Search

Does conventional blastocyst morphological evaluation correlate with euploidy (as assessed by comprehensive chromosome screening (CCS) of trophectoderm (TE) biopsies) and implantation potential?A 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.Combined 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.This 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.PGS 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.Among 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 (P0.01) 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.The 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.This 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.No funding was obtained for the study. All authors have no conflicts to declare.None.

Objective To determine whether the process of oocyte vitrification affects oocyte viability in in vitro fertilization (IVF) patients between 30 and 39 years of age. Design Prospective controlled study. Setting Private IVF practice. Patient(s) A total of 30 women assigned and 22 qualified. Intervention(s) 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. Main Outcome Measure(s) Oocyte survival, fertilization, day-3 embryo quality, blastocyst formation, clinical pregnancy, implantation, and live-birth rates. Result(s) 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. Conclusion(s) 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.

The aim of this study was to determine whether the denuding procedure causes the polar body to move within the perivitelline space. Only those patients undergoing IVF who had unused in-vitro matured (IVM) oocytes were included in this study. IVM oocytes were initially viewed under a non-invasive, polarized light microscope. A laser was used to mark the location of the polar body on the zona. Oocytes were subjected to the denuding procedure with a 150 microm, 135 microm and 125 mum diameter pipette. After each pipetting, the oocytes were viewed again to determine whether the polar body had moved. After denuding, the oocyte was left to culture overnight and viewed 24 h later. After denuding with the 150 microm, 135 microm and 125 microm pipettes and after 24 h in culture, the average angle between the spindle and polar body was 15.4 +/- 10.4 degrees , 16.1 +/- 10.1 degrees , 20.9 +/- 11.7 degrees , and 26.7 +/- 18.2 degrees , respectively (P = 0.0021). Slight changes in angles were noted between denuding with the different diameter pipettes. The largest changes in angles were seen after 24 h in culture.

Objective To develop a mouse model to investigate the possible causes for increased success rates when lysed cells are removed from thawed embryos. Design Experimental study. Setting Clinical IVF laboratory. Intervention(s) Assisted hatching, cell lysis, and removal of lysed cells. Main Outcome Measure(s) Embryonic growth rate and morphology. Result(s) The mouse embryos were divided into three groups; control (no cell lysis), group 1 (cell lysis and removal), and group 2 (cell lysis only). There was no significant difference in the initial number of blastomeres in each group or the number of cells lysed artificially in groups 1 and 2. The rate of embryonic development showed a significant delay in group 2 (7.97 ± 4.92; control, 10.42 ± 8.18; group 1, 5.74 ±4.42; group 2). The embryo morphology on day 4 was significantly improved in group 1 and the control group when compared with group 2. Conclusion(s) Mouse embryos with artificially lysed cells after thawing had poorer developmental quality and growth rates compared with control embryos. However, removal of lysed cells restored the embryo's developmental potential to that of the control. Cell number and morphology was also significantly improved compared with embryos without lysed cell removal. These findings are consistent with human embryo development after thawing when lysed cells are present and thus mechanical lysis seems to be an appropriate method by which to further study frozen–thawed lysed cell removal.

Objective To evaluate the effect of degenerated (lysed) blastomere removal on implantation and pregnancy rates in cleavage-stage cryo–embryo transfer (ET) cycles. Design Randomized clinical trial. Setting Private reproductive medical center. Patient(s) A total of 88 patients who received frozen–thawed ET, divided into two groups. Intervention(s) Embryo freezing and thawing; opening of the zona pellucida and removal of cryodamaged blastomeres (in the study group), followed by same-day ET. Main Outcome Measure(s) Extent of survival of cleavage-stage embryos after the freeze–thaw procedure; embryo implantation and clinical pregnancies. Result(s) Oocyte number per patient, fertilization rate, embryo development rate (and quality), and freezing rates were similar in the two groups in the fresh cycle. In the control group, a total of 55 embryos (25%) of the 217 thawed remained fully intact, and 53 (26%) of the 207 in the study group remained intact. The average number of embryos transferred per group was similar (control, 3.4 ± 0.9; study, 3.3 ± 0.9). Implantation rates were 12% and 26% in the control and study groups, respectively. The clinical pregnancy rate was 23% in the control group and 64% in the study group when lysed cell removal was performed. Conclusion(s) The results show that pregnancy and implantation rates are higher in the study group; therefore, the removal of degenerated blastomeres may be beneficial to all patients who undergo cleavage-stage, frozen–thawed ET.

The objective of the present study was to compare a traditionally used bovine-derived hyaluronidase (Hyase) with the newly developed recombinant human-derived enzyme product (Cumulase) in intracytoplasmic sperm injection (ICSI) procedures using a sibling oocyte model in a prospective randomized design. The results of the study demonstrate that Cumulase is safe and effective in an ICSI treatment program and can provide comparable if not improved parameters, including fertilization and embryo developmental rates.

STUDY QUESTION Does comprehensive chromosome screening (CCS) of cells sampled from the blastocyst trophectoderm (TE) accurately predict the chromosome complement of the inner cell mass (ICM)? SUMMARY ANSWER Comprehensive chromosome screening of a TE sample is unlikely to be confounded by mosaicism and has the potential for high diagnostic accuracy. WHAT IS KNOWN ALREADY 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. STUDY DESIGN, SIZE, DURATION 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. MATERIALS, SETTING, METHODS 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. Fisher's exact test was used to test for random allocation of aneuploid cells between TE and ICM. MAIN RESULTS AND THE ROLE OF CHANCE 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). Fisher's exact test for the 66 aneuploidies recorded showed no preferential allocation of abnormal cells between ICM and TE (P = 0.33). LIMITATIONS, REASONS FOR CAUTION 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. WIDER IMPLICATIONS OF THE FINDINGS 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. STUDY FUNDING/COMPETING INTEREST(S) No specific funding was sought or obtained for this study.

Our objective with this chapter is to give a brief overview of the conventional in vitro fertilization (IVF) procedure and some of the factors that determine success. The decision-making process of whether to do conventional IVF, intracytoplasmic sperm injection (ICSI), or split-ICSI is very complex. We present the influencing factors in a manner that allows thorough evaluation of every case, resulting in an educated, well thought through decision of which insemination method to use. We believe it will assist embryologists in deciding which method of fertilization to use in their labs.

Cryopreservation of human embryos was introduced more than 2 decades ago, mainly as an approach to save supernumerary embryos produced after ovarian hyperstimulation. The Society for Assisted Reproduction Technology and the European Society of Human Reproduction and Embryology both continuously publish statistics on embryo freezing and birth rates in the United States and Europe, respectively. These are valuable data for seeing the overall impact of cryopreservation on clinical infertility treatment. However, the data does not distinguish between methods of freezing, rendering it impossible to determine the current success rates resulting from slow freezing alone. Many factors are causing clinics to switch to vitrification instead of traditional slow freezing. Several studies suggest that vitrification results in significantly higher survival and developmental rates than slow freezing. A further driving force for an increased used of vitrification is the practicality of the technique. Apart from being much more cost effective, the procedure requires significantly less time when compared to the long equilibration methods required by typical slow-freezing protocols. Despite these clear advantages of vitrification, many labs still use slow freezing, mainly because of the wealth of clinical data that supports the technique. Switching a well-established, effective method for a new protocol also seems intimidating, thereby preventing implementation of newer technologies. We predict that, in the future, vitrification may become the sole method used for clinical embryo cryopreservation.

This chapter reviews the current status of assisted reproduction techniques in the light of the best evidence available. In order to optimize the results of assisted reproduction, various laboratory modifications have been suggested. These include performing Intracytoplasmic sperm injection (ICSI) rather than in vitro fertilization (IVF) for all oocytes, using co-culturing techniques, assisted hatching techniques, as well as selecting the embryos with the best potential for implantation based on their morphology, their metabolism, or by prolonging their culture in vitro to the blastocyst stage. Embryo transfer (ET) is arguably the most critical step in assisted reproduction and the least successful. Various attempts have been made to improve endometrial receptivity in order to increase the clinical outcome of IVF and ICSI. These include various regimens of luteal support, the use of corticosteroids, the removal of hydrosalpinges, diminishing uterine contractions as well as enhancing the endometrial blood flow.

The inorganic analysis of archaeological bone material can potentially provide a wealth of information about the chronology, diet and palaeoenvironment of past populations: for example, strontium and uranium levels are used in palaeodietary and dating studies, respectively. However, the extent to which the chemical composition of bone is subject to diagenetic change during burial is open to controversy due, in part, to differences in analytical technique, bone types and burial conditions. To investigate this problem, archaeological human bone material from a number of different geological environments including Pompeii and a 12th century British ecclesiastical site, together with material from two seawater burials (The “Mary Rose” and a 6th century Mediterranean wreck) have been studied using the nuclear microprobe facility at the University of Oxford. Results using microbeam PIXE show that bone is subject to contamination from a wide range of trace elements depending on the burial conditions. Elemental maps are presented to demonstrate the distribution of trace element accumulation under different burial conditions, and the significance of this work to future trace element studies is discussed.

The impact, process of attachment and subsequent spreading of drops of carbon tetrachloride at mercury/solution interfaces have been recorded photographically. The effect of the history of the drop, prior to attachment at the interface, on the subsequent spreading phenomena is discussed. The reproducibility of dynamic contact angles is demonstrated. The influence of anionic and cationic surface-active agents on the spreading of carbon tetrachloride drops at a mercury/solution interface has been studied. The rate of change of contact angle with time depended on the magnitudes of the interfacial free energies involved, and these depended on the nature and concentration of the surface-active agent. The effects of various factors such as gravity, temperature and natures of the phases on the spreading process have been considered.

The surface areas (A) per molecule of several typical anionic and cationic surface-active agents at carbon tetrachloride/water, and mercury/water interfaces have been determined by the application of Gibb's adsorption equation. Whereas a limiting value for A at the carbon tetrachloride/water interface was of the order 54 A2, values at the mercury/water interface were three to five times higher. This was probably due to the incomplete orientation of the molecules at the mercury/water interface. Spreading in the mercury/carbon tetrachloride/solution system was controlled by the magnitudes of the interfacial forces. Because of the rapid rate of establishment of equilibrium excesses at the interfaces, compared with the rate of interfacial expansion (or contraction) these forces were effectively constant. The spreading processes were exponential and followed first-order kinetics of the form: dθ dt = K(θ eq − θ t ) , where θeq is the value of θ at t∞, θt the value at time t, and K a constant only for a particular concentration of surface-active agent; it is proportional to the rate of the spreading process. The energy of activation of the spreading process was found to increase with increase in concentration of each surface-active agent. At a unique and reproducible “critical” concentration of each of the ionic surface-active agents, the mechanism of the spreading process changed abruptly, and the magnitude of the energy of activation then effectively remained constant, i.e., independent of concentration. At low surface concentrations of the ionic surface-active agents (apparently chemisorbed on the mercury surface) a mechanism is proposed in which these molecules are compressed ahead of the spreading drop. As the surface concentration increased, however, a situation was reached where this compression mechanism was no longer possible, due to the dense packing of surface-active molecules. After this stage, the drop proceeded to spread over the adsorbed layer of surface-active molecules. It has been shown that spreading coefficients can not only predict the position of the equilibrium stage but in some cases the speed of a spreading process as well. Better agreement may have been obtained if the difficulties involved in a change in mechanism of the spreading process had not occurred.

The process of attachment of carbon tetrachloride drops at mercury/solution of surface-active agent interfaces has been studied by means of high-speed cine photography. At smooth interfaces drops do not apparantly make single point attachments, but attach over relatively large basal areas. The contact angle on attachment depends on the difference in the works of adhesion of the two liquids for the mercury surface. In the present system the initial stages of spreading were mainly controlled by the mercury/ solution and mercury/carbon tetrachloride interfacial tensions. The contact angle during this stage was almost of 90°, and remained at this value as the base area increased. The drop maintained its ellipsoidal, preattachment shape until the base area equalled the maximum cross-sectional area, when drop distortion occurred and the contact angle (measured through the aqueous phase) exceeded 90°. The rapid processes of attachment and initial spreading (0.1 sec) and the manner in which the contact angle changed with time, have been shown to be very reproducible. The passage of the drop from an ellipsoidal shape to that of the cap of a sphere was marked by the induction of oscillations in the vertical plane, which produced vibrations in the spreading plane. For a given system, the effect of induced oscillations on the dynamic contact angle depended on the magnitude of the interfacial driving force causing spreading.

The surface tension of mercury in air has been found, by the drop-weight method, to be 476.0 ± 2.0 dynes/cm at 25°C. The values of the surface tensions of seven different grades of mercury, which varied from commercial to polarographic, were measured and found to have the same values (within experimental error). The sample grades were analyzed and were found to contain very similar base metal impurity levels, i.e.