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.     

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.     

Blastomere development after embryo biopsy: a new model to predict embryo development and to select for transfer

One of the most important and unsolved problems in in-vitro fertilization is to decide which embryos are more suitable to implant and therefore should be transferred. We analysed the in-vitro development of isolated biopsied blastomeres and compared it to the development of the original embryo, in order to find a relationship that could show the embryo's potential future development and so increase implantation rates. A total of 66 normally fertilized human embryos were biopsied at the 6- to 10-cell stages. At day 6, blastomeres were counted by nuclear labelling. A total of 33 embryos (50%) reached the blastocyst stage. Of the isolated blastomeres, 63% divided and 53% cavitated over 3 days in culture. Of the blastomeres taken from embryos that developed to the blastocyst stage, 88% divided, 79% cavitated, 76% divided and cavitated and 9% neither divided nor cavitated. In those from arrested embryos, 39% divided (P < 0.001), 21% cavitated (P < 0.001), 15% divided and cavitated (P < 0.001) and 55% neither divided nor cavitated (P < 0.001). Blastomeres biopsied from embryos that reached the blastocyst stage showed a significantly higher proportion of division and cavitation than those originated from arrested embryos. Culture of the isolated blastomeres can demonstrate those embryos more likely to develop to the blastocyst stage and that are probably more suitable to implant. Cryopreserving biopsed embryos and culturing blastomeres would increase implantation rates. Embryos can then be selected according to the blastomere development and thawed for transfer in a future cycle.     

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.     

Predictors of ongoing implantation in IVF in a good prognosis group of patients

BACKGROUND: The aim of this study was to investigate whether, in a large randomized trial, it is possible to identify specific maternal and/or embryo variables that could independently correlate with ongoing implantation in IVF/ICSI. METHODS: In a Scandinavian study, 661 women were randomized to elective single embryo transfer or double embryo transfer. Women aged <36 years undergoing their first or second IVF cycle and with at least two good quality embryos were eligible. Only one cycle per subject was included. In the present study, cycles with 0 or 100% ongoing implantation (n = 520) were analysed regarding maternal and embryo variables. RESULTS: In this selected study group, the ongoing implantation rate was 195/734 (26.6%). In the univariate analysis, first IVF cycle, conventional IVF as fertilization method and 4-cell embryos showed a statistically higher ongoing implantation rate than did second IVF cycle, ICSI and non-4-cell embryos. In the multivariate analysis the same variables correlated independently to ongoing implantation. In addition, ovarian sensitivity correlated independently to ongoing implantation. CONCLUSION: This information should be used when selecting the number of embryos for transfer with the overall aim to reduce the rate of multiple births while maintaining a satisfactory birth rate.     

Minimizing embryo expulsion after embryo transfer: a randomized controlled study

BACKGROUND: The aim of this work was to modify the embryo transfer technique to prevent expulsion of the embryos by exerting gentle mechanical pressure on the cervix using the vaginal speculum. METHODS: A total of 639 infertile patients undergoing ICSI were prospectively randomized into two groups using sealed dark envelopes. In the study group (n=325) the screw of the vaginal speculum was loosened in order to exert a gentle pressure on the portiovaginalis of the cervix before ejecting the embryos, and was maintained for 7 min afterwards. In the control group (n=314) no pressure was applied on the cervix during embryo transfer and the vaginal speculum was removed after transferring the embryos. RESULTS: The clinical pregnancy rate was significantly higher in the study group than in the control group [207/325 (67%) versus 150/314 (47.8%); odds ratio (OR) 1.39; 95% confidence interval (CI) 1.11-1.74]. The implantation rate was also significantly higher in the study group [304/913 (33.3%) versus 198/920 (21.5%); OR 1.54; 95% CI 1.26-1.89]. CONCLUSIONS: Applying gentle mechanical pressure on the portiovaginalis of the cervix using the vaginal speculum during and after transferring the embryos significantly improved clinical pregnancy and implantation rates.     

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.     

Uterine evaluation by microhysteroscopy in IVF candidates

Over an eight-month period, microhysteroscopy was performedon 180 women, candidates for in-vitro fertilization (IVF). Onehundred and two of them were suspected of having uterocervicalpathology (group I), and 78 were not (group II). Group I hadmicrohysteroscopic abnormalities in 60.8% and group II in 21.8%.Microhysteroscopy showed 30.6% of 36 positive hysterographiesto be false positive and 37.5% of 144 negative hysterographiesto be false negative, producing a confirmation rate of 63.9%.Admission to the IVF programme depended on microhysteroscopicnormality. One hundred and one women were accepted directly,and 20 refused (‘primary decision’). Fifty-ninewere deferred with treatable hysteroscopic abnormalities, pendingmedical (22) and surgical (37) treatment, 14 via the microhysteroscope.Of the 59 women deferred, 51 were finally admitted and 8 rejectedafter a microhysteroscopic check (‘secondary decision’).Finally, 152 of the 180 candidates (84.4%) were admitted. Microhysteroscopyhelped to determine the optimal route for embryo transfer in23 women with cervical pathology. Term pregnancy rates per embryotransfer were similar with hysteroscopic findings primarilynormal, or normal after successful treatment (11.2 and 10.8%respectively). Microhysteroscopy seems useful for selectionof cases for IVF, accurately determining utero-cervical pathologyor normality, aiding in surgical correction of abnormalitiesand facilitating the process of embryo transfer. We suggestthat microhysteroscopy should be performed routinely on allIVF candidates.     

Implantation: Uterine fluid human decidua-associated protein 200 and implantation after embryo transfer

Uterine fluid samples from 109 patients undergoing in-vitrofertilization and embryo transfer were obtained so as to examinethe relationship between the uterine fluid concentration ofhuman decidua-associated protein (hDP) 200 and the implantationrate. The sampling was performed on the day of embryo transferwith a Wallace catheter, used for the testing of cervical patencybefore embryo replacement. The implantation rate, as well asthe pregnancy rate, demonstrated a significantly positive correlationwith the concentration of hDP 200 in the uterine fluid, measuredjust before embryo transfer. These results indicate that hDP200, identified as a rheumatoid factor secreted by the endometrium,may be involved in the implantation process.     

Ultrasound-guided embryo transfer maximizes the IVF results on day 3 and day 4 embryo transfer but has no impact on day 5

BACKGROUND: The use of ultrasound-guided embryo transfer has been reported to affect success rates in some centres but not others. In a prospective study, we examined the influence of ultrasound guidance in embryo transfer performed on different days after oocyte retrieval. METHODS: Two different methods of embryo transfer were evaluated in 1069 consecutive transfers. The ultrasound-guided embryo transfer was used in 433 cases, whereas 636 embryo transfers were performed with the tactile assessment ('clinical feel') method. RESULTS: Ultrasound-guided embryo transfer yielded a higher overall pregnancy rate than the 'clinical feel' approach, 47 versus 36% (P < 0.001). This difference was statistically significant where embryos were transferred after 3 or 4 days of culture, 45.9 versus 37.1% (P = 0.001) and 42.3 versus 27% (P = 0.035) respectively but not significant (P = 0.112) on day 5 embryo transfer (56.3 versus 45.7%). Likewise, the implantation rate was significantly different between the two groups on day 3 and 4 embryo transfer, 23.3 versus 15.8% (P < 0.01) and 21.6 versus 15.7% (P < 0.05%) respectively but no statistical difference was noted on day 5 embryo transfer, 26.7 versus 23.6%. CONCLUSION: Ultrasound assistance in embryo transfer on day 3 and 4 significantly improved pregnancy rates in IVF but had no impact on day 5.     

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.     

The probability of pregnancy after embryo transfer is affected by the age of the patient, cause of infertility, number of embryos transferred and the average morphology score, as revealed by multiple logistic regression analysis

Because the process of conception is affected by many variables,a multiple logistic regression analysis was performed to assess(i) the impact and relative weight of both patient and embryovariables and (ii) their possible effects on the probabilityof a vital pregnancy after embryo transfer. A statistical modelwas constructed predicting the probability of pregnancy afterembryo transfer. The variables that contributed significantlyto the predictive value of the model were the age of the patient,the cause of infertility, the number of embryos transferredand the average morphology score of the transferred embryos.Embryo variables appeared to have a significant but modest valuein predicting the probability of pregnancy after embryo transfer.Other variables, such as the thickness of the endometrium, werefound to have no prognostic value. Moreover, we found that theireffect could be explained by the variables already includedin the model.     

Stimulation of embryo hatching and implantation by prostacyclin and peroxisome proliferator-activated receptor delta activation: implication in IVF

BACKGROUND: Successful IVF depends in part on quality embryos. Recent work suggests that prostaglandin I(2) (PGI(2) or prostacyclin) promotes the development of embryos in vitro and enhances their implantation potential. The mechanism underlying the effects of PGI(2) is unclear. It has been reported that peroxisome proliferator-activated receptor delta (PPARdelta) mediates the effects of PGI(2) at the implantation sites. METHODS: The expression of PPARdelta in the preimplantation embryos was examined by RT-PCR, western blot analysis and immunohistochemistry. Synthetic PPARdelta ligand (L-165041) and PPARdelta targeted (PPARdelta(-/-)) embryos were used to reveal the roles of PPARdelta in PGI(2)-stimulated and spontaneous embryo development. RESULTS: Preimplantation embryos express PPARdelta, which is essential for the enhancing effect of PGI(2) and the spontaneous progression of preimplantation embryos. Enhanced blastocyst hatching by PGI(2) (P < 0.05) was abrogated by PPARdelta deletion. Blastocyst formation and embryo hatching were impaired in PPARdelta(-/-) embryos. PPARdelta deletion significantly reduced embryo cell proliferation (P < 0.01); PPARdelta activation increased embryo cell proliferation (P < 0.05). PPARdelta activation enhanced the implantation of wild-type (WT) embryos (P < 0.05); PPARdelta deletion reduced embryo implantation (P < 0.05). CONCLUSIONS: PPARdelta is essential for spontaneous and PGI(2)-stimulated embryo development and blastocyst hatching. The implantation of cultured embryos is enhanced by PPARdelta activation. PPARdelta represents a novel therapeutic target to improve IVF outcome.