Identification of viable embryos in IVF by non-invasive measurement of amino acid turnover

BACKGROUND: IVF is limited by low success rates and an unacceptably high multiple pregnancy rate. These outcomes would be improved significantly if a single embryo of high viability could be replaced in each treatment cycle, but widespread acceptance of such a policy is hindered by the lack of predictive factors for embryo selection. We have conducted a retrospective clinical study of a novel non-invasive method of embryo selection based on the depletion/appearance of amino acids in the culture medium. METHODS: Fifty-three cycles of IVF treatment using ICSI were studied. Embryos were cultured for 24 h in 4 microl drops of medium containing a physiological mixture of 18 amino acids. The spent medium was analysed for amino acid content by high performance liquid chromatography. RESULTS: The turnover of three amino acids, Asn, Gly and Leu, was significantly correlated with a clinical pregnancy and live birth. These correlations were independent of known predictors, such as female age, basal levels of FSH, embryo cell number and embryo morphological grade. CONCLUSIONS: Non-invasive assay of amino acid turnover has the potential to improve significantly the prospective selection of the most viable embryos, or single embryo, for replacement in an IVF cycle.     

Metabolism of human embryos following cryopreservation: implications for the safety and selection of embryos for transfer in clinical IVF

BACKGROUND: Cryopreservation of supernumerary embryos is routinely performed in human-assisted reproduction, providing a source of embryos which can be thawed for use in subsequent treatment cycles. However, the viability of cryopreserved embryos has traditionally relied on morphological assessment, which is a poor predictor of embryo health since freezing leads to a significant overall reduction in implantation potential, and its long-term efficacy is unknown. This study describes how the post-thaw metabolism of human embryos can be used to predict future development to the blastocyst stage. METHODS: HPLC was used to analyse the post-thaw amino acid metabolism of human embryos from day 2 to day 3 of development. RESULTS: It was possible to predict with 87% accuracy which frozen-thawed embryo would develop to the blastocyst stage. Developmentally competent embryos were more metabolically quiescent than their arresting counterparts. Amino acid turnover was also capable of distinguishing between the developmental potential of the best, Grade I embryos P < 0.05. CONCLUSIONS: The data suggests that cryopreservation in IVF is a safe procedure and that amino acid turnover can be used to select which cryopreserved embryo will develop to the blastocyst stage, irrespective of their post-thaw grade.     

DNA damage and metabolic activity in the preimplantation embryo

BACKGROUND: Embryos with greater viability have a lower or ‘quieter’amino acid metabolism than those which arrest. We have hypothesizedthis is due to non-viable embryos possessing greater cellular/moleculardamage and consuming more nutrients, such as amino acids forrepair processes. We have tested this proposition by measuringphysical damage to DNA in bovine, porcine and human embryosat the blastocyst stage and relating the data to amino acidprofiles during embryo development. METHODS: Amino acid profiles of in vitro-derived porcine and bovine blastocystswere measured by high-performance liquid chromatography andthe data related retrospectively to DNA damage in each individualblastomere using a modified alkaline comet assay. Amino acidprofiles of spare human embryos on Day 2–3 were relatedto DNA damage at the blastocyst stage. RESULTS: A positive correlation between amino acid turnover and DNA damagewas apparent when each embryo was examined individually; a relationshipexhibited by all three species. There was no relationship betweenDNA damage and embryo grade. CONCLUSIONS: Amino acid profiling of single embryos can provide a non-invasivemarker of DNA damage at the blastocyst stage. The data are consistentwith the quiet embryo hypothesis with viable embryos (lowestDNA damage) having the lowest amino acid turnover. Moreover,these data support the notion that metabolic profiling, in termsof amino acids, might be used to select single embryos for transferin clinical IVF.     

The Graduated Embryo Score (GES) predicts blastocyst formation and pregnancy rate from cleavage-stage embryos

BACKGROUND: Embryo morphology and cleavage rates alone do not consistently identify embryos with high implantation potential following IVF. Blastocyst transfer has been reported to improve success rates by identifying potentially superior quality embryos. Algorithms for predicting IVF outcomes based on the presence of early developmental milestones have been proposed. Here we introduce the Graduated Embryo Score (GES). METHODS: Nucleolar alignment along the pronuclear axis, regular cleavage and degree of fragmentation at the first cell division, and cell number and morphology on day 3 were weighted to create a possible GES of 100 for each of 1245 fertilized embryos derived from 109 patients aged <40 years. The GES was correlated with IVF outcome. RESULTS: Of 983 embryos for extended culture, 349 (36%) developed to blastocyst and 180 (18%) were good quality (grade I-II). When ranked by cell number and morphology alone, 34% of embryos with > or =7 cells and <20% fragmentation formed good quality blastocysts. Using GES, embryos scoring 90-100 had 64% blastocyst formation compared with 31% scoring 70-85 and with 11% scoring 30-65. Embryos scoring 70-100 had 44% blastocyst development compared with 9% scoring 0-65. Fifty-six patients (51%) conceived on-going gestations from 294 transferred embryos. In patients with at least one transferred embryo scoring > or =70, the pregnancy rate was 59% compared with 34% if all embryos scored <70. The overall implantation rate was 28%. Among embryos scoring 70-100, an implantation rate of 39% was seen, compared with 24% among embryos scoring 0-65. CONCLUSIONS: Predicting which cleaved embryos will form blastocysts could permit the high success rates associated with blastocyst transfer to be achieved from day 3 embryo transfer.     

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.     

Sequential assessment of individually cultured human embryos as an indicator of subsequent good quality blastocyst development

BACKGROUND: It is of fundamental importance for IVF clinics to determine the most viable embryos for transfer. The challenge for ART clinics is to transfer fewer embryos, thereby minimizing the risk of multiple-infant births, while still maintaining the greatest chance of pregnancy for their patients. In this study, an investigation was made to determine if developmental markers on the day of fertilization (day 1) can predict good subsequent blastocyst development. METHODS AND RESULTS: A total of 1550 individually cultured 2PN embryos from 191 patients undergoing IVF/ICSI treatment at the Yale University Center for Reproductive Medicine and Infertility from February to December 2001 was included. The results showed a significant positive relationship between early-cleaving 2-cell embryos and subsequent good quality > or =4-cell, > or =7-cell and blastocyst development (P < 0.05). PN symmetry (the relative size of the PN to each other), when checked at the time fertilization, is also a significant indictor of good quality > or =4-cell, > or =7-cell stage embryos and blastocysts. Combined, a developing embryo showing PN symmetry with early cleavage and subsequent good > or =4-cell and > or =7-cell cleavage, has a one in two chance of developing into a good-quality blastocyst. CONCLUSION: Early embryo assessment can be used as an indicator of subsequent good blastocyst development.     

Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro

BACKGROUND: The absence of reliable markers for the identification of viable embryos for transfer at the early cleavage stage is likely to contribute to the generally low implantation rates and high incidence of multiple gestation in IVF treatment. In this study, we investigate the relationship between timing of first cleavage and the incidence of blastocyst formation in vitro. METHODS: Couples (n = 70) with at least one embryo remaining after transfer were included in the analyses. All embryos (n = 579) were examined for early cleavage at 25 h after insemination. Following embryo transfer, the remaining embryos (n = 426) were cultured until day 7 of development, and assessed for blastocyst formation. RESULTS: Eighty-five embryos (14.7%) cleaved to the 2-cell stage within 25 h of insemination; 26 of these were selected for transfer on day 2. Of the 59 embryos remaining in culture, 19 (32.2%) developed to the blastocyst stage; this was a significantly higher number than was observed in embryos (61/367; 16.6%) that failed to cleave within 25 h of insemination (P < 0.01). Within these two groups of embryos the proportion of hatched blastocysts was 11/59 (18.6%) and 26/367 (7.1%) respectively (P < 0.005). CONCLUSIONS: These findings indicate that early cleavage is indicative of increased developmental potential in human embryos and may be useful as an additional criterion in the selection of embryos for transfer.     

Extended embryo culture in human assisted reproduction treatments

In order to evaluate the niche of extended embryo culture in an IVF programme, retrospective analysis of non-selected IVF patients, who underwent ovarian stimulation from April 1998 to June 1999 in a single private practice assisted reproductive technology centre, was performed. Embryos were cultured for 48 h in S1/G1.2 medium followed by 48 to 72 h of culture in S2/G2.2 to day 5 or day 6. Only fertilized oocytes exhibiting two pronuclei from donor and non-donor IVF and intracytoplasmic sperm injection (ICSI) cases were examined to determine the relationship between embryo cell number on day 3 and subsequent rate of blastocyst formation. Results indicated that a proportional relationship existed between the number of blastomeres present in day 3 embryos and the rate of blastocyst formation. Fifty-four per cent of embryos that had six cells on day 3 formed blastocysts, while 76% of those embryos with eight cells formed blastocysts. Blastocyst development did not increase further when embryos had more than eight cells on day 3, indicating that embryos with greater cell numbers on day 3 are not always predictive of a greater likelihood of blastocyst formation. Fertilized oocytes exhibiting two pronuclei from donors produced significantly more blastocysts (67%) than those from IVF patients (52%; P < 0.01), and had a significantly higher implantation rate (54%) compared with IVF patients (30%; P < 0.01). Furthermore, blastocyst cryopreservation resulted in significantly higher implantation rates than cryopreserved cleavage stage embryos (P < 0.001).     

Amino acids promote human blastocyst development in vitro

Supplementation of culture media with amino acids has been shown to benefit preimplantation embryo development in several species. This randomized study analysed the in-vitro development of human embryos obtained after IVF in the presence or absence of a combination of amino acids from the 2- to 4-cell stage to the blastocyst stage. A total of 129 human embryos was randomly distributed between three serum-free chemically defined sequential media: (i) glucose-free Earle's balanced salt solution (EBSS) with glutamine (Gln) prior to morula stage, supplemented with glucose for blastocyst formation; (ii) glucose-free EBSS with glutamine and non-essential amino acids (AA) for cleavage stage development, and supplemented with all 20 AA for blastocyst formation (Earle's+AA); and (iii) a sequential commercial medium containing amino acids (K-SCIM). Embryos were individually cultured for successive periods of 24 h. On day 6 of development, blastocysts were differentially labelled and the numbers of trophectoderm and inner cell mass cells, mitoses and dead cells were examined. Blastocyst development was similar for the three sequential media. The mixture of AA significantly increased total blastocyst cell numbers from 61.8 +/- 4.2 with Earle's+Gln to 99.3 +/- 8.4 with Earle's+AA and 100.2 +/- 9.4 with K-SCIM (P = 0.005). This increase was present in both the trophectoderm and inner cell mass lineages (P < 0.02). Furthermore, the dead cell index was significantly lower with Earle's+AA (P = 0.047).     

Embryo evaluation by analysing blastomere nuclei

BACKGROUND: To create a more effective selection standard for early embryos, we developed a new grading system consisting of conventional morphological evaluation in combination with analysis of blastomere nuclei. METHODS: A total of 744 embryos used during 459 cycles of embryo transfer on day 2 and blastocyst transfer were subjected to retrospective analysis. The overall implantation rate was 15.5% (115/744). Morphological evaluation of the embryos was performed on day 2 by referring to both the size of blastomere and fragmentation (conventional method) and the nucleic features of the blastomeres--either multinucleated or anucleic (nuclei counting method). The implantation rate for every transferred embryo and blastocyst was examined. RESULTS: Although a high implantation rate was observed with the highest quality embryos as judged by either the conventional method (24.1%; 57/237) or the nuclei counting method (26.1%; 104/399), the nuclei counting method predicted implantation rate better than the conventional method. The embryos that were considered to be high quality according to the conventional method, but low quality according to the nuclei counting method, had a limited implantation success rate of 6.3% (4/66). Also, after blastocyst transfer, implantation occurred most often when high quality embryos evaluated by the nuclei counting method were used (25.5%; 25/98), while the blastocysts from low quality embryos seldom implanted (3.2%; 2/63). CONCLUSIONS: When choosing which embryo to transfer, the normality of blastomere nuclei may be a more important index of quality than standard fragmentation features and/or blastomere uniformity analysis. When choosing among embryos, if nucleic status is identical, then embryos with the least fragmentation should be chosen. Moreover, in blastocyst transfer, a blastocyst whose nuclei were judged normal on day 2 should be selected on day 5 over any other blastocysts.     

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

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

Pregnancy and birth resulting from transfer of a blastocyst observed to have one pronucleus at the time of examination for fertilization.

This case report describes a successful full-term pregnancy and birth after the transfer of a zona-free blastocyst derived from an oocyte observed at fertilization check as having only one distinct pronucleus (PN). The patient had previously undergone four in-vitro fertilization (IVF) cycles and three frozen embryo transfer cycles, with one pregnancy resulting. In this IVF cycle, 7/19 oocytes were fertilized exhibiting two distinct PN; however, all these oocyctes failed to develop in culture and had arrested or totally fragmented by day 6 after insemination. One oocyte (1/19) displayed only one PN 18 h after insemination, but culture of this oocyte led to development of an early cavitating blastocyst by day 6. Since no other embryos were available for transfer to the patient, this embryo was transferred, resulting in a full-term pregnancy with delivery of a normal healthy boy. Observation of a single PN at the normal time of fertilization assessment would not appear to be an absolute indicator of developmental incompetence. In-vitro culture to 6 days post-insemination provides the opportunity to assess embryological development after activation of the embryonic genome. Formation of a morphologically normal blastocyst may be an indicator of a fertilized embryo with normal developmental capacity.     

Improved development of human embryos in vitro by a human oviductal cell co-culture system.

This study was undertaken to determine the effect of co-culture with human oviductal cells on human embryos. Spare embryos from gamete intra-Fallopian transfer (GIFT), pronuclear stage transfer (PROST) and in-vitro fertilization/embryo transfer (IVF/ET) programmes were either cultured in serum-supplemented Earle's balanced salt solution alone, or co-cultured in the same solution with oviductal cells from the pronuclear stage (day 1 post-insemination) or two- to four-cell stage (day 2 post-insemination). The co-cultured embryos appeared to have a higher developmental potential (higher rate of blastocyst formation and lower fragmentation rate), although there was no statistical difference in their rate of development, degree of fragmentation and stages attained, when compared with conventionally cultured embryos. The percentage of hatching blastocysts was significantly higher (P less than 0.05, Fisher's exact test) for embryos co-cultured from day 1 post-insemination (38%) than for embryos which had not been co-cultured (7%). The blastocyst hatching rate for embryos co-cultured from day 2 post-insemination was 15%. It was therefore concluded that co-culture of human embryos with oviductal cells could improve the development of the embryos in vitro. The degree of improvement was more pronounced when the co-culture started at an earlier stage.     

High outcome predictability after IVF using a combined score for zygote and embryo morphology and growth rate

BACKGROUND: A scoring system has been developed to determine preimplantation embryo quality, and used to select embryos for transfer into the uterus of patients. METHODS AND RESULTS: The system was used to study early embryo development and to test whether these scores alone can accurately predict IVF outcome. Following zygote and embryo scores through early development, the data showed that a top quality zygote does not necessarily indicate that the resulting embryo will be top quality after in-vitro culture. The embryo quality score can change dramatically when embryos are cultured to day 2 or 3 post-fertilization. Pregnancy rates and implantation rates were compared with the cumulative and separated zygote and embryo scores. Analysis of the predictability of scoring systems suggested that morphological scores alone are relatively unpredictive of IVF outcome. When weighted for in-vitro growth rate, scores were highly predictive, more so than the rate of development alone. CONCLUSIONS: These data suggested that a combination of in-vitro growth rate and morphological analysis both of zygotes and embryos was highly indicative of outcome after IVF. The results can be adopted to the single embryo transfer approach to IVF.     

Cryopreservation of biopsied embryos at the blastocyst stage

BACKGROUND: The availability of an efficient cryopreservation program is especially important in the case of embryos that have undergone blastomere biopsy for PGD. Unfortunately, the freezing/thawing of biopsied embryos has given disappointing results when performed at the cleavage stage. In this study, embryos diagnosed as normal after PGD were grown to the blastocyst stage, frozen and thawed for successive frozen embryo transfer. METHODS: A total of 34 patients performed a thawing cycle in which 47 blastocysts were thawed. The cryopreservation solutions were based on HEPES-buffered medium supplemented with human serum albumin (HSA), sucrose and 1,2-propanediol. The same protocol was applied to embryos from 88 IVF/ICSI patients, which underwent 92 thawing cycles with 150 thawed blastocysts. RESULTS: The survival rate was similar in the two groups (53% after PGD and 58% in IVF/ICSI cycles), as well as the cumulative pregnancy rate per patient (59% after PGD versus 47% in IVF/ICSI cycles), despite a higher maternal age and a lower proportion of embryos available for transfer or cryopreservation in the PGD group. CONCLUSIONS: Neither the survival rate nor the subsequent development and chances of implantation, differed between embryos frozen at the blastocyst stage following biopsy and those frozen intact.     

Case report: successful pregnancy after vitrification of a human blastocyst that had completely escaped from the zona pellucida on day 6

This case report describes a successful pregnancy after vitrification of a human hatched blastocyst. A 31-year-old woman, after failed stimulated and thaw cycles, underwent short-treatment protocol stimulation, and oocytes were recovered transvaginally with ultrasound guidance. Eight mature oocytes were obtained and six were fertilized with conventional IVF. Consecutive embryo transfer was performed, in which two cleaved embryos were transferred on day 3 and a single blastocyst was transferred on day 5, but no implantation occurred. On day 6, one of the non-transferred embryos developed into a blastocyst that had completely escaped from the zona pellucida. The zona-free hatched blastocyst was vitrified using a cryotop procedure after artificial shrinkage, which in our clinical experience has proved to be effective for zona-intact blastocysts. Six months after the previous retrieval cycle, the cryopreserved hatched blastocyst survived the warming process and was transferred to the patient's uterus. Implantation resulted in a healthy pregnancy; the pregnancy is ongoing at 33 weeks. This is the first report of a pregnancy after vitrification of a human blastocyst that had completely escaped from the zona pellucida.     

A retrospective analysis of the in-vitro development of 'spare' human in-vitro fertilization preimplantation embryos using 'in-house' prepared medium and 'Medi-Cult' commercial medium

In-house prepared medium was used routinely in our in-vitrofertilization (IVF) facility prior to the introduction of thecommercial ‘Medi-Cult’ products. A comparative studyof the in-vitro development of embryos cultured in two [T6 andEarle's balanced salt solution (EBSS)] humaninactivated serum(HlS)-supplemented media from days 0 to 5 showed that 44.7%(46/103) of the embryos developed to the blastocyst stage inthe T6 medium compared with 22.3% (23/103) in EBSS. Followingthe introduction of the commercial Medi-Cult IVF M2 medium,which is used routinely to culture fertilized eggs from days0 to 2, new baseline data were required for the in-vitro developmentof ‘spare’ embryos from days 2 to 5. When Medi-CultM3 medium was used, 35.6% (37/104) of the ‘spare’day 2 embryos achieved the blastocyst stage. However, if morphologicallysimilar (four normal nucleated blastomeres with no fragmentation)day 2 embryos were selected, an increase in the blastocyst rateto 50.0% (33/66) was achieved. This compared favourably withthe 45.0% blastocyst rate (published in the Medi-Cult literature)for M2/M3 medium cultured human embryos. A small series of experimentswith T6 $ HIS medium and human serum albumin (HSA)- supplementedHam's F-10, MCDB 302 and M3 media was undertaken to identifya suitable medium which could be used for the culture of M2medium day 2 embryos. Results show that M2 medium cultured embryosplaced in Ham's F-10 medium supplemented with 10 mg/ml HSA gavean acceptable 37.8% (14/45) blastocyst rate. Therefore, thismedium could be substituted for M3 medium in an emergency. Atotal of 483 IVF embryos donated by patients, which were surplusto the therapeutic IVF programme, were used for these studiesover a period of 30 months. Late day 2 IVF spare embryos wereassigned an embryo score based on a high-power phase-contrastmicroscopic examination prior to being placed in culture. Theembryo score provides an effective in-vitro parameter with whichembryos from different patients can be compared. The cleavageand development of individual embryos were monitored on days2 to 5. In some cases, the continuing normal development andviability of the day 5 cultured embryo were assessed by monitoringthe hatching, attachment and outgrowth of the cavitated blastocyst.     

Successful pregnancy following replacement of embryos previously refrozen at blastocyst stage: case report

We present the first reported clinical pregnancy following transfer of embryos that had been subjected to two freeze-thaw cycles: the first at day 3 after insemination, and the second after culturing to the blastocyst stage. A 25-year-old woman undergoing IVF treatment for male factor infertility opted for intracytoplasmic sperm injection (ICSI). ICSI treatment resulted in the successful production of 19 early cleavage embryos, all of which were frozen. After thawing, the embryos were cultured to the blastocyst stage. Thereafter, the blastocysts were refrozen and again thawed prior to embryo transfer. Embryos surviving a day 3 freeze-thaw cycle developed to the blastocyst stage and survived the second freeze-thaw cycle. Successful clinical pregnancy resulted following two sequential freeze-thaw cycles. This finding shows that it is possible to refreeze supernumerary blastocysts for subsequent transfer.     

Fertilization and early embryology: Selection of viable mouse blastocysts prior to transfer using a metabolic criterion

The success rate of human in-vitro fertilization (IVF) remainslow, with only -10% of embryos transferred resulting in a termpregnancy. A major contributor to this embryonic loss Is poorembryo development in vitro. Such poor development can be attributedto both chromosomal and anatomic anomalies in oocytes afterovarian stimulation and to suboptimal embryo culture conditions.The low success rate of IVF is compounded by an inability toselect those embryos most likely to implant after transfer (viable).Currently morphology is used almost exclusively as the solecriterion to decide which embryos are replaced. This procedureis not only subjective but has a poor correlation with subsequentdevelopmental competence. Therefore, the development of techniquesto quantify embryo viability prior to transfer will significantlyincrease pregnancy rates. We report here that the non-invasiveassessment of glycolytic activity (percentage of glucose convertedto lactate) in individual mouse blastocysts prior to transfercan be used successfully to identify viable embryos. Blastocystswith a low glycolytic activity, close to that of in-vivo developedblastocysts, had a significantly higher viability than thosewith abnormally elevated levels of glycolysis. Using glycolyticactivity as a marker of viability resulted in a four fold increasein the pregnancy rate compared with embryos selected at randomfor transfer. We propose that the success of clinical IVF canbe increased significantly by employing quantitative tests forviability.     

Multiple-birth risk associated with IVF and extended embryo culture: USA, 2001

BACKGROUND: Multiple births are associated with serious adverse infant and maternal outcomes. The objective of this study was to assess the multiple-birth risk (MBR) associated with IVF and determine whether the risk is impacted by stage of embryo development at transfer. METHODS: A population-based sample of 50 819 IVF transfers utilizing day 3 or day 5 embryos performed in the USA in 2001 on women aged 20-40 years was used to assess MBR and live-birth rate (LBR), stratified by patient age, supernumerary embryo availability, and number of embryos transferred. RESULTS: Although significantly more day 5 than day 3 transfers used < or =2 embryos (69.2 versus 27.7%), the former were not associated with decreased MBR. MBR was high when >1 embryo was transferred, irrespective of embryo development stage. LBR were generally maximized with 2 embryos transferred, and for some (day 5 transfers, patients aged 35-37 years) with one embryo. Electing to transfer a single day 5 embryo appeared efficacious for some patients: women aged 20-37 years with supernumerary embryos cryopreserved had LBR of 31.6-39.5%. CONCLUSIONS: MBR is high when > or =2 embryos are transferred. Single embryo transfer is the only way to prevent many multiple births and associated adverse health outcomes.