Unsuitability of multinucleated human blastomeres for preimplantation genetic diagnosis

Multinucleated blastomeres (MNBs) were detected in 30.4% of230 cleaved but subsequently arrested human embryos, and in66.7% of 21 cleaved embryos rejected after preimplantation geneticdiagnosis. A total of 71 MNBs from both groups of embryos wasanalysed by fluorescence in-situ hybridization (FISH) usingsimultaneous X and Y chromosome detection. The sex chromosomeanalyses of these MNBs suggests the existence of at least threemechanisms of MNB generation. In 95% of the embryos in whichmononucleated and multinucleated blastomeres were analysed,the sex of the MNBs corresponded with the sex of the entireembryo. However, the number of sex chromosomes per MNB and theirdistribution in each nucleus varied greatly, indicating theirunsuitability for aneuploidy diagnosis at the preimplantationstage.     

Sperm aneuploidy in fathers of children with paternally and maternally inherited Klinefelter syndrome

BACKGROUND: It is unclear whether frequency of sperm aneuploidy is associated with risk of fathering children with trisomy. METHODS: We recruited 36 families with a boy with Klinefelter syndrome (KS), interviewed the fathers about their exposures and medical history, received a semen sample from each father, and collected blood samples from the mother, father and child. We applied a multicolour fluorescent in-situ hybridization assay to compare the frequencies of sperm carrying XY aneuploidy and disomies X, Y and 21 in fathers of maternally and paternally inherited KS cases. RESULTS: Inheritance of the extra X chromosome was paternal in 10 and maternal in 26 families. Fathers of paternal KS cases produced higher frequencies of XY sperm (P = 0.02) than fathers of maternal KS cases. After controlling for age, the major confounding variable, the difference between the two groups was no longer significant (P less-than-or-equal 0.2). Also, there were no significant differences between the parental origin groups for disomy X, Y or 21. CONCLUSIONS: Men who fathered a child with a Klinefelter syndrome produced higher frequencies of XY sperm aneuploidy, which is explained, in part, by both paternal age and parent of origin.     

Prognostic role of preimplantation genetic diagnosis for aneuploidy in assisted reproductive technology outcome

BACKGROUND: Preimplantation genetic diagnosis (PGD) for aneuploidy is recommended to couples at risk of generating chromosomally abnormal embryos. The aim of this study was to demonstrate that PGD for aneuploidy has an important role in the prognosis of subsequent treatments. METHODS: A total of 389 couples underwent their first PGD for aneuploidy due to either female age >or=38 years (n = 266) or >or=3 previous unsuccessful cycles (n = 123). After the first PGD followed by an unsuccessful treatment cycle, 141 couples underwent 175 subsequent PGD cycles. These patients were divided into three groups depending on the number of euploid embryos available for transfer in their first PGD cycle: group A included patients where no euploid embryos were diagnosed; group B included patients who had only one euploid embryo; and group C included patients with at least two normal embryos resulting from chromosomal analysis. RESULTS: In subsequent cycles, group A patients underwent significantly fewer transfers (45%) compared with group B (69%, P < 0.05) and group C patients (85%, P < 0.001). The pregnancy rate per transfer was significantly decreased in group A (15%) compared with group B (36%; P < 0.02) and group C (30%; P < 0.03). Accordingly, the live birth rate per patient was significantly lower in group A compared with group C (8.5% versus 30%; P < 0.005). CONCLUSIONS: The outcome of the first PGD for aneuploidy may have a predictive role for subsequent attempts.     

Cryo-thawed embryos obtained from conception cycles have double the implantation and pregnancy potential of those from unsuccessful cycles

BACKGROUND: The purpose of this study was to evaluate the influence of fresh IVF/ICSI cycle outcome on the prognosis of the related frozen embryo replacement (FER) cycle. METHODS: 459 FER cycles, involving 2049 cleavage stage embryos with no or up to 10% fragmentation, were performed for which the outcome of the fresh cycle was recorded. The cycles were divided into two groups; group A included cycles in which cryopreserved embryos were obtained from fresh cycles in which conception occurred. Group B were cycles in which cryopreserved embryos originated from unsuccessful fresh cycles. RESULTS: Groups A and B were comparable with respect to mean (+/- SD) age at cryopreservation (33 +/- 3.9 versus 33.2 +/- 4 years, P = not significant), mean number of oocytes retrieved and fertilized normally in the fresh cycle (11 +/- 5.2 versus 11.2 +/- 4.8, P = not significant) and mean age at the cryo-thawed transfer (34.5 +/- 4.2 versus 33.9 +/- 4 years, P = not significant). No significant difference was found between the two groups with regard to mean number of embryos cryopreserved (6.5 +/- 3.9 versus 6.2 +/- 3.6) and subsequently thawed (4.5 +/- 2.5 versus 4.5 +/- 1.8) per cycle and number of cryo-thawed embryos transferred per cycle (2.0 +/- 0.7 versus 2.1 +/- 0.8). However, the implantation rate per transferred embryo in group A was double that in group B (23 versus 11.2%, P < 0.0001). Moreover, the clinical pregnancy and ongoing pregnancy rates per cycle were significantly higher in group A compared with group B (34.8 and 27.3% versus 15.6 and 13.1%, P < 0.0001 and P = 0.0003 respectively). The difference in FER cycle outcome could not be explained by confounding variables. CONCLUSIONS: After thawing, cryopreserved embryos originating from conception IVF/ICSI cycles achieve double the implantation and pregnancy rates of those obtained from unsuccessful cycles.     

Preimplantation diagnosis of aneuploidy using fluorescent in-situ hybridization: evaluation using a chromosome 18-specific probe

Fluorescent detection of in-situ hybridization (FISH) with achromosome 18-specific probe (P5041 B.5 D18Z1) has been usedto assess the use of this method for preimplantation diagnosisof aneuploidy. Interphase nuclei (n = 802) have been analysedfrom 59 normally fertilized embryos developing in vitro at thenormal rate between days 2 and 7 post-insemination. The efficiencyof hybridization in control cells, as assessed by the proportionwith two signals in normal female lymphocytes was 88.9% (n =353) and with three signals in a trisomic (48, XXX+18) fibroblastcell line 74.0% (n = 290). Fifty-four of the human embryos wereconsidered to be diploid on the basis that the majority of nucleihad two signals. Some nuclei in these embryos had one or nosignal, especially on day 2, and tetraploid nuclei were alsowidespread. Among the remaining five embryos, one 5-cell embryoon day 2 had three hybridization signals in 4/5 nuclei and wastrisomic for chromosome 18, one 4-cell embryo on day 2 had onlyone signal in 4/4 nuclei and was monosomic, and the three otherembryos were aneuploid mosaics and/or had multi-nucleated blastomeres.Analysis of the incidence of interphase nuclei with more orless than the diploid number of hybridization signals indicatesthat more than a single nucleus will be necessary for accuratepreimplantation diagnosis of aneuploidy.     

Fertilization and early embryology: The presence of multinucleated blastomeres in human embryos is correlated with chromosomal abnormalities

The purpose of the present study was to determine whether thepresence of one or more multinucleated blastomeres during earlyembryonic development is associated with chromosomal abnormalitiesIn sibling blastomeres of that embryo. Embryos with multinucleatedcells (n = 47) detected on day 2 or 3 of development were comparedto dividing embryos without multinucleation. Arrested embryoswere excluded from this study. Chromosome abnormalities weredetected using fluorescent in-situ hybridization (FISH) withX, Y, 18 and 13/21 chromosome- specific probes. Of 47 embryosincluded in this study, 76.6% were chromosomafly abnormal, comparedto 50.9% in the control group (P < 0.001). Excluding aneuploidy,which is originated in the gametes and not the embryo, the differenceswere even higher, with 74.5% of multinucleated embryos beingchromosomafly abnormal compared to 32.3% of non-multinucleatedembryos (P < 0.001). Day of multinucleation appearance, numberof nuclei per cell, number of multinucleated cells per embryoand developmental quality of the embryos as well as the typeof fertilization (intracytoplasmic sperm injection versus standardinsemination) were not found to affect the rate of chromosomalabnormalities In embryos with multinucleated cells. These resultssuggest that embryos with multinucleated cells may not be suitablefor replacement and should be excluded unless no other embryosare available.     

Morphologically normal spermatozoa of patients with secretory oligo-astheno-teratozoospermia have an increased aneuploidy rate

BACKGROUND: Normal morphology is a major criterion for selecting spermatozoa to be injected. Given that teratozoospermia is one of the most critical parameters associated with sperm aneuploidy, the purpose of this study was to evaluate the aneuploidy rate of morphologically normal spermatozoa of patients with oligo-astheno-teratozoospermia (OAT). METHODS: Ten patients with secretory OAT and six age-matched normozoospermic men with a normal karyotype were enrolled. After assignment to normal or abnormal category, the location of each spermatozoon was recorded using an electronic microstage locator. Slides were then subjected to triple-colour fluorescence in situ hybridization for chromosomes X, Y and 12. RESULTS: OAT patients had a lower number of morphologically normal and abnormal spermatozoa carrying the X chromosome, compared with normozoospermic men. They also exhibited increased XY and XX disomy rates. Morphologically abnormal spermatozoa from normozoospermic men also had an increased XX disomy rate compared with normally shaped spermatozoa obtained from the same men. The total sperm aneuploidy rate of morphologically abnormal spermatozoa of normozoospermic men was 4.4-fold higher than that of spermatozoa with normal morphology. The total aneuploidy rates of spermatozoa with normal or abnormal head shape from OAT patients were similar to each other and to that of abnormally shaped spermatozoa from normozoospermic men, but they were higher than the rate found in normally shaped spermatozoa of normal men. CONCLUSIONS: Normally shaped spermatozoa of OAT patients have an increased aneuploidy rate.     

Transvaginal ultrasound-guided embryo transfer improves pregnancy and implantation rates after IVF.

BACKGROUND: Attempts are constantly being made to improve clinical pregnancy rates after IVF and embryo transfer. Since November 1998, we have gradually been adopting transvaginal ultrasound guidance during embryo transfer. We retrospectively examined the efficacy of this method on pregnancy and implantation rates. METHODS: The results of 846 cycles from our IVF-embryo transfer programme were analysed and comparisons were made between those carried out using ultrasound guidance and those by the clinical touch method. RESULTS: Higher pregnancy and implantation rates (28.9 and 15.2% respectively) were found in the group using the transvaginal ultrasound guidance during embryo transfer compared with those in the group using the clinical touch method (13.1 and 7.0% respectively). The differences were statistically significant (P < 0.01). There was no significant difference in ectopic pregnancy rates between the two groups. CONCLUSION: The use of transvaginal ultrasound-guided embryo transfer significantly improved both pregnancy and implantation rates. Although technically difficult, we suggest its use may maximize the chances of achieving a successful pregnancy outcome.     

Fertilization and early embryology: Diagnosis of major chromosome aneuploidies in human preimplantation embryos

A short fluorescence in-situ hybridization (FISH) procedureusing fluorochrome and digoxigenin labelled DNA probes was developedfor application in human preimplantation embryos in order toanalyse the five chromosomes most involved in human aneuploidy(X, Y, 18, 13 and 21). The chromosomes were fluorescent-stainedand detected simultaneously in 157 blastomeres from 30 humanembryos. Successful FISH analysis was achieved in 93% of theblastomeres. Aberrations for these chromosomes were found in70% of abnormally developing monospermic embryos. The majorityof normally developing monospermic embryos obtained from olderpatients were also chromosomally abnormal. By analysing allor most of the cells from these embryos, true mosaicism wasdistinguished from technique failure. Mosaic embryos, polyploidembryos with ploidies as high as 8n, haploid embryos, embryosmonosomic for 13/21 and for X, and embryos trisomic for 13/21and 18, were common in abnormally developing embryos. In contrast,aneuploidy was the main chromosome abnormality found in normallydeveloping monospermic embryos.     

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

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

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.     

Chromosomal mosaicism throughout human preimplantation development in vitro: incidence, type, and relevance to embryo outcome

BACKGROUND: A large percentage of in-vitro generated cleavage stage human embryos are chromosomally mosaic, consisting of both normal (diploid) and abnormal (non-diploid) cells. The present study characterized mosaicism at each stage of cleavage division and examined its effect on preimplantation development in vitro. METHODS: A total of 216 normally fertilized (two-pronucleate) embryos which were not selected for transfer to the patients were analysed for chromosomal abnormalities using multi-colour fluorescence in-situ hybridization DNA probes specific for three to five of nine different chromosomes (X, Y, 2, 7, 13, 16, 18, 21, 22). RESULTS: Overall, 48.1% of embryos were mosaic. The frequency of mosaic embryos increased from 15.2 to 49.4 to 58.1%, from the 2-4-cell to 5-8-cell to morula stages respectively, and the types of non-diploid cells detected were mostly aneuploid or chaotic. The incidence of mosaicism at the blastocyst stage was 90.9%; however, most of the mosaicism comprised diploid and polyploid cells. Arrested mosaic embryos had a higher incidence of chaotic abnormalities, and higher proportions of abnormal cells compared with the non-arrested group. CONCLUSIONS: Post-zygotic errors leading to mosaicism may occur, and persist throughout preimplantation development in vitro. Our results suggest that mosaicism involving multiple chromosomal imbalances and/or imbalances affecting a high proportion of cells in an embryo appear to impair development to the blastocyst stage.     

Abnormalities for chromosomes 13 and 21 detected in spermatozoa from infertile men

Sperm samples from infertile men with oligozoospermia or teratozoospermiawere studied by multicolour fluorescence in-situ hybridization(FISH) using DNA probes for chromosomes 13 and 21. A total of90 809 sperm nuclei from nine infertile men and 182 799 spermnuclei from 18 control donors were analysed. There was a highlysignificant increase in the frequency of spermatozoa disomicfor chromosome 13 in infertile patients (0.28%) compared tocontrol donors (0.13%) (two-tailed Z statistic P <0.0001and for chromosome 21 (0.48% in infertile men versus 0.37% incontrols, P <0.0001). Also there was a significantly increasedfrequency of diploid spermatozoa in infertile men (0.85%) comparedto control donors (0.66%) (P <0.0001). Our previous studieson these same infertile patients demonstrated increased frequenciesof sperm disomy for chromosomes 1 and XY. This suggests thatinfertile men, who are prime candidates for intracytoplasmicsperm injection, may be at a very small increased risk of aneuploidoffspring.     

Number of embryos for transfer after IVF and ICSI: a Cochrane review

BACKGROUND: The most common complication of IVF is multiple pregnancy, which occurs in 25% of pregnancies following the transfer of two embryos. Single embryo transfer can minimize twin pregnancies but could also lower live birth rates. Our aim was to perform a systematic review of randomized trials to determine the effectiveness of single versus double embryo transfer. METHODS: Cochrane Collaboration review methods were followed. Randomized controlled trials comparing single and double embryo transfers were identified by searching Medline, EMBASE and the Cochrane register of controlled trials. Contents of specialist journals and proceedings from meetings of relevant societies were hand searched. Data were pooled with Rev Man software using the Peto-modified Mantel-Hanzel method. RESULTS: Pooled results from four trials indicate that although double embryo transfer leads to a higher live birth rate per woman [odds ratio (OR) 1.94, 95% confidence interval (CI) 1.47-2.55] in a fresh IVF cycle, comparable results are obtained by subsequent transfer of a frozen embryo (OR 1.19, 95% CI 0.87-1.62). The multiple pregnancy rate is significantly higher (OR 62.83, 95% CI 8.52-463.57) after double embryo transfer. CONCLUSIONS: Single embryo transfer significantly reduces the risk of multiple pregnancy, but also decreases the chance of live birth in a fresh IVF cycle. Subsequent replacement of a single frozen embryo achieves a live birth rate comparable with double 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.     

Smoking habits of parents and male: female ratio in spermatozoa and preimplantation embryos

BACKGROUND: Previous observations have addressed a decreased male:female ratio associated with smoking. Our aim was to assess whether this effect is observed at the spermatozoa or at the early embryo development. METHODS: We retrospectively assessed smoking intake habits of 56 couples included in our preimplantation genetic diagnosis (PGD) program. Three groups were established according to male or female cigarette consumption per day: non-smokers, smokers (1-19 cigarettes per day) and heavy smokers (> or =20 cigarettes per day). Fluorescence in-situ hybridization (FISH) was performed on ejaculated sperm samples to analyse chromosomes X and Y. On day 3, embryos were also analysed. Additionally, sperm samples from four heavy smoking and four non-smoking donors were prospectively analysed before and after capacitation. RESULTS: FISH on spermatozoa revealed no statistical differences in the Y:X ratio between the three groups. However, in the PGD study, in male heavy smokers, the XY:XX embryo ratio was decreased compared with non-smokers (22:47 versus 80:71; P = 0.0057). The smoking condition of the female partner had no significant effect on embryo XY:XX ratio, but for non-smoking females with a heavy smoking partner, the ratio was decreased (P = 0.0018) compared with non-smoking males. In heavy smoking donors a decreased of Y:X ratio was observed after swim-up with a statistically significant difference of ratios (P = 0.021). CONCLUSIONS: Smoking habits of males do not have an effect on the percentage of X- and Y-bearing spermatozoa on ejaculated samples. However, male heavy smokers produce an increased incidence of female embryos that could be related to an enrichment of X spermatozoa after swim-up in patients with high tobacco consumption.     

Influence of early ICSI-derived embryo sHLA-G expression on pregnancy and implantation rates: a prospective study

BACKGROUND: We have previously reported the retrospective observation that when at least one embryo, transferred on day 3, expressed sHLA-G above the geometric mean (sHLA-G+) 46 h post-ICSI, there was a marked improvement in both pregnancy (PR) and implantation (IR) rates. METHODS: The media surrounding individual embryos derived from ICSI performed on oocytes from 482 women < or =43 years of age were tested for sHLA-G expression by specific ELISA. RESULTS: We report here prospective results showing improved IVF results following the transfer of 'good quality' embryos (7-9 cells with <20% fragmentation) by preferentially including at least one sHLA-G+ embryos. PR and IR for women < or =38 years were 63% and 32% when one transferred embryo was sHLA-G+, and 69% and 36% when at least two embryos were sHLA-G+. When none of the embryos transferred was sHLA-G+, PR and IR were 25% and 13%, respectively. Comparable PR and IR for women 39-43 years were 29% and 11% when none of the transferred embryos were sHLA-G+; 38% and 15% when at least one sHLA-G+ embryo was transferred; and 61% and 26% when at least two 2 sHLA-G+ embryos were transferred. The data were stratified by patient age. CONCLUSIONS: PR and IR increased with the addition of each sHLA-G+ embryo, regardless of age. While there are significant barriers to routine embryo sHLA-G testing, we believe that if implemented, this would provide a mechanism for optimizing IVF PR while minimizing the risk of multiple pregnancies.     

The implantation of every embryo facilitates the chances of the remaining embryos to implant in an IVF programme: a mathematical model to predict pregnancy and multiple pregnancy rates

BACKGROUND: We aimed to assess the validity of a theoretical mathematical model to predict the pregnancy rate and the multiple pregnancy rate in IVF/oocyte donation programmes on the basis of the implantation rate and the number of transferred embryos. METHODS: A total of 1835 embryo transfers corresponding to three different programmes in two centres with different implantation rates were analysed. Pregnancy and multiple pregnancy rates observed in the aforementioned programmes were compared with those obtained following different mathematical models. Four models were tested: binomial model, ground model, maternal variability model and collaborative model. The goodness of fit was performed by means of the maximum likelihood fit method. RESULTS: The binomial model could not predict the pregnancy rate, and especially the multiple pregnancy rate. The multiple pregnancy rate predicted following the binomial model was much lower than observed, up to 40-fold reduced. Ground model and maternal variability model adjusted to the data with more precision, but were still not accurate. Finally, the collaborative model reproduced with very great accuracy both pregnancy rate and the multiple pregnancy rate. A collaborative parameter of 22% was found, implying that the implantation probability of each embryo is increased by 22% for every embryo previously implanted. CONCLUSIONS: Embryonic implantation does not follow a binomial law, showing that the implantation is not independent from the number of embryos implanted. The best fit to the data is obtained following a collaborative model by which the implantation of one embryo is facilitated by the implantation of other embryo(s). The mathematical formula of the collaborative model predicts very accurately the pregnancy rate and the multiple pregnancy rate in IVF/oocyte donation programmes, based on the implantation rate of this specific programme and the number of embryos transferred up to five embryos. We recommend using the aforementioned formula to quantify the pregnancy rate and the risk of multiple pregnancy in the counselling of the infertile couple at embryo transfer. Such a formula is freely available at www.ifca.unican.es/matorras/mathpreg/.     

Morphological evaluation of human embryos and derivation of an embryo quality scoring system specific for day 3 embryos: a preliminary study

A scoring system specific for day 3 embryos has not been extensively explored. Most IVF laboratories continue to grade embryos solely on the basis of cell number and percentage fragmentation as was traditionally done for day 2 embryos. Additional morphological features, some unique to day 3 embryos, may be useful in selecting embryos most likely to blastulate and implant. The objective of this study was to derive an embryo scoring system for day 3 transfers which is predictive of positive pregnancy outcomes. A total of 316 transferred embryos from 93 patients was recorded on videotape and evaluated. The following parameters were used to grade the embryos: cell number, fragmentation pattern (FP), cytoplasmic pitting, compaction, equal sized blastomeres, blastomere expansion and absence of vacuoles. The clinical pregnancy rate was 41.9%, with an implantation rate of 18% per embryo transferred. The mean number of embryos transferred per patient was 3.4. Three formulae were derived to score embryo quality in each transfer based on the average score of individual embryos transferred. In the first scoring system, cell number alone was used to predict pregnancy outcome. The second scoring system was based on blastomere number and the observed FP. The third scoring system utilized both blastomere number and FP but also combined this with five morphological criteria to yield a final day 3 embryo quality (D3EQ) score. We found the D3EQ score to be prognostic of pregnancy outcome. This study suggests that although cell number and FP are certainly predictors of positive pregnancy outcomes, additional parameters specific to day 3 embryos should be used to stratify a cohort of embryos further.