Background: Studies have suggested that embryo-endometrial developmental asynchrony caused by slow-growing embryos can be corrected by freezing the embryo and transferring it back in a subsequent cycle. Therefore, we hypothesized that live birth rates (LBR) would be higher in frozen embryo transfer (FET) compared with fresh embryo transfers. Objective: To compare LBR between fresh and FET cycles. Materials and Methods: A cross-sectional analysis of 10,744 single autologous embryo transfer cycles that used a single cleavage stage embryo was performed. Multivariate analysis was performed to compare LBR between FET and fresh cycles, after correcting for various confounding factors. Sub-analysis was also performed in cycles using slow embryos. Results: Both LBR (19.13% vs 14.13%) and clinical pregnancy (22.48% vs 16.25%) rates (CPR) were higher in the fresh cycle group (p < 0.00). Multivariate analysis for confounding factors also confirmed that women receiving a frozen-thawed embryo had a significantly lower LBR rate compared to those receiving a fresh embryo (OR 0.76, 95% CI 0.68-0.86, p < 0.00). In the sub-analysis of 1,154 cycles using slow embryos, there was no statistical difference in LBR (6.40% vs 6.26%, p = 0.92) or CPR (8.10% vs 7.22%, p = 0.58) between the two groups. Conclusion: This study shows a lower LBR in FET cycles when compared to fresh cycles. Our results suggest that any potential gains in LBR due to improved embryo-endometrial synchrony following FET are lost, presumably due to freeze-thaw process-related embryo damage.

Storing female reproductive potential can offer enhanced prospects for future conception in women whose fertility is threatened by cytotoxic therapies. Human female reproductive potential can be cryopreserved and stored at very low temperatures as embryos or gametes. Gamete (oocyte) cryopreservation circumvents potential issues associated with ownership when future use is being considered and may, therefore, be more generally acceptable as an approach. Advances in the technology, in particular the clinical application of vitrification, have significantly improved the outcomes from mature oocyte cryopreservation, which are now comparable to those from embryo cryopreservation. In cases where mature oocyte cryopreservation is not feasible, ovarian cortex containing primordial follicles can be cryopreserved, and over 100 births have now been reported following grafting of stored ovarian tissue. Ovarian tissue cryopreservation is now an established approach to preserve future fertility for young women; however, the efficiency is difficult to determine particularly for the prepubertal tissue with a scarcity of data.

The ability to store human embryos in a viable state at very low temperatures has been critical to the evolution of responsible practice in clinical Assisted Reproductive Technology (ART). It has encouraged a reduction in the frequency of simultaneous multiple embryo transfer and thereby reduced the risks associated with multiple pregnancy while maintaining high cumulative pregnancy rates from single oocyte collection cycles. In this chapter, we describe a simple slow freezing procedure for human early cleavage stage embryos that results in a high proportion of post-thaw embryos surviving and retaining their implantation potential.

The ability to store human embryos in a viable state at very low temperatures has been critical to the evolution of responsible practice in clinical Assisted Reproductive Technology (ART). It has encouraged a reduction in the frequency of simultaneous multiple embryo transfer and thereby reduced the risks associated with multiple pregnancy while maintaining high cumulative pregnancy rates from single oocyte collection cycles. In this chapter, we describe a simple slow freezing procedure for human early cleavage stage embryos that results in a high proportion of post-thaw embryos surviving and retaining their implantation potential.

High survival rates and clinical outcomes similar to those from fresh oocytes and blastocysts have been observed with open oocyte vitrification systems. It has been suggested that the extremely fast cooling rates that are only achieved with open systems are necessary for human oocyte and blastocyst vitrification. However, there is a potential risk of introducing contamination with open systems. The aim of this study was to assess whether similar survival and subsequent implantation rates could be achieved using a closed vitrification system for human oocytes and blastocysts.

BACKGROUND:Vitrification is now a commonly applied technique for cryopreservation in assisted reproductive technology (ART) replacing, in many cases, conventional slow cooling methodology. This review examines evidence relevant to comparison of the two approaches applied to human oocytes and embryos at different developmental stages.METHODS:Critical review of the published literature using PubMed with particular emphasis on studies which include data on survival and implantation rates, data from fresh control groups and evaluation of the two approaches in a single setting.RESULTS:Slow cooling is associated with lower survival rates and compromised development relative to vitrification when applied to metaphase II (MII) oocytes, although the vitrification results have predominantly been obtained using direct contact with liquid nitrogen and there is some evidence that optimal protocols for slow cooling of MII oocytes are yet to be established. There are no prospective randomized controlled trials (RCTs) which support the use of either technique with pronuclear oocytes although vitrification has become the method of choice. Optimal slow cooling, using modifications of traditional methodology, and vitrification can result in high survival rates of early embryos, which implant at the same rate as equivalent fresh counterparts. Many studies report high survival and implantation rates following vitrification of blastocysts. Although slow cooling of blastocysts has been reported to be inferior in some studies, others comparing the two approaches in the same clinical setting have demonstrated comparable results. The variation in the extent of embryo selection applied in studies can lead to apparent differences in clinical efficiency, which may not be significant if expressed on a 'per oocyte used' basis.CONCLUSIONS:Available evidence suggests that vitrification is the current method of choice when cryopreserving MII oocytes. Early cleavage stage embryos can be cryopreserved with equal success using slow cooling and vitrification. Successful blastocyst cryopreservation may be more consistently achieved with vitrification but optimal slow cooling can produce similar results. There are key limitations associated with the available evidence base, including a paucity of RCTs, limited reporting of live birth outcomes and limited reporting of detail which would allow assessment of the impact of differences in female age. While vitrification has a clear role in ART, we support continued research to establish optimal slow cooling methods which may assist in alleviating concerns over safety issues, such as storage, transport and the use of very high cryoprotectant concentrations.

Previous reports of slow cooling of human mature oocytes have shown a reduced clinical efficiency relative to fresh oocytes. This study reports that equivalent fertilization and implantation rates to those obtained using fresh oocytes and cryopreserved embryos can be achieved with human mature oocytes dehydrated in 1.5 M propanediol and 0.2 M sucrose at 37°C and cryopreserved using slow cooling rates.

Increasing the proportions of embryos and blastomeres which survive cryopreservation would be expected to make a significant contribution to the outcome of assisted reproduction treatment. Despite this, the methodology used for slow cooling of human cleavage-stage embryos has remained largely unchanged for over two decades. Previous studies have demonstrated the value, in terms of cryosurvival, of increasing the extent of intracellular dehydration by increasing the concentration of non-permeating cryoprotectant prior to slow cooling of oocytes and embryos which have been biopsied for preimplantation genetic diagnosis. The present study extends the use of this approach to the slow cooling of non-biopsied day-2 embryos. Dehydration in the presence of 0.2 mol/l sucrose significantly increased the proportions of surviving embryos, surviving blastomeres and fully intact embryos (92.6%, 91.1%, and 80.4%, respectively) relative to those observed after dehydration in 0.1 mol/l sucrose (78.5%, 74.1%, and 54.6%, respectively, all P < 0.001). Post-thaw resumption of mitosis in vitro and implantation were not adversely affected by the increased prefreeze dehydration. This improved method for slow cooling of cleavage-stage embryos should have a major impact on clinical outcome.

Although early developmental markers are frequently used to select embryos for transfer in human assisted reproduction, their value as independent predictors of outcome is often unclear. In this study, the value of using early syngamy and first cleavage as predictors of implantation potential of Day 2 embryos was investigated by examining their interrelationships with subsequent development, female age and implantation. Implantation rates were higher when syngamy occurred before 23-24 h post insemination even when all embryos analysed were transferred 42 h post insemination at the 4-cell stage (25.8 v. 11.9% for the later syngamy group; P < 0.01). Although there was a significant (r = 0.682; P < 0.001) relationship between earlier entry into syngamy and female age, earlier syngamy was still associated with a significantly higher implantation rate in Day 2 embryos with four blastomeres in women under 36 years of age (31.4 v. 15.4% for the later syngamy group; P < 0.05). The ability of timing of syngamy to predict implantation independent of other variables was confirmed by multiple logistic regression analysis. Although related to both subsequent embryo development and female age, early entry into syngamy is a predictor of implantation potential independent of both correlates in human Day 2 in vitro-fertilised embryos.

Rapid recent advances in in-vitro fertilization (IVF) and in-vitro maturation (IVM) of human oocytes are leading to vast changes in the practice of IVF. This article attempts to define the various approaches to these new methods.

Clinical application of oocyte cryopreservation may be in the context of fertility preservation for women about to undergo cytotoxic therapies or may be as an alternative to embryo cryopreservation in routine assisted reproduction. The clinical efficiency of oocyte cryopreservation will be a consequence of the cumulative impact of pre-freeze oocyte quality, postthaw survival and subcellular effects of cryopreservation protocols, which impact on early embryo quality and post-transfer viability, together with the degree of selection which is applied to the available biological material. Any valid assessment must include reference to all the above aspects, particularly when comparing cryopreserved oocytes with non-frozen controls or cryopreserved embryos. Cumulative pregnancy rates from oocyte collections may provide the most relevant index of success. Survival of human oocytes cryopreserved using current methodology is similar to that achieved with early-cleavage-stage embryos. Although evidence suggests that developmental potential may be compromised when current oocyte cryopreservation protocols are applied, there is a paucity of rigorously controlled studies in the literature.

The clinical role of oocyte cryopreservation in assisted reproduction, as an adjunct to sperm and embryo cryopreservation, has been comparatively slow to evolve as a consequence of theoretical concerns related to efficacy and safety. Basic biological studies in the 1990's alleviated many of these concerns leading to more widespread adoption of the technology. While a number of babies were born from the approach validated in the 1990's, its perceived clinical inefficiency led to the search for improved methods. Introduction of elevated dehydrating sucrose concentrations during cryopreservation increased survival and fertilization rates, but there is no well-controlled evidence of improved clinical outcome. Similarly, the use of sodium-depleted cryopreservation media has not been demonstrated to increase clinical efficiency. More recently, and in the absence of basic biological studies addressing safety issues, the application of vitrification techniques to human oocytes has resulted in reports of a number of live births. The small number of babies born from clinical oocyte cryopreservation and the paucity of well-controlled studies currently preclude valid comparisons between approaches. Legal restrictions on the ability to select embryos from cryopreserved oocytes in Italy, where many of the available reports originate, also obscure attempts to assess oocyte cryopreservation objectively.