David H. Edgar

Clinical outcomes following cryopreservation of blastocysts by vitrification or slow freezing: a population-based cohort study

STUDY QUESTION What are the clinical efficacy and perinatal outcomes following transfer of vitrified blastocysts compared with transfer of fresh or of slow frozen blastocysts? SUMMARY ANSWER Compared with slow frozen blastocysts, vitrified blastocysts resulted in significantly higher clinical pregnancy and live delivery rates with similar perinatal outcomes at population level. WHAT IS KNOWN ALREADY Although vitrification has been reported to be associated with significantly increased post-thaw survival rates compared with slow freezing, there has been a lack of general consensus over which method of cryopreservation (vitrification versus slow freezing) is most appropriate for blastocysts. STUDY DESIGN, SIZE, DURATION A population-based cohort of autologous fresh and initiated thaw cycles (a cycle where embryos were thawed with intention to transfer) performed between January 2009 and December 2011 in Australia and New Zealand was evaluated retrospectively. A total of 46 890 fresh blastocyst transfer cycles, 12 852 initiated slow frozen blastocyst thaw cycles and 20 887 initiated vitrified blastocyst warming cycles were included in the data analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS Pairwise comparisons were made between the vitrified blastocyst group and slow frozen or fresh blastocyst group. A Chi-square test was used for categorical variables and t-test was used for continuous variables. Cox regression was used to examine the pregnancy outcomes (clinical pregnancy rate, miscarriage rate and live delivery rate) and perinatal outcomes (preterm delivery, low birthweight births, small for gestational age (SGA) births, large for gestational age (LGA) births and perinatal mortality) following transfer of fresh, slow frozen and vitrified blastocysts. MAIN RESULTS AND THE ROLE OF CHANCE The 46 890 fresh blastocyst transfers, 11 644 slow frozen blastocyst transfers and 19 978 vitrified blastocyst transfers resulted in 16 845, 2766 and 6537 clinical pregnancies, which led to 13 049, 2065 and 4955 live deliveries, respectively. Compared with slow frozen blastocyst transfer cycles, vitrified blastocyst transfer cycles resulted in a significantly higher clinical pregnancy rate (adjusted relative risk (ARR): 1.47, 95% confidence intervals (CI): 1.39-1.55) and live delivery rate (ARR: 1.41, 95% CI: 1.34-1.49). Compared with singletons born after transfer of fresh blastocysts, singletons born after transfer of vitrified blastocysts were at 14% less risk of being born preterm (ARR: 0.86, 95% CI: 0.77-0.96), 33% less risk of being low birthweight (ARR: 0.67, 95% CI: 0.58-0.78) and 40% less risk of being SGA (ARR: 0.60, 95% CI: 0.53-0.68). LIMITATIONS, REASONS FOR CAUTION A limitation of this population-based study is the lack of information available on clinic-specific cryopreservation protocols and processes for slow freezing-thaw and vitrification-warm of blastocysts and the potential impact on outcomes. WIDER IMPLICATIONS OF THE FINDINGS This study presents population-based evidence on clinical efficacy and perinatal outcomes associated with transfer of fresh, slow frozen and vitrified blastocysts. Vitrified blastocyst transfer resulted in significantly higher clinical pregnancy and live delivery rates with similar perinatal outcomes compared with slow frozen blastocyst transfer. Comparably better perinatal outcomes were reported for singletons born after transfer of vitrified blastocysts than singletons born after transfer of fresh blastocysts. Elective vitrification could be considered as an alternative embryo transfer strategy to achieve better perinatal outcomes following Assisted Reproduction Technology (ART) treatment. STUDY FUNDING/COMPETING INTERESTS No specific funding was obtained. The authors have no conflicts of interest to declare.

The developmental potential of cryopreserved human embryos.

Using rigorously matched non-frozen controls we have shown that cryopreservation does not alter the implantation potential of early cleavage stage (day 2) human embryos if no blastomere loss occurs. Thawed intact 4-cell embryos have a significantly higher implantation (fetal heart) rate (16.9%) than similar 2-cell embryos (7.2%). This difference is not due to blastomere number per se since increasing the cell number in frozen embryos by allowing an extended period in culture prior to freezing does not alter their intrinsic developmental potential. Blastomere loss, which occurred in almost half of all thawed embryos, is directly related to a reduction in developmental potential. We estimate that approximately 30% of the expected fresh embryo implantations are lost as a consequence of cryopreservation. Both preimplantation and peri-implantation losses may contribute to this outcome.

The effects of cryopreservation regimens on the morphology of human ovarian tissue.

Human ovarian tissue consisting of stroma, pre-granulosa cells and oocytes, has been frozen using a variety of cooling rates and dehydration regimens. The differential survival of the various cell types under these conditions highlights the difficulty in defining optimum protocols for the cryopreservation of multicellular tissue.

How should the clinical efficiency of oocyte cryopreservation be measured

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.

Embryonic factors affecting outcome from single cryopreserved embryo transfer.

Multiple pregnancy minimization by single embryo transfer is becoming more prevalent, but is less common in the case of cryopreserved embryos. This study defines embryonic characteristics in single cryopreserved embryo transfers associated with success rates equivalent to those achieved when transferring two cryopreserved embryos. In a retrospective analysis of 6916 cryopreserved day-2 embryo transfer procedures, transfer of two cryopreserved embryos resulted in higher clinical pregnancy rates when compared with transfer of a single thawed embryo but was also associated with elevated multiple pregnancy rates (26.7% in women under 36). Optimal outcome (implantation rate of 30.9%) from single cryopreserved embryo transfer (SCET) in women under 36 was associated with cryopreservation at the 4-cell stage, loss of fewer than two blastomeres and subsequent cleavage of at least two surviving blastomeres. In comparison, transfer of two cryopreserved embryos in women under 36 resulted in pregnancy and implantation rates of 25.5 and 16.1% respectively. Interestingly, in cryopreserved 4-cell stage embryos, loss of a single blastomere did not reduce implantation potential and cleavage of only a single post-thaw blastomere was not indicative of increased implantation potential. Establishment of these critical thresholds provides a rational basis for SCET.

The first Australian experience of heterotopic grafting of cryopreserved ovarian tissue: evidence of establishment of normal ovarian function.

Cryostorage of reproductive potential, in the form of ovarian cortex, for young women about to undergo cytotoxic therapies has been offered clinically for some time. However, the prospects of re‐establishing reproductive function using this tissue remain unclear. We now report reproducible follicular development, oocyte retrieval and embryo development following heterotopic grafting of cryopreserved ovarian cortex which had been stored for over 10 years.

Use of assisted reproductive technology to reduce the risk of transmission of HIV in discordant couples wishing to have their own children where the male partner is seropositive with an undetectable viral load

The advances in treatment of HIV and the introduction of polymerase chain reaction assay for the virus now make it acceptable for HIV discordant couples where the male partner is seropositive to attempt to conceive through artificial insemination by husband (AIH) or via in vitro fertilisation. With undetectable viral load and washed sperm, there is minimal risk of transmission of HIV to the female partner, children, other patients, or staff. We describe the development of a programme of AIH for HIV discordant couples and the reasoning behind offering such a programme.

The application and impact of cryopreservation of early cleavage stage embryos in assisted reproduction

The contribution of cryopreserved embryos to the overall outcomes achieved by a clinical assisted reproduction programme has increased in importance with the trend towards reducing the numbers of fresh embryos transferred following in vitro fertilisation. Although cryopreservation appears to fully preserve developmental potential in early cleavage stage embryos that survive intact, it results in a reduction in potential when blastomere loss occurs during freezing and thawing. Overall, it can be estimated that cryopreservation results in approximately a 30% reduction in the potential for pregnancy in a population of embryos. Both blastomere survival and post-thaw resumption of mitosis can act as markers of implantation potential in frozen/thawed embryos. Application of strict criteria for freezing embryos and transferring thawed embryos may enhance apparent success rates, but may also result in some pregnancy potential being discarded. The role of embryo cryopreservation in minimising the incidence of multiple pregnancy must be balanced with the need for efficiency in the quest to establish pregnancy.

CLINICAL ASSISTED REPRODUCTION: The Influence of Prefreeze Growth Rate and Blastomere Number on Cryosurvival and Subsequent Implantation of Human Embryos

Purpose: To determine whether the relatively low implantation rate of cryopreserved Day 2 embryos with only 2 blastomeres can be increased as a consequence of increasing their blastomere content by extending the prefreeze culture time.Methods: Of a total of 3480 Day 2 embryos studied, 1921 (55.2%) had reached the 4-cell stage by 40 h postinsemination (FAST) and were transferred or cryopreserved. The remaining embryos that underwent subsequent cell division by 46 h (INTERMEDIATE; 18.3% of total) or 66 h (SLOW; 20.3% of total) were also cryopreserved whereas the 6.2% that remained arrested at 66 h were discarded. Thawed embryos from each category were assessed for survival, postthaw cleavage, and implantation.Results: The proportion of thawed embryos that survived, the proportion of surviving embryos that underwent postthaw cleavage, and the implantation rate of transferred embryos were all reduced in the slower growing cryopreserved embryos.Conclusions: The growth rate, and not the number of blastomeres per se, is a critical factor in predicting the developmental potential of cryopreserved embryos.

Live birth following transfer of a cryopreserved embryo generated from a cryopreserved oocyte and a cryopreserved sperm: Case report

As is the case with non-frozen oocytes, the efficient and successful use of cryopreserved oocytes in human assisted reproduction is, in part, dependent on the ability to apply selection criteria when choosing the ‘best’ embryos for transfer from a cohort. In many cases this, in turn, will necessitate the cryopreservation of non-transferred embryos to minimise the risk of multiple pregnancy. It is therefore important to establish that an embryo, generated by fertilization of a frozen-thawed oocyte, can be capable of surviving subsequent cryopreservation while retaining the potential for normal development. In this case report, we document the delivery of a normal male infant following transfer of a frozen-thawed embryo, generated by the fertilization of a frozen-thawed oocyte by a frozen-thawed sperm.