BACKGROUND: There are currently an estimated 250,000 female cancer survivors of reproductive age living in the US. Loss of fertility is an issue many cancer survivors face after treatment, as all forms of cancer therapy can cause infertility. Methods to preserve fertility can be initiated prior to cancer therapy. These methods include embryo cryopreservation, oocyte cryopreservation, fertility sparing surgery, ovarian tissue cryopreservation, ovarian transposition, and medical therapy. LITERATURE REVIEW: Although the clinical guidelines state that oncologists should discuss the risk of infertility with every patient of reproductive age and should refer every patient who is interested in or ambivalent towards fertility preservation to reproductive endocrinologists, studies have shown that a significant proportion of female cancer patients report never receiving information about fertility. Even fewer female cancer patients are referred to reproductive endocrinologists for further discussion and/or potential treatment. PROPOSED PROJECT: Oncologists at Boston Medical Center will be recruited to participate in a study that measures the effect of an educational intervention on referral rate to reproductive endocrinology. The knowledge gained from the intervention will be assessed with a pre- and post-test. The proportion of female patients age 18-45 referred to reproductive endocrinology will be evaluated through the Electronic Medical Record System. The correlation between knowledge gain and change in referral rates will also be assessed. CONCLUSION: Fertility after cancer treatment is an essential issue to consider for young cancer survivors. These patients benefit from being referred to reproductive endocrinologists so that they can get information about fertility preservation and undergo treatment in a timely fashion. Improving and/or reinforcing oncologist knowledge about this topic will increase the rate at which they initiate this conversation and therefore the number of female patients who are referred to reproductive endocrinology. SIGNIFICANCE: Providing female cancer patients with information about and opportunities to undergo fertility preservation will maximize their options. This will lead to a higher quality of life after cancer therapy.

Cryopreservation of fish germ cells has important applications in aquaculture, conservation of endangered species and human genomic studies. Although investigations on cryopreservation of fish sperm and embryos have been carried out extensively, cryopreservation of fish oocytes has not been studied systematically. The objective of the present study was to develop successful cryopreservation protocol for zebrafish oocytes at temperature of liquid nitrogen (-196°C), or if unachieved, to investigate the limiting factors associated with fish oocytes cryopreservation. In this study, the effects of cryoprotectants exposure and enzymatic treatments on oocytes survival were studied, and new viability tests for zebrafish oocytes were developed. The effects of controlled slow cooling with different cryoprotective agents, in different freezing media and at different cooling rates on cryosurvival of zebrafish (D. rerio) oocytes were investigated. Cryomicroscopic observations on zebrafish oocytes were also carried out. Three reliable vital tests -trypan blue (TB) staining, ATP assay, and in vitro maturation followed by germinal vesicle breakdown observation (GVBD) were found suitable for assessment of oocytes viability. Vitellogenesis (stage III) was found to be the optimal developmental stage for cryopreservation. Methanol was found to be the best CPA for zebrafish oocytes. Combination of 4M methanol and 0.2M glucose in potassium chloride (KCI) buffer was found to be the optimal cryoprotective solution. Controlled slow cooling at 0.3°C/min rate, combined with seeding at -12.5°C and plunge to liquid nitrogen (LN) at-40°C were found to be the optimal conditions for cryopreservation of stage III oocytes. However, even with the optimal protocol, TB-assessed viability, Le. the ratio of oocytes with intact plasma membrane after cooling to -196°C was 19.6±8%. Furthermore, GVBD experiments showed that none of the cryopreserved oocytes can be matured in vitro, and their ATP levels were decreased dramatically, indicating that successful cryopreservation of fish oocytes at liquid nitrogen temperature still remains elusive. Cryomicroscopic observations demonstrated, that the damages of oocytes are associated with intracellular ice formation (lIF). IIF occurred simultaneously with extracellular ice formation (ElF) in nearly 100% of the cases, and formation of lethal hexagonal type of ice was observed. This study was the first systematic attempt to cryopreserve fish oocytes at liquid nitrogen temperature. The results provided will undoubtedly assist successful protocol design for cryopreservation of fish oocytes in the future.

Made available in DSpace on 2014-08-20T13:32:51Z (GMT). No. of bitstreams: 1 tese_milton_carvalho_macedo_junior.pdf: 497444 bytes, checksum: 51c0892c0419a147e202b85034bef566 (MD5) Previous issue date: 2007-01-30 / The knowledge of the reproductive potential of a male is of economic and reproductive importance, mainly if used in programs of artificial insemination. The conventional tests for semen quality evaluation do not have the capacity to measure the fertilizing potential of a sample, although they indicate if that one is fertile or not. The in vitro penetration test, appears in this context as an alternative laboratorial test to categorize of males capacity of fecundation, as mimics in vitro what happens in vivo. However this test has its use limited for for difficult execution and high cost. With the objective to simplify the test, the present work evaluated alternatives for reduction of the execution time, use of cryopreserved oocytes in different methods and its association with in vivo fertility through heterospermic insemination and following diagnostic fertility. It was concluded that: 1) It is possible to use the incubation system BOTTLE, to replace the conventional incubation system, eliminating the need for using expansive CO2 incubator; 2) Oocytes can be cryopreserved in CRIOVIAL, together with the medium of fecundation and mineral oil, eliminating the use of stereoscopic lupa to manipulate oocytes in the day of the execution of the test; 3) It is viable to reduce co-incubation time of oocytes and sperm for 6 hours, if sperm concentration is of 2 X 106 per ml of way of used fecundation; 4) The paternity diagnosis disclosed that when inseminating females swine with one pool of semen of four males, using a dose of 2,8 X 109 spermatozoa, all the males had the same participation in the paternity of the produced pigs / O conhecimento do potencial reprodutivo de um macho é de importância econômica e reprodutiva, principalmente se ele é utilizado em programa de inseminação artificial. Os testes convencionais que avaliam a qualidade seminal não possuem a capacidade de medir o potencial fertilizante de uma amostra e, apenas indicam se a mesma é fértil ou não. O teste de penetração espermática in vitro, aparece neste contexto como um teste laboratorial alternativo para categorizar os machos férteis, quanto a sua capacidade de fecundação, pois mimetizam in vitro o que acontece in vivo. No entanto, este teste tem seu uso limitado por ser de difícil execução e por utilizar equipamentos de alto custo. Com o objetivo de simplificar o teste, o presente trabalho avaliou alternativas para diminuição do tempo de execução, utilização de oócitos criopreservados de diferentes maneiras e sua associação com fertilidade in vivo através de inseminação heterospérmica e diagnóstico de paternidade dos leitões nascidos. Foi concluído que: 1) É possível utilizar o sistema de incubação FRASCO, em substituição ao sistema de incubação convencional, eliminando a necessidade de uso da onerosa incubadora de CO2; 2) Pode-se congelar oócitos em CRIOTUBO, juntamente com o meio de fecundação e óleo mineral, eliminado a utilização de lupa esteriomicrocópica para manipular oócitos no dia da execução do teste; 3) É viável reduzir o tempo de co-incubação de oócitos e espermatozóides para 6 horas, desde que a concentração espermática utilizada seja de 2 X 106 espermatozóide para cada ml de meio de fecundação utilizado; 4) O diagnóstico de paternidade revelou que ao inseminar fêmeas suínas com um pool de sêmen de quatro machos, utilizando dose inseminante de 2,8 X 109 espermatozóides, todos os machos tiveram participação semelhante na taxa de paternidade dos leitões nascidos