O-225. Early cleavage of in-vitro fertilized human embryos to the 2-cell stage: a novel indicator of embryo quality and viability

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

Sperm nuclear DNA damage and altered chromatin structure: effect on fertilization and embryo development

In the first part of this report we investigate whether chromatin anomalies in human spermatozoa can influence fertilization after intracytoplasmic sperm injection (ICSI). We have examined the sperm chromatin packaging quality using the chromomycin A3 (CMA3) fluorochrome and the presence of DNA damage in spermatozoa using in-situ nick translation. When comparing the spermatozoa of patients undergoing in-vitro fertilization (IVF) and ICSI distinct differences are evident in that ICSI males have a higher CMA3 fluorescence, indicating spermatozoa with loosely packed chromatin, and more spermatozoa containing endogenous DNA nicks. When examining the unfertilized oocytes of ICSI patients we found that men who had a high percentage of anomalies in their chromatin, i.e. >30% CMA3 fluorescence and >10% nicks, had more than double the number of unfertilized oocytes containing spermatozoa that had remained condensed. The observation that failed fertilized oocytes, injected with spermatozoa from patients with a higher percentage of sperm nuclear anomalies, contain more condensed spermatozoa indicates that a selection process against these spermatozoa may be in place at the time of fertilization. In the second part of the study we show that spare ICSI embryos have significantly lower rates of development to the blastocyst stage compared with those developed after routine IVF. These results show that a greater understanding of the molecular basis of male infertility is therefore needed to broaden our knowledge on the effect that abnormal spermatozoa have on fertilization and embryo developmen

Paternal effect on genomic activation, clinical pregnancy and live birth rate after ICSI with cryopreserved epididymal versus testicular spermatozoa

<p>Abstract</p> <p>Background</p> <p>This study takes an in depth look at embryonic development, implantation, pregnancy and live birth rates with frozen epididymal and testicular sperm from obstructed (OA) and non-obstructed (NOA) patients.</p> <p>Methods</p> <p>Paternal effect of sperm source on zygote formation, embryonic cleavage, and genomic activation were examined. Additional outcome parameters monitored were clinical pregnancy rate (CPR), implantation rate (IR) and live birth rate.</p> <p>Results</p> <p>In this report, we retrospectively analyzed 156 ICSI cycles using cryopreserved epididymal sperm (ES; n = 77) or testicular sperm (TESE; n = 79). The developmental potential of embryos did not appear to be influenced by the type of surgically retrieved sperm. The average number of blastomeres observed on Day 3 was not different among different groups; 7.5 +/- 1.7 (ES), 7.6 +/- 2.1 (TESE-OA) and 6.5 +/- 2.3 (TESE-NOA). Compaction and blastulation rates, both indicators of paternal genomic activation, were similar in embryos derived from ICSI with ES or TESE from OA as well as NOA men. The only parameter significantly affected in NOA-TESE cases was the fertilization rate. CPR and IR with cryopreserved TESE (TESE-OA 59%, 34%, and TESE-NOA 37%, 20%) were also not statistically different, from that achieved with cryopreserved ES (61% and 39%). Live birth rates also appeared to be independent of sperm type. The 87 clinical pregnancies established using cryopreserved TESE and ES, resulted in the birth of 115 healthy infants. No congenital anomalies were noted.</p> <p>Conclusion</p> <p>Zygotic activation seems to be independent of sperm origin and type of azoospermia.</p

Impact of GnRH analogues on oocyte/embryo quality and embryo development in in vitro fertilization/intracytoplasmic sperm injection cycles: a case control study

<p>Abstract</p> <p>Background</p> <p>Despite the clinical outcomes of ovarian stimulation with either GnRH-agonist or GnRH-antagonist analogues for in vitro fertilization (IVF) being well analysed, the effect of analogues on oocyte/embryo quality and embryo development is still not known in detail. The aim of this case-control study was to compare the efficacy of a multiple-dose GnRH antagonist protocol with that of the GnRH agonist long protocol with a view to oocyte and embryo quality, embryo development and IVF treatment outcome.</p> <p>Methods</p> <p>Between October 2001 and December 2008, 100 patients were stimulated with human menopausal gonadotrophin (HMG) and GnRH antagonist in their first treatment cycle for IVF or intracytoplasmic sperm injection (ICSI). One hundred combined GnRH agonist + HMG (long protocol) cycles were matched to the GnRH antagonist + HMG cycles by age, BMI, baseline FSH levels and by cause of infertility. We determined the number and quality of retrieved oocytes, the rate of early-cleavage embryos, the morphology and development of embryos, as well as clinical pregnancy rates. Statistical analysis was performed using Wilcoxon's matched pairs rank sum test and McNemar's chi-square test. P < 0.05 was considered statistically significant.</p> <p>Results</p> <p>The rate of cytoplasmic abnormalities in retrieved oocytes was significantly higher with the use of GnRH antagonist than in GnRH agonist cycles (62.1% vs. 49.9%; P < 0.01). We observed lower rate of zygotes showing normal pronuclear morphology (49.3% vs. 58.0%; P < 0.01), and higher cell-number of preembryos on day 2 after fertilization (4.28 vs. 4.03; P < 0.01) with the use of GnRH antagonist analogues. The rate of mature oocytes, rate of presence of multinucleated blastomers, amount of fragmentation in embryos and rate of early-cleaved embryos was similar in the two groups. Clinical pregnancy rate per embryo transfer was lower in the antagonist group than in the agonist group (30.8% vs. 40.4%) although this difference did not reach statistical significance (P = 0.17).</p> <p>Conclusion</p> <p>Antagonist seemed to influence favourably some parameters of early embryo development dynamics, while other morphological parameters seemed not to be altered according to GnRH analogue used for ovarian stimulation in IVF cycles.</p

Promising System for Selecting Healthy In Vitro–Fertilized Embryos in Cattle

Conventionally, in vitro–fertilized (IVF) bovine embryos are morphologically evaluated at the time of embryo transfer to select those that are likely to establish a pregnancy. This method is, however, subjective and results in unreliable selection. Here we describe a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse cinematography in our developed microwell culture dish and analyzes embryonic metabolism. The system can noninvasively identify prognostic factors that reflect not only blastocyst qualities detected with histological, cytogenetic, and molecular analysis but also viability after transfer. By assessing a combination of identified prognostic factors—(i) timing of the first cleavage; (ii) number of blastomeres at the end of the first cleavage; (iii) presence or absence of multiple fragments at the end of the first cleavage; (iv) number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle; and (v) oxygen consumption at the blastocyst stage—pregnancy success could be accurately predicted (78.9%). The conventional method or individual prognostic factors could not accurately predict pregnancy. No newborn calves showed neonatal overgrowth or death. Our results demonstrate that these five predictors and our system could provide objective and reliable selection of healthy IVF bovine embryos

Blastocyst development from supernumerary embryos after intracytoplasmic sperm injection: a paternal influence?

The success of intracytoplasmic sperm injection (ICSI) warrants further study on the role of paternal factors in early human embryogenesis. To investigate whether poor sperm parameters can influence embryo development, we examined the development of ICSI-fertilized embryos to the blastocyst stage. We present results of blastocyst development from supernumerary ICSI embryos after co-culture on monkey kidney epithelial cells. In addition, we compare the development of supernumerary embryos to the blastocyst stage after ICSI and in-vitro fertilization (IVF). Of 168 supernumerary ICSI embryos, 45 (26.8%) developed to blastocysts. Sperm concentration and morphology did not influence blastocyst development. In contrast, blastocysts arose from spermatozoa that had a significantly higher (P = 0.015) forward progressive motility compared with spermatozoa from those patients who failed to produce blastocysts (42.7% versus 28.2%, respectively). Overall the rate of embryo development to the blastocyst stage after ICSI was lower (26.8%) than that after IVF (47.3%). When the rate of blastocyst development was calculated for patients with three or more supernumerary embryos, it remained significantly higher for the IVF patients than for the ICSI patients (45.6% versus 30.0%). There was no significant difference in the mean cell number and quality of the supernumerary embryos between the IVF and ICSI patients. This study confirms previous reports that have postulated that abnormal spermatozoa may manifest a negative paternal effect on preimplantation embryo developmen

Early cleavage of human embryos to the two-cell stage after intracytoplasmic sperm injection as an indicator of embryo viability

In-vitro fertilization (IVF) embryos are selected for transfer on the basis of morphology and rate of development. However, when a number of embryos have similar characteristics, the selection of the best embryos is left to chance. Recently, we proposed a simple, novel method to overcome this problem, based on pre-selection of embryos cleaving early to the two-cell stage. In this study we have adopted the same method to choose embryos fertilized after intracytoplasmic sperm injection (ICSI). Fertilized embryos that had cleaved to the two-cell stage by 27 h post-injection were designated as 'early cleavage' embryos, while those that had not yet reached the two-cell stage were designated as 'no early cleavage'. In all cases, the early cleavage embryos were transferred when available. Early cleavage was observed in 54 (61.4%) of the 88 cycles assessed. There were significantly (P = 0.04) more clinical pregnancies in the early cleavage group, 14/54 (25.9%), compared with the no early cleavage group 2/34 (3.2%). No differences between the groups were found when comparing key parameters (age, stimulation protocol and semen characteristics) of the couples. Using the ICSI technique, we have shown that early cleavage to the two-cell stage is not influenced by the timing of fertilization, and is more likely due to intrinsic factors within the oocyte or embryo that promote embryo cleavage after fertilizatio

The rate of development and time of transfer play different roles in influencing the viability of human blastocysts

Improved embryo culture protocols now render more feasible the possibility of obtaining human blastocysts after in-vitro fertilization. In this study we present: (i) results of blastocyst development from supernumerary embryos after co-culture on green monkey kidney epithelial cells and (ii) pregnancy rates after transfer of frozen blastocysts. In addition, we have examined the influence of the day of blastocyst freezing and the day of transfer after the luteinizing hormone (LH) peak on pregnancy and implantation rates. Of 423 supernumerary embryos, 200 developed to the blastocyst stage (47.3%). By days 5 and 6, 67% of the blastocysts had reached the blastocyst stage, and were frozen, compared to 28.5% by day 7. When we compared the cases where only blastocysts frozen on days 5 and 6 were transferred compared to those frozen and transferred on or after day 7 the pregnancy rates were 7/18 (38.9%) and 1/16 (6.2%) respectively. In contrast, when we examined the influence of the day of transfer we found that pregnancies were established from day 5 up to day 9 post LH peak. Based on these results, we suggest that every attempt should be made to increase the development rate of supernumerary embryos to the blastocyst stage, as it appears that the quality of blastocysts transferred, as shown in this study by rate of development, plays a more crucial role than the timing of transfe