or three embryos. Not surprisingly, the rate of twin pregnancy after IVF is high (about 24% of all pregnancies). Routine transfer of one, rather than two, embryos would be expected to result in a much lower rate of twin pregnancies at the cost of a lower take-home baby rate. The aim of this study was to compare hypothetical costs to society incurred by pregnancies achieved with IVF protocols based on the transfer of one or two embryos. We compared actual (for two-embryo transfers) and hypothetical (for one-embryo transfers) take-home baby rates; risks of twin pregnancies; and costs of sick leave and hospitalization during pregnancy, deliveries, neonatal intensive care, and handicap care after transfer of one or two embryos. The study showed that even when more treatments might be needed to achieve similar baby take-home rates after transfer of one compared with two embryos, the lower twin pregnancy rate of the former approach caused it to be more cost-efficient than the latter. In conclusion, IVF costs are the sum of fertilization treatment costs and the costs for health care of the pregnant women and their offspring. Considering the association of the latter costs with numbers of embryos transferred, studies of one- embryo transfer protocols are urgently needed.      

The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in-vitro fertilization and embryo transfer programme.

In order to achieve a clinical pregnancy rate higher than that achieved following initial adoption of in-vitro fertilization embryo transfers, more than one embryo is transferred. This has led to a substantial increase in unwanted multiple pregnancy rates with IVF as compared with natural conception. What is therefore required is a simple, clinically useful embryo scoring system, to reflect embryo developmental potential, which will enable the selection of the optimal number of embryos to transfer in order to achieve the maximum pregnancy rate with a low incidence of high order multiple pregnancies. We believe that the Cumulative Embryo Score (CES) achieves these aims. On the day of embryo transfer the grade of each embryo transferred was multiplied by the number of blastomeres to produce a score for each embryo, and summation of the scores obtained for all the embryos transferred gave the CES. The grouped pregnancy rates obtained rose as the CES increased to maximum of 42. A continued increase in the CES above 42 did not result in any further rise in the pregnancy rate. However, an analysis of all our IVF pregnancies showed that the multiple pregnancy rate continued to rise above a CES of 42. By restricting the CES per embryo transfer to 42, 78% of triplet pregnancies and 100% of the quadruplet IVF pregnancies could have been predicted and potentially avoided.     

Early pregnancy wastage following late implantation of embryos after in-vitro fertilization and embryo transfer

Serial plasma concentrations of human chorionic gonadotrophin (HCG), progesterone and oestradiol were measured in pregnancies after in-vitro fertilization and embryo transfer. The first detection day of HCG after embryo transfer (8.4 +/- 1.1) and the HCG doubling time (DT) of 64 normal singleton pregnancies were compared to those of 14 first-trimester miscarriages. The same parameters were evaluated in nine late-implanted conceptions, seven of which resulted in early pregnancy wastage. The HCG DT of late-implanted pregnancies was consistent with that of singleton term pregnancies in the first 12 days, while first-trimester miscarriages which had implanted at the usual time had a significantly longer DT from implantation onwards. The reduced trophoblastic secretory rate suggests poor embryo quality in these cases. A decreased progesterone/oestradiol ratio was observed in late-implanted pregnancies but because of the small number of individuals, no definite conclusion can be drawn. More patients with delayed implantation should be tested to justify this observation.     

Pregnancy: Heterotopic pregnancies after in-vitro fertilization and embryo transfer

A total of 20 cases of heterotopic pregnancy were encounteredamong 2650 clinical pregnancies (0.75%) resulting from in-vitrofertilization/embryo transfer at Bourn Hall Clinic (Cambridge,UK) during the period July 1984-July 1993. The aetiology ofheterotopic pregnancy in the series is multifactorial, withtubal damage as the main factor. Transvaginal ultrasonographyshowed a high sensitivity for making correct diagnoses of heterotopicpregnancies compared with transabdominal ultrasonography (93.3versus 50.0%). The mean plasma human chorionic gonadotrophin(HCG) concentration on day 13 after embryo transfer was similarto those of uncomplicated intrauterine pregnancies and hencewas of no diagnostic value. The serial plasma HCG concentrationsof patients who delivered were significantly higher than forthose who aborted their intrauterine pregnancies (P < 0.01),although the sample of data available was too small to makefirm inferences. It does appear that serial HCG concentrationsmay have a predictive value of fair accuracy regarding the outcomeof the intrauterine pregnancy in heterotopic pregnancies. Theclinical presentations of the 20 cases at first examinationwere quite variable, with 45% (9/20) of patients asymptomatic.Tubal pregnancy in one patient resolved spontaneously, two caseswere treated by an injection of potassium chloride into thegestational sac and the remaining 17 cases were treated by salpingectomy.In 10 patients the intrauterine pregnancy resulted in live birthand the remaining 10 patients aborted spontaneously.     

Delayed embryo transfer in an in-vitro fertilization programme: how to avoid working on Sunday.

It has previously been demonstrated that use of gonadotrophin releasing hormone analogues allows the administration of HCG to be delayed and makes it possible to avoid oocyte retrievals at weekends. In this study, we demonstrate that it is also possible to avoid embryo transfers on Sundays, by delaying transfers of Friday retrievals until Monday, without any apparent change in the results. This facility should improve the cost-effectiveness of assisted reproduction programmes.     

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.     

Uterine rupture in first or second trimester of pregnancy after in-vitro fertilization and embryo transfer

We report five cases of early rupture of cornual pregnancy withhistory of previous salpingectomy and cornual resection followingin-vitro fertilization (IVF) and embryo transfer. We discussthe predisposing factors, diagnostic and therapeutic modalitiesin these patients. A high index of suspicion is required foran early diagnosis. It is imperative that the physicians whocare for the patients be fully aware of the possibility of sucha complication in a high risk population; therefore, appropriatecounselling and close follow-up might help to avoid such obstetricalcatastrophes, by termination of pregnancy, either surgicallyor medically.     

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).     

Reply: Pregnancy outcome after blastocyst transfer as compared to early cleavage stage embryo transfer

Sir, We appreciate the interest of van der Ven and Montag in ourstudy and their useful comments. The primary aim of our retrospective cohort study was neonataloutcome. Hence, the purpose     

Early cleavage of in-vitro fertilized human embryos to the 2-cell stage: a novel indicator of embryo quality and viability

A number of non-invasive methods have been proposed to evaluate embryo viability in human in-vitro fertilization programmes. In addition to biochemical analyses, a common method for the selection of embryos prior to transfer involves assessment of embryo quality and morphology. We propose a new method to evaluate embryo viability based on the timing of the first cell division. Fertilized embryos that had cleaved to the 2-cell stage 25 h post-insemination were designated as 'early cleavage' embryos while the others that had not yet reached the 2-cell stage were designated as 'no early cleavage'. In all cases the early cleavage embryos were transferred when available. Early cleavage was observed in 27 (18.9%) of the 143 cycles assessed. There were significantly (chi2 = 4.0; P = 0.04) more clinical pregnancies in the early cleavage group, 9/27 (33.3%), compared with the no early cleavage group, 17/116 (14.7%). No difference was found when comparing key parameters (age, stimulation protocol and semen characteristics) of couples belonging to both groups, pointing to an intrinsic property or factor(s) within the early cleaving embryos. We propose 'early cleavage' as a simple and effective non-invasive method for selection and evaluation of embryos prior to transfer.      

Pregnancy outcome after blastocyst transfer as compared to early cleavage stage embryo transfer

BACKGROUND: Retrospective cohort study to evaluate differences in outcome when embryo transfer was performed either on day 2-3 (cleavage stage, CS-group) or on day 4-5 (blastocyst stage, BS-group). METHODS: A total of 1259 consecutive cycles yielding 500 live born babies performed at a single centre in Bregenz, Austria, were included. Main outcome measures were implantation and (multiple) pregnancy rates and neonatal outcome including birth defects. RESULTS: Total Pregnancy rate was 44% vs 28% (P < 0.001) and the total 'take home baby rate' was 37% vs 22% in the BS-group and the CS-group, respectively. Rate of multiple gestations (34% vs 17%, P = 0.001) was significantly higher among the BS-group, resulting in a higher rate of preterm deliveries < 36 weeks (26% vs 17%, P = 0.045). Female factor causing infertility (40% vs 21%, P < 0.001) was significantly higher among the BS-group. For the CS-group, rate of singleton pregnancies (83% vs 66%, P = 0.001) and idiopathic cause of infertility (34% vs 22%, P = 0.012) were significantly higher. No statistically significant differences were found in sex, Caesarean section rate, Apgar score and umbilical artery pH-values, total mean birth weight, admission rate to intensive care unit, days of hospitalization and number of minor and major birth defects. CONCLUSIONS: Our data suggest that blastocyst transfer may lead to a higher pregnancy rate with an overall better take-home baby rate (THBR) at the cost of higher rates of multiples and preterm deliveries.     

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.     

Pregnancy: The prediction of ectopic pregnancy after in-vitro fertilization and embryo transfer

Data from 135 patients who suffered ectopic pregnancies andfrom 135 patients who progressed to singleton deliveries afterin-vitro fertilization and embryo transfer have been analysedretrospectively. The ectopic pregnancies represent all suchcases observed at Bourn Hall Clinic between 1983 and 1993. Thedelivered group was randomly selected from the same time period.The ectopic pregnancies included 20 heterotopic, eight ovarianand six bilateral tubal pregnancies; the remainder were singletontubal pregnancies. The aim of this study was to identify thevariables which differed systematically for the two groups ofpatients and to explore whether such variables could be usedto predict ectopic pregnancy at an early stage. The mean plasmaconcentration of human chorionic gonadotrophin and progesteronefor the ectopic pregnancy group was significantly lower thanthat for the singleton delivery group (P < 0.001). However,there was such a degree of overlap that it was impossible todevise a cut-off concentration for either hormone which wouldoffer a clinically useful predictor of ectopic pregnancy. Nevertheless,using the discriminant function analysis of these data, togetherwith the history of pelvic inflammatory disease, we could predictup to 90% of cases of ectopic pregnancy by day 23 after embryotransfer, long before ultrasound imaging would be useful.     

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.     

Cleavage stage of human embryos two days after fertilization in vitro and their developmental ability after transfer into the uterus

In 126 selected patients treated with clomiphene and human menopausalgonadotrophin, 154 embryos were transferred into the uterus41–50 h after in-vitro insemination. Fifty per cent ofembryos implanted. The rate of cleavage was inversely relatedto the success of embryo transfer; implantation failures occurredfor 69 and 46% of embryos with more than or less than four cells,respectively {p <0.05). Cleavage rate was not related tothe viscosity of the cumulus cell mass, serum supplementationof culture medium, nor to the age or genetic factors of thewoman. There was a trend towards smaller follicles, and a significant(p <0.05) tendency for patients developing numerous folliclesto yield faster-cleaving embryos. These relationships were notfound in 50 other embryos obtained from patients treated withpure FSH.