A major increase in oocyte cryopreservation cycles in the USA, Australia and New Zealand since 2010 is highlighted by younger women but a need for standardized data collection.

STUDY QUESTION: What are the cohort trends of women undergoing oocyte cryopreservation (OC)? SUMMARY ANSWER: There has been a dramatic increase in OC cycles undertaken each year since 2010, and the demographics of women accessing OC has shifted to a younger age group, but so far very few women have returned to use their cryopreserved oocytes in treatments. WHAT IS KNOWN ALREADY: Although OC, as a method of fertility preservation, is offered around the world, global data are lacking on who is accessing OC, who is returning to thaw oocytes and whether these trends are changing. STUDY DESIGN, SIZE, DURATION: A trinational retrospective cohort study was performed of 31 191 OC cycles and 972 oocyte thaw (OT) cycles undertaken in the USA (2010-2016) and 3673 OC and 517 OT cycles undertaken in Australia/New Zealand (Aus/NZ; 2010-2015). PARTICIPANTS/MATERIALS, SETTING, METHODS: Data were obtained from the USA Society for Assisted Reproductive Technology (SART) national registry and the Australian and New Zealand Assisted Reproduction Database (ANZARD). De-identified data were requested on all autologous oocyte freeze-all cycles and all cycles where autologous oocytes were thawed to be used in a treatment cycle for the time periods of interest. MAIN RESULTS AND THE ROLE OF CHANCE: In both the USA and Aus/NZ, there has been a dramatic rise in the number of OC cycles performed each year (+880% in the USA from 2010 to 2016 and +311% in Aus/NZ from 2010 to 2015). Across both regions, most women undergoing OC were aged in their late 30s, but the average age decreased over time (USA: 36.7 years vs 34.7 years in 2010 and 2016, respectively). The number of women returning for thaw cycles was low (USA: 413 in 2016, Aus/NZ: 141 in 2015) and most thaw cycles (47%) across both regions involved oocytes that were frozen for <6 months. In the USA, a higher proportion of cycles resulted in a live birth when only thawed oocytes were used, compared to cycles that combined thawed oocytes with fresh oocytes (25% vs 11%, respectively; P < 0.001). Age at retrieval influenced live birth rate in the USA; 38% of thaw cycles started in women who stored oocytes when aged ≤35 years resulted in a live birth, whereas only 16% resulted in a live birth for women who stored oocytes when aged ≥36 years. Similar data were unobtainable from Aus/NZ. LIMITATIONS, REASONS FOR CAUTION: There were limitations associated with both the SART and ANZARD data outputs received. The format in which the ANZARD data were provided, and the inconsistencies seen amongst cycle reporting in the SART dataset, restricted data interpretation. For example, both datasets did not provide a clear indication as to why women were undergoing OC and it was not possible to accurately calculate duration of storage for thaw cycles in the USA. We also did not obtain details on embryo quality from either database and acknowledge that embryo quality and subsequent outcome (embryo freezing or discard) would be of interest, especially when considering the efficacy of OC. WIDER IMPLICATIONS OF THE FINDINGS: The data show that there is widespread demand for OC, and it is increasingly undertaken by younger women; however, the limitations encountered in the dataset support the need for a shift to a more uniform approach to data collection and presentation by large databases, worldwide. STUDY FUNDING/COMPETING INTEREST(S): This study received funding from the Fertility Society of Australia to support the ANZARD data extraction. M.J. is supported by an Australian Government Research Training Program Scholarship stipend. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Cryopreservation and long-term maintenance of bovine embryo-derived cell lines.

The aim of this study was to develop methods for cryopreservation and long-term maintenance of putative bovine embryonic stem cells (ESCs). Putative bovine ESC (bESC) lines (n=3) isolated in conventional medium were used to compare slow-freezing and vitrification. After warming, vitrified cells (96.9%) demonstrated significantly (P<0.05) better survival than frozen-thawed cells (81.5%) and formed significantly more colonies with good morphology (vitrification: 93/93, 100.0%; slow-freezing: 74/106, 69.81%; P<0.05). The effect of inhibitors of differentiation (PD184352, SU5402, CHIR99021) on ESC maintenance was assessed on putative bESC lines established in N2B27-3i medium (n=8) or conventional medium (n=1) after culture over 30 passages (>240 days). All cell lines expressed ALP, SSEA1, SSEA4, OCT4, REX1 and SSEA1. OCT4 expression was confirmed by relative real-time PCR and was upregulated in early passages of putative bESCs cultured in N2B27-3i (2.9±0.89-fold higher at Passage (P) 2-4), whereas the converse was observed later (P22-26; 2.2±0.1-fold increase in conventional medium). Putative bESC lines isolated in N2B27-3i medium (n=3) or conventional medium (n=1) were vitrified at P18 and, after warming, were cultured for a further 12 passages. These cells survived vitrification and expressed OCT4, REX1, SSEA1, ALP, SSEA1 and SSEA4. These results demonstrate that putative bESC lines that express pluripotent markers can be cultured long term and retain expression of pluripotent markers after vitrification.

Ontogeny of the oestrogen receptors ESR1 and ESR2 during gonadal development in the tammar wallaby, Macropus eugenii.

Oestrogen has wide ranging effects in development mediated mainly via the two oestrogen receptors, alpha (ESR1, also known as ERalpha) and beta (ESR2, also known as ERbeta). Oestrogen is the key factor that directs the indifferent gonad to become an ovary in many non-mammalian vertebrates. Oestrogen is not required for early ovarian differentiation in mammals but can disrupt normal testicular development in eutherians. Surprisingly, exogenous oestrogen can cause sex reversal of an XY gonad in two marsupials, the North American opossum and the tammar wallaby. To understand the mechanism by which oestrogen induces sex reversal, we characterised the genes for ESR1 and ESR2 and examined their expression during gonadal differentiation in the tammar wallaby, Macropus eugenii. Both receptors were expressed in the somatic cells and germ cells of the indifferent gonad in both XX and XY foetuses throughout all stages of development, and persisted in these cells into adulthood. ERs were also present in many other tissues including kidney, pituitary and mammary gland. ER mRNA was not significantly altered by exogenous oestrogen in cultured XY gonads but the receptors translocated to the nucleus in its presence. These findings confirm that there is conserved expression of the ERs in the indifferent gonad despite the lack of available ligand during early gonadal development. The receptors can respond to exogenous estrogen at this early stage and are capable of transducing signals in the early mammalian gonad. However, the selective forces that maintained conserved ER expression in this tissue remain unknown.

Growth and histology of ovarian follicles after cold storage in the tammar wallaby.

Cold storage is a simple method for storing and transporting tissues and organs. The reliability of this method for maintaining structure and function of marsupial ovarian tissue was assessed using histological techniques and follicle culture. Tammar wallaby ovaries were placed in cold storage (phosphate-buffered saline at 4 degrees C) for 24 or 48 h. Although necrotic changes were evident in the germinal epithelium, cortex and interstitial tissue after cold storage, there was little evidence of necrotic changes in ovarian follicles and oocytes appeared normal. Secondary follicles isolated from ovarian tissue after cold storage grew by a similar amount to non-stored follicles when cultured for 4 days in vitro, but no follicles from any group developed to tertiary follicles. Cold storage for up to 24 h had little obvious effect on the structure of ovarian tissue and follicles isolated from this tissue maintained their structure during culture. However, degeneration in culture increased with storage time and was significantly higher after cold storage for 48 h. As demonstrated in the tammar wallaby, cold storage has potential as a method for storage and transport of marsupial ovaries up to 24 h.

Intra-cytoplasmic sperm injection in a marsupial.

Here we report the first use of intra-cytoplasmic sperm injection (ICSI) in a marsupial, the tammar wallaby (Macropus eugenii), to achieve in vitro fertilization and cleavage. A single epididymal spermatozoon was injected into the cytoplasm of each mature oocyte collected from Graafian follicles or from the oviduct within hours of ovulation. The day after sperm injection, oocytes were assessed for the presence of pronuclei and polar body extrusion and in vitro development was monitored for up to 4 days. After ICSI, three of four (75%) follicular and four of eight (50%) tubal oocytes underwent cleavage. The cleavage pattern was similar to that previously reported for in vivo fertilized oocytes placed in culture, where development also halted at the 4- to 8-cell stage. One-third of injected oocytes completed the second cleavage division, but only a single embryo reached the 8-cell stage. The success of ICSI in the tammar wallaby provided an opportunity to examine the influence of the mucoid coat that is deposited around oocytes passing through the oviduct after fertilization. The presence of a mucoid coat in tubal oocytes did not prevent fertilization by ICSI and the oocytes cleaved in vitro to a similar stage as follicular oocytes lacking a mucoid coat. Cell-zona and cell-cell adhesion occurred in embryos from follicular oocytes, suggesting that the mucoid coat is not essential for these processes. However, blastomeres were more closely apposed in embryos from tubal oocytes and cell-cell adhesion was more pronounced, indicating that the mucoid coat may be involved in maintaining the integrity of the conceptus during cleavage.