STUDY QUESTIONDoes conventional blastocyst morphological evaluation correlate with euploidy (as assessed by comprehensive chromosome screening (CCS) of trophectoderm (TE) biopsies) and implantation potential?SUMMARY ANSWERA moderate relation between blastocyst morphology and CCS data was observed but the ability to implant seems to be mainly determined by the chromosomal complement of preimplantation embryos rather than developmental and morphological parameters conventionally used for blastocyst evaluation.WHAT IS KNOWN ALREADYCombined with improving methods for cryopreservation and blastocyst culture, TE biopsy and CCS is considered to be a promising approach to select euploid embryos for transfer. Understanding the role of morphology in blastocyst stage preimplantation genetic screening (PGS) cycles may help in further optimizing the cycle management and clinical outcomes.STUDY DESIGN, SIZE, DURATIONThis is a multicenter retrospective observational study performed between January 2009 and August 2013. The study includes the data analysis of 956 blastocysts with conclusive CCS results obtained from 213 patients following 223 PGS cycles. Single frozen embryo transfer (FET) cycles of 215 euploid blastocysts were performed where it was possible to track the implantation outcome of each embryo transferred.PARTICIPANTS/MATERIALS, SETTING, METHODSPGS was offered to infertile patients of advanced maternal age (>35 years) and/or with a history of unsuccessful IVF treatments (more than two failed IVF cycles) and/or previous spontaneous abortion (more than two spontaneous miscarriages). Prior to TE biopsy for CCS, blastocyst morphology was assessed and categorized in four groups (excellent, good, average and poor quality). The developmental rate of each embryo reaching the expanded blastocyst stage was defined according to the day of biopsy post-fertilization. Day 5 and Day 6 biopsied blastocysts were defined as faster and slower growing embryos, respectively. A novel blastocyst biopsy method, not requiring the opening of the zona pellucida at the cleavage stage of embryo development, was used. Linear regression models were used to test the relationship between blastocyst morphology and developmental rate CCS data and FET cycle outcomes of euploid blastocysts.MAIN RESULTS AND THE ROLE OF CHANCEAmong the embryological variables assessed (morphology and developmental rate), only blastocyst morphology was predictive of the CCS data. The euploidy rate was 56.4, 39.1, 42.8 and 25.5% in the excellent, good, average and poor blastocyst morphology groups, respectively. A diagnosis of complex aneuploidy was also associated with blastocyst morphology (P < 0.01) with 6.8, 15.2, 17.4 and 27.5% of excellent, good, average and poor quality embryos, respectively, showing multiple chromosome errors. Faster and slower growing embryos showed a similar aneuploidy rate. Regression logistic analysis showed that none of the parameters used for conventional blastocyst evaluation (morphology and developmental rate) was predictive of the implantation potential of euploid embryos. The implantation potential of euploid embryos was the same, despite different morphologies and developmental rates.LIMITATIONS, REASONS FOR CAUTIONThe study is limited by its retrospective nature. A higher sample size or a prospective randomized design could be used in future studies to corroborate the current findings.WIDER IMPLICATIONS OF THE FINDINGSThis study provides knowledge for a better laboratory and clinical management of blastocyst stage PGS cycles suggesting that the commonly used parameters of blastocyst evaluation are not good enough indicators to improve the selection among euploid embryos. Accordingly, all poor morphology and slower growing expanded blastocysts should be biopsied and similarly considered for FET cycles. This knowledge will be of critical importance to achieve similar cumulative live birth rates in PGS programs compared with conventional IVF, avoiding the potential for exclusion of low quality but viable embryos from the biopsy and transfer procedures. Future research to identify non-invasive biomarkers of reproductive potential may further enhance selection among euploid blastocysts.STUDY FUNDING/COMPETING INTEREST(S)No funding was obtained for the study. All authors have no conflicts to declare.TRIAL REGISTRATION NUMBERNone.

Does comprehensive chromosome screening (CCS) of cells sampled from the blastocyst trophectoderm (TE) accurately predict the chromosome complement of the inner cell mass (ICM)?Comprehensive chromosome screening of a TE sample is unlikely to be confounded by mosaicism and has the potential for high diagnostic accuracy.The effectiveness of chromosome aneuploidy screening is limited by the technologies available and chromosome mosaicism in the embryo. Combined with improving methods for cryopreservation and blastocyst culture, TE biopsy and CCS is considered to be a promising approach to select diploid embryos for transfer.The study was performed between January 2011 and August 2011. In the first part, a new ICM isolation method was developed and tested on 20 good morphology blastocysts. In the main phase of the study, fluorescence in situ hybridization (FISH) was used to reanalyse the ICMs and TEs separated from 70 embryos obtained from 26 patients undergoing blastocyst stage array comparative genome hybridization (aCGH) PGS cycles.The isolated ICM and TE fractions were characterized by immunostaining for KRT18. Then, non-transferrable cryopreserved embryos were selected for the FISH reanalysis based on previous genetic diagnosis obtained by TE aCGH analysis. Blastocysts either diploid for chromosome copy number (20) or diagnosed as single- (40) or double aneuploid (10) were included after preparing the embryo into one ICM and three equal-sized TE sections. Accuracy of the aCGH was measured based on FISH reanalysis. Chromosomal segregations resulting in diploid/aneuploid mosaicism were classified as low-, medium- and high- grade and categorized with respect to their distribution (1TE, 2TE, 3TE, ICM or ALL embryo). Linear regression model was used to test the relationship between the distributions and the proportion of aneuploid cells across the four embryo sections. Fishers exact test was used to test for random allocation of aneuploid cells between TE and ICM.All ICM biopsy procedures displayed ICM cells in the recovered fraction with a mean number of ICM cells of 26.2 and a mean TE cell contamination rate of 2. By FISH reanalysis of previously aCGH-screened blastocysts, a total of 66 aneuploidies were scored, 52 (78.8) observed in all cells and 14 (21.2) mosaic. Overall, mosaic chromosomal errors were observed only in 11 out of 70 blastocysts (15.7) but only 2 cases were classified as mosaic diploid/aneuploid (2.9). Sensitivity and specificity of aCGH on TE clinical biopsies were 98.0 and 100 per embryo and 95.2 and 99.8 per chromosome, respectively. Linear regression analysis performed on the 11 mosaic diploid/aneuploid chromosomal segregations showed a significant positive correlation between the distribution and the proportion of aneuploid cells across the four-blastocyst sections (P 0.01). In addition, regression analysis revealed that both the grade and the distribution of mosaic abnormal cells were significantly correlated with the likelihood of being diagnosed by aCGH performed on clinical TE biopsies (P 0.019 and P 0.01, respectively). Fishers exact test for the 66 aneuploidies recorded showed no preferential allocation of abnormal cells between ICM and TE (P 0.33).The study is limited to non-transferable embryos, reanalyzed for only nine chromosomes and excludes segmental imbalance and uniparental disomy. The prevalence of aneuploidy in the study group is likely to be higher than in the general population of clinical PGD embryos.This study showed high accuracy of diagnosis achievable during blastocyst stage PGS cycles coupled with 24-chromosomes molecular karyotyping analysis. The new ICM isolation strategy developed may open new possibilities for basic research in embryology and for clinical grade derivation of human embryonic stem cells.No specific funding was sought or obtained for this study.

To determine whether the process of oocyte vitrification affects oocyte viability in in vitro fertilization (IVF) patients between 30 and 39 years of age.Prospective controlled study.Private IVF practice.A total of 30 women assigned and 22 qualified.Denudation of oocytes, cryopreservation of oocytes using vitrification method in a medium with 15% ethylene glycol (EG), 15% dimethylsulfoxide (DMSO), and 0.5 M sucrose.Oocyte survival, fertilization, day-3 embryo quality, blastocyst formation, clinical pregnancy, implantation, and live-birth rates.After denudation of oocytes, mature sibling oocytes were randomly allocated to the fresh and vitrified groups. The survival rate was 79.6% after vitrification/warming. Overall, no statistically significant differences were found in fertilization, day-3 embryo quality, or blastocyst formation rates between the fresh and vitrified groups. The positive β-human chorionic gonadotropin, clinical pregnancy rate, and implantation rate were 13 (59.0%) of 22, 10 (45.4%) of 22, and 16 (30.1%) of 53 for the vitrified group. The overall efficiency in achieving a live birth was 11 (5.9%) of 186 per vitrified oocyte.The impact of vitrification can be reduced to a minimal level, making it possible to achieve high pregnancy and implantation rates in this age group of IVF patients.

ObjectiveTo evaluate if PGS with array-CGH combined with blastocyst stage biopsy is a more efficient approach than the traditional FISH analyzes combined with day-3 embryo biopsy or no biopsy (control).DesignRetrospective matched control study.Materials and Methods30 patients had biopsy on blastocyst; (cryopreserved and ET in subsequent cycle) using aCGH (PGS/CGH group); 72 patients had PGS using FISH (PGS/FISH group; biopsy on day-3 with day-5 fresh ET) and 103 IVF patients having the same demographic were selected without PGS (Control; day-5 fresh ET). Clinical and laboratory data were analyzed by One-way ANOVA (with Bonferroni post-hoc test) and Fisher's exact tests with P<0.05.ResultsIn the PGS/CGH, PGS/FISH and Control groups, ages of female patients were 37.4 (+/-2.9), 38.4 (+/-2.4) and 38.5 (+/-2.1) (NS); the mean number of zygotes obtained were 11.9 (+/-5.4), 11.3 (+/-4.9) and 10.0 (+/-4.4) (NS); the mean number of embryos biopsied were 8.3 (+/-3.5), 10.3 (+/-4.2) and 0 (P<0.05); and the mean number of embryos transferred were 1.6 (+/-0.9), 1.6 (+/-0.8) and 2.1 (+/-1.3) (P<0.05 between PGS/FISH and Control). Proportion of euploid embryos were significantly higher (P<0.05) in the PGS/CGH group (40.6%) compared to the PGS/FISH group (26.3%). Clinical pregnancy rate was significantly higher in the PGS/CGH group (60.9%, P<0.05) compared to PGS/FISH (25.4%) and not different to Control (41.2% P=0.057). Implantation rate was significantly higher (P<0.05) in the PGS/CGH group (45.9%) compared either to PGS/FISH (24.8%) or to Control (29.2%). Logistic regression analysis adjusted for female age and number of embryos transferred shown that day5 CGH improved clinical pregnancy chance by 2.45 (95%CI 1.12 to 6.71) compared to control.ConclusionThese results show that PGS using aCGH with blastocyst biopsy provides higher implantation and pregnancy rates than day-3/FISH or control and also confirms that day-3 biopsy with FISH does not improve outcomes. ObjectiveTo evaluate if PGS with array-CGH combined with blastocyst stage biopsy is a more efficient approach than the traditional FISH analyzes combined with day-3 embryo biopsy or no biopsy (control). To evaluate if PGS with array-CGH combined with blastocyst stage biopsy is a more efficient approach than the traditional FISH analyzes combined with day-3 embryo biopsy or no biopsy (control). DesignRetrospective matched control study. Retrospective matched control study. Materials and Methods30 patients had biopsy on blastocyst; (cryopreserved and ET in subsequent cycle) using aCGH (PGS/CGH group); 72 patients had PGS using FISH (PGS/FISH group; biopsy on day-3 with day-5 fresh ET) and 103 IVF patients having the same demographic were selected without PGS (Control; day-5 fresh ET). Clinical and laboratory data were analyzed by One-way ANOVA (with Bonferroni post-hoc test) and Fisher's exact tests with P<0.05. 30 patients had biopsy on blastocyst; (cryopreserved and ET in subsequent cycle) using aCGH (PGS/CGH group); 72 patients had PGS using FISH (PGS/FISH group; biopsy on day-3 with day-5 fresh ET) and 103 IVF patients having the same demographic were selected without PGS (Control; day-5 fresh ET). Clinical and laboratory data were analyzed by One-way ANOVA (with Bonferroni post-hoc test) and Fisher's exact tests with P<0.05. ResultsIn the PGS/CGH, PGS/FISH and Control groups, ages of female patients were 37.4 (+/-2.9), 38.4 (+/-2.4) and 38.5 (+/-2.1) (NS); the mean number of zygotes obtained were 11.9 (+/-5.4), 11.3 (+/-4.9) and 10.0 (+/-4.4) (NS); the mean number of embryos biopsied were 8.3 (+/-3.5), 10.3 (+/-4.2) and 0 (P<0.05); and the mean number of embryos transferred were 1.6 (+/-0.9), 1.6 (+/-0.8) and 2.1 (+/-1.3) (P<0.05 between PGS/FISH and Control). Proportion of euploid embryos were significantly higher (P<0.05) in the PGS/CGH group (40.6%) compared to the PGS/FISH group (26.3%). Clinical pregnancy rate was significantly higher in the PGS/CGH group (60.9%, P<0.05) compared to PGS/FISH (25.4%) and not different to Control (41.2% P=0.057). Implantation rate was significantly higher (P<0.05) in the PGS/CGH group (45.9%) compared either to PGS/FISH (24.8%) or to Control (29.2%). Logistic regression analysis adjusted for female age and number of embryos transferred shown that day5 CGH improved clinical pregnancy chance by 2.45 (95%CI 1.12 to 6.71) compared to control. In the PGS/CGH, PGS/FISH and Control groups, ages of female patients were 37.4 (+/-2.9), 38.4 (+/-2.4) and 38.5 (+/-2.1) (NS); the mean number of zygotes obtained were 11.9 (+/-5.4), 11.3 (+/-4.9) and 10.0 (+/-4.4) (NS); the mean number of embryos biopsied were 8.3 (+/-3.5), 10.3 (+/-4.2) and 0 (P<0.05); and the mean number of embryos transferred were 1.6 (+/-0.9), 1.6 (+/-0.8) and 2.1 (+/-1.3) (P<0.05 between PGS/FISH and Control). Proportion of euploid embryos were significantly higher (P<0.05) in the PGS/CGH group (40.6%) compared to the PGS/FISH group (26.3%). Clinical pregnancy rate was significantly higher in the PGS/CGH group (60.9%, P<0.05) compared to PGS/FISH (25.4%) and not different to Control (41.2% P=0.057). Implantation rate was significantly higher (P<0.05) in the PGS/CGH group (45.9%) compared either to PGS/FISH (24.8%) or to Control (29.2%). Logistic regression analysis adjusted for female age and number of embryos transferred shown that day5 CGH improved clinical pregnancy chance by 2.45 (95%CI 1.12 to 6.71) compared to control. ConclusionThese results show that PGS using aCGH with blastocyst biopsy provides higher implantation and pregnancy rates than day-3/FISH or control and also confirms that day-3 biopsy with FISH does not improve outcomes. These results show that PGS using aCGH with blastocyst biopsy provides higher implantation and pregnancy rates than day-3/FISH or control and also confirms that day-3 biopsy with FISH does not improve outcomes.

Using sibling oocytes, the objective of this study was to compare the intracytoplasmic sperm injection (ICSI) fertilization rates to those achieved with conventional IVF in patients with high rates of oocyte immaturity. This study was observational in nature, and included 91 patients who were treated using split insemination techniques. The fertilization rates for the ICSI group and the IVF group were 41.1 +/- 15.0% and 53.2 +/- 19.8%, respectively (P <: 0.0001). There was no significant difference in day-3 embryo quality between the two groups. There was a significantly higher number of embryos frozen in the IVF group than in the ICSI group: 357 (84.8%) and 297 (76.7%), respectively (P = 0.037). Furthermore, the number of embryos either transferred or frozen was significantly higher in the IVF group than the ICSI group: 459 of 1173 (39.1%) and 385 of 1268 (30.4%), respectively (P < 0.0001). These data indicate that conventional IVF results in a higher fertilization rate than ICSI. Furthermore, IVF provided more embryos available for transfer or cryopreservation when compared with ICSI, thereby optimizing the patient's cycle.

Recent clinical reports not only show that cryopreserved embryos can be successfully used for human fertility treatment, but also that cryopreserved oocytes may be used successfully as an adjunct to human assisted reproductive technologies. Vitrification is known to establish a glass-like solid state during the cooling process. The high concentration of cryoprotectants and an extremely rapid rate of cooling are responsible for the formation of the solid state, and also prevent formation of intracellular ice crystals. Hence, in theory, vitrification should minimize cryo-injuries, and therefore has great promise for oocyte and embryo cryopreservation. This article describes two pregnancies from vitrified-warmed blastocysts obtained after intracytoplasmic sperm injection fertilization of vitrified-wanned oocytes. Vitrification was employed to cryopreserve the oocytes and the subsequent blastocysts. The results present the intriguing implication that vitrification may serve as an efficient method for clinical oocyte cryopreservation and embryo re-cryopreservation.

This study aimed to evaluate oocyte vitrification efficiency using in-vivo matured (IVO) versus rescued in-vitro matured (IVM) oocytes. The results show that oocyte survival (85% versus 81%), fertilization (86% versus 76%) and cleavage rate (98% versus 89%) was not significantly different in IVO oocytes compared with rescued IVM sibling oocytes. The fertilized oocytes from IVO and IVM groups were cultured to blastocyst stage; however, embryo development was significantly reduced in the rescued IVM group (72% versus 15%). Embryo transfer was only performed with the embryos derived from IVO oocytes on day 5; 42 blastocysts were transferred to 18 recipients; 16 of 18 recipients had positive beta-human chorionic gonadotrophin and a total of 26 fetal cardiac activities were detected in 15 recipients (implantation: 26/42, 61.9%). Ten of the 15 recipients have delivered 19 healthy babies, and the other five pregnancies are still ongoing. These data indicate that the combination of oocyte vitrification and rescued IVM not only yield a new strategy to extend the pool of total fertilizable oocytes, but also demonstrate that the efficiency of vitrified/warmed oocytes can be comparable to fresh oocytes with regard to clinical outcomes.

Objectives: The recovery of immature human oocytes is a common occurrence in routine IVF. The effect of artificially removing a germinal vesicle (GV) or Metaphase I (MI) stage oocyte from its follicular environment is largely unknown, but potential damage to cell cycle regulatory mechanisms is likely. Here, we assessed chromosomal status of the embryos that developed from MI oocytes which had undergone ICSI within four hours post egg retrieval.Design: A prospective observational study involving 69 ICSI-IVF cycles. Fertilization, embryo development and chromosome status of microinjected MI ooctes was assessed by fluorescent in situ hybridization (FISH).Materials and Methods: Oocytes were exposed to hyaluronidase for 1 minute immediately following egg retrieval and returned to culture. After thirty minutes coronal cells were removed, and oocytes were assessed for maturity. Intracytoplasmic sperm injection (ICSI) was carried out on all MI oocytes within 3–6 hours post egg retrieval, and all MI injected and fertilized eggs were cultured separately. Seventy-two hours post egg retrieval, embryos derived from MI injected oocytes were scored and reserved for blastocyst culture if they were at least 6 cells and had less than 20% fragmentation. Embryos which did not meet blastocyst culture criteria underwent zona removal, disaggregation and fixation. Five color fluorescent in situ hybridization (FISH) for chromosomes X,Y,13,18,21 was later carried out for aneuploidy assessment of the fixed cells.Results: One hundred and eighteen MI oocytes underwent ICSI and 31 of them displayed 2PN (26.5%), 2 of them showed 1PN, 4 showed 3PN and 81 eggs did not display any pronuclei. Sixteen embryos (that had 2PN fertilization) were fixed and results were obtained on fifteen. A total of 93 cells were fixed, of which 60 (66.6%) provided results. Nine cells that were fixed were multinucleated (15%). Only one embryo had a normal chromosomal content, while the remaining fourteen embryos were chaotic mosaics.Conclusion: The results of the present study indicate that ICSI of MI oocytes not only results in a lower than normal fertilization rate, but also produces a very high proportion of chromosomally abnormal early cleavage embryos. It may be hypothesized that premature activation of an oocyte which has not completed meiosis I may alter cell cycle regulatory pathways and checkpoints which are responsible for preventing further cleavage and development of an abnormal cell. Moreover, the presence of the sperm centrosome prior to completion of meiosis I may alter the bipolarity of the first meiotic spindle resulting in an abnormal separation of the homologues which will consequently lead to a chaotic mosaic embryo, as it was observed in our study. These results indicate that ICSI of MI oocytes should not be used for clinical purposes. However, it may be an important model to study timing of oocyte activation and spindle modifications. Objectives: The recovery of immature human oocytes is a common occurrence in routine IVF. The effect of artificially removing a germinal vesicle (GV) or Metaphase I (MI) stage oocyte from its follicular environment is largely unknown, but potential damage to cell cycle regulatory mechanisms is likely. Here, we assessed chromosomal status of the embryos that developed from MI oocytes which had undergone ICSI within four hours post egg retrieval. Design: A prospective observational study involving 69 ICSI-IVF cycles. Fertilization, embryo development and chromosome status of microinjected MI ooctes was assessed by fluorescent in situ hybridization (FISH). Materials and Methods: Oocytes were exposed to hyaluronidase for 1 minute immediately following egg retrieval and returned to culture. After thirty minutes coronal cells were removed, and oocytes were assessed for maturity. Intracytoplasmic sperm injection (ICSI) was carried out on all MI oocytes within 3–6 hours post egg retrieval, and all MI injected and fertilized eggs were cultured separately. Seventy-two hours post egg retrieval, embryos derived from MI injected oocytes were scored and reserved for blastocyst culture if they were at least 6 cells and had less than 20% fragmentation. Embryos which did not meet blastocyst culture criteria underwent zona removal, disaggregation and fixation. Five color fluorescent in situ hybridization (FISH) for chromosomes X,Y,13,18,21 was later carried out for aneuploidy assessment of the fixed cells. Results: One hundred and eighteen MI oocytes underwent ICSI and 31 of them displayed 2PN (26.5%), 2 of them showed 1PN, 4 showed 3PN and 81 eggs did not display any pronuclei. Sixteen embryos (that had 2PN fertilization) were fixed and results were obtained on fifteen. A total of 93 cells were fixed, of which 60 (66.6%) provided results. Nine cells that were fixed were multinucleated (15%). Only one embryo had a normal chromosomal content, while the remaining fourteen embryos were chaotic mosaics. Conclusion: The results of the present study indicate that ICSI of MI oocytes not only results in a lower than normal fertilization rate, but also produces a very high proportion of chromosomally abnormal early cleavage embryos. It may be hypothesized that premature activation of an oocyte which has not completed meiosis I may alter cell cycle regulatory pathways and checkpoints which are responsible for preventing further cleavage and development of an abnormal cell. Moreover, the presence of the sperm centrosome prior to completion of meiosis I may alter the bipolarity of the first meiotic spindle resulting in an abnormal separation of the homologues which will consequently lead to a chaotic mosaic embryo, as it was observed in our study. These results indicate that ICSI of MI oocytes should not be used for clinical purposes. However, it may be an important model to study timing of oocyte activation and spindle modifications.

Objectives: Removal of a single blastomere for preimplantation genetic diagnosis (PGD) from day-3 embryo is a well established method. However, it is unknown whether the method of choice for zona pellucida (ZP) drilling for the biopsy procedure will influence further embryonic development. The most commonly applied technique for perforation of the ZP is zona drilling using acidified Tyrode's solution (AT), while only a few centers employ laser-assisted hatching (LAH). Because of the lack of studies comparing the two methods, many centers are reluctant to switch to the simpler and faster laser drilling. Thus, in the present study we assessed two zona drilling methods in terms of blastocyst development rates using sister embryos. Design: Prospective, randomized study. Sister embryos of 14 patients were randomly assigned to (1) AT zona drilling or to (2) laser zona drilling on day 3. After biopsy, subsequent embryo culture until the blastocyst stage (day 5) was performed. Materials and Methods: A total of 14 IVF-PGD cycles were included into the study that was performed between January and March of 2003. Patients underwent controlled ovarian hyperstimulation using recombinant FSH after pituitary down-regulation with long leuprolide acetate protocol. Embryos from the same patients (eligible for biopsy: >5 cells and <30% fragmentation) were randomly divided into two groups on day 3. In Group 1 embryos were submitted to Acidified Tyrode's zona drilling and in Group 2 embryos underwent laser-assisted hatching using a 1.48 μm diode laser (Octax). In both groups an opening of the zona of 15 to 20 micro-meter was formed followed by the removal of a single blastomere. Embryos were cultured sequentially in Sage cleavage and blastocyst medium until day 5. For statistical analysis One-way ANOVA, Kruskal-Wallis and chi-square tests were applied whenever appropriate. Results: The mean age (±S.D.) of the women in this study was 36.9 (±3.9). A total of 285 oocytes were recovered, and 175 oocytes fertilized normally (61%). There was an equal distribution of embryos in both groups based on cell stage, and percent fragmentation. Fourteen patients achieved pregnancy (50%) after transferring 30 blastocysts. The results are summarized in Table 1. Tabled 1 Conclusions: Blastocyst development rates (and blastocyst quality) were similarly high in both the AT group and in LAH group, indicating that laser hatching does not impair embryonic development to the blastocyst stage. Implantation rates were not compared because patients often had a combination of AT and LAH embryos used for transfer. Given that an equal number of LAH and AT embryos were transferred resulting in a 50% pregnancy rate, the data suggests that zona drilling by laser micro-beam does not produce additional risks for embryonic development beyond the blastocyst stage. Additionally, the results of the present study demonstrate that LAH is a suitable alternative to the use of acidified Tyrode's solution for zona drilling.

Objectives: Different factors may contribute to the success of frozen-thawed, cleavage stage embryo transfer (ET) including intactness/damage rate of embryos after the thawing procedure. It is observed that pregnancy and implantation rates are usually lower in cryo-ET cycles, even when most contributing factors appear to be optimal, simply because of the cryodamage to the embryos. The freezing-thawing procedure may impact embryos in different negative ways such as hardening of the zona pellucida and degeneration of blastomeres which may lead to impaired embryonic development and consequently to lower implantation and pregnancy rates. Therefore, the objective of the present study was to investigate the combined effect of laser-assisted hatching and the removal of degenerated blastomeres on implantation and pregnancy rates in cryo-ET cycles where cleavage stage embryos were transferred. Design: A total of 76 cryo-ET cycles were randomized prospectively into two groups. In 38 cycles, laser-assisted hatching and removal of degenerated blastomeres were performed on the frozen-thawed embryos (study group). In the other 38 cycles, there was no intervention performed on the thawed embryos before ET (control group). Materials and Methods: The study was performed between September 2002 and March 2003. Patients in the fresh cycle underwent controlled ovarian hyperstimulation using recombinant FSH after pituitary down-regulation with long leuprolide acetate protocol. Supernumerary embryos eligible for freezing (5 cells or more and <25% fragmentation) were cryopreserved on day 3 using the propenediol slow-freezing method (Vitrolife). Embryos were thawed approximately 80 hours after hCG administration in a leuprolide acetate and oral micronized estradiol replacement cycle. Embryos from the study group were submitted to laser-assisted hatching, using a 1.48 μm diode laser (Octax), followed by the removal of degenerated blastomeres. Embryos were transferred 3 to 5 hours after the thawing using the Edwards-Wallace catheter. For statistical analysis One-way ANOVA, Kruskal-Wallis and chi-square tests were applied whenever appropriate. Results: Results are summarized in the table. Oocyte number per patient, fertilization and embryo developmental rate (and quality) were similar in the two groups in the fresh cycle. Tabled 1 Conclusions: The results of the present study suggest that opening of the zona pellucida (by laser-assisted hatching) and removal of the degenerated blastomeres of the frozen-thawed embryos increases implantation and pregnancy rates. It may be hypothesized that impaired embryo viability after cryopreservation is related, at least in part, to the altered features of the zona pellucida and to the presence of damaged blastomeres. Based on the outcome of this study, the combined procedure of laser hatching and removal of degenerated blastomeres should be recommended to all patients who undergo cleavage stage, frozen-thawed embryo transfer.

Objective: Historically, embryo transfers following embryo biopsy and preimplantation diagnosis (PGD) were performed on day 3 or 4. With the development of sequential media and blastocyst transfer, patients undergoing PGD now have the option of a day 4 or day 5 transfer. The objective of this study was to compare the pregnancy and implantation rates between patients who received a day 4 or day 5 embryo transfer following PGD for aneuploidy Design: Retrospective Analysis Materials/Methods: Embryo biopsy was performed in Ca2+/Mg2+ -free medium on day 3 and a diagnosis was determined using fluorescent in situ hybridization (FISH) for chromosomes X,Y,13,18 and 21. On the morning of day 4 all biopsied embryos were transferred into blastocyst culture medium. Patients who did not want their embryos cultured until day 5 had an embryo transfer on day 4. Embryos which were not transferred remained in culture until day 6. Results: Blastocyst transfer was offered to 50 patients post preimplantation genetic diagnosis. Twenty-two patients chose to have a day 5 transfer, while the remaining 40 requested to have a day 4 transfer. In the blastocyst transfer group 56% (13/23) of the patients had a positive pregnancy test of which 52% (12/23) are ongoing. The average age in this group was 38 and the implantation rate was 27% (16/59). Patients who received a day 4 embryo transfer had a 33.3%% (9/27) pregnancy rate of which 26% (7/27) are ongoing. The average age in this group was 39 and the implantation rate was 12% (8/66). Conclusions: The removal of a cell or cells from a day 3 embryo for the purpose of PGD does not preclude the subsequent in vitro development of that embryo to the blastocyst stage. The transfer of blastocysts instead of day 4 embryos post PGD may allow the embryo to better withstand the trauma of embryo transfer procedure. In this study blastocysts replaced to the uterus had a higher implantation rate than embryos transferred on day 4. Culture to the blastocyst stage may allow for selection of embryos for ET when multiple chromosomally normal embryos are available, or allow extra time for performing the genetic diagnosis or rechecking results. Many smaller IVF centers cannot justify the cost of equipment needed for PGD, though a micromanipulator is available. Cells removed from embryos can be shipped to a reference laboratory for analysis, allowing 48 hours for reporting results before embryo transfer.

Feasibility of cryopreservation of mature human oocytes using 1, 2-propanediol and sucrose was studied initially utilizing 1 and 2 day old unfertilized oocytes. Of these 285 aged oocytes 55% survived thawing, and 41% of 128 oocytes inseminated by single sperm intracytoplasmic injection (ICSI) fertilized normally. Limited embryonic development occurred in 51% of these embryos (n = 27) observed for the next 4 days. Cryosurvival of fresh donated oocytes (n = 81) was poorer (n = 20; 24.7%), while fertilization (n = 13; 65%) and embryo development (100%) was good prior to uterine transfer on day 3. Eight oocyte recipient cycles were undertaken, in which cryopreserved donated oocytes were thawed and inseminated by ICSI. Five of these cycles reached embryo transfer, and three pregnancies were initiated though none went successfully to term. Oocyte cryopreservation will ultimately facilitate oocyte donation procedures; however, cryosurvival of fresh frozen oocytes must be improved to at least the degree observed with aged unfertilized oocytes.

: Feasibility of cryopreservation of mature human oocytes using 1, 2-propanediol and sucrose was studied initially utilizing 1 and 2 day old unfertilized oocytes. Of these 285 aged oocytes 55% survived thawing, and 41% of 128 oocytes inseminated by single sperm intracytoplasmic injection (ICSI) fertilized normally. Limited embryonic development occurred in 51% of these embryos (n = 27) observed for the next 4 days. Cryosurvival of fresh donated oocytes (n = 81) was poorer (n = 20; 24.7%), while fertilization (n = 13; 65%) and embryo development (100%) was good prior to uterine transfer on day 3. Eight oocyte recipient cycles were undertaken, in which cryopreserved donated oocytes were thawed and inseminated by ICSI. Five of these cycles reached embryo transfer, and three pregnancies were initiated though none went successfully to term. Oocyte cryopreservation will ultimately facilitate oocyte donation procedures; however, cryosurvival of fresh frozen oocytes must be improved to at least the degree observed with aged unfertilized oocytes.