Recent drastic advances of cryobiology and cryotechnology have enabled the cryopreservation of mammalian embryos and gametes. Improved vitrification methods have made it possible to preserve pre-fertilized oocytes with little loss of viability. Using a highly efficient vitrification method with ultra-rapid cooling (the Cryotop method), nearly 100% post-thaw survival rates have been obtained for human oocytes. These oocytes also have similar developmental ability in vitro after ICSI and IVC, and also to the term after ET. High post-thaw survival and pregnancy rates by the Cryotop method have been repeated at many IVF faculties throughout the world. The Cryotop method has contributed to the establishment of oocyte banks for unmarried cancer patients to preserve their fertility after chemo- and radio-therapy. Successful human oocyte banks have been established worldwide, resulting in the birth of more than 300 healthy babies so far.

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Ovarian tissue cryopreservation is an effective method for protecting against infertility as well as preservating endangered animal species. The technique is particularly sought after as a strategy against ovarian failure caused by aggressive chemotherapy in young women with cancer. There are two uses for cryopreserved ovarian tissues after thawing: grafting and culture. Grafting carries the risk of reintroduction of disease. This article describes the status of ovarian cryopreservation in humans and the other animals and also details the successful birth of a pup from preantral follicle oocytes derived from a mouse cryopreserved ovary followed by in vitro growth, in vitro maturation and in vitro fertilization-embryo transfer (IVF-ET).

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Since the 1950's, cryopreservation of human semen has been recognized as an efficient procedure for infertility therapy, and research has mainly focused on long-term banking of donor semen for artificial insemination (AID). Because assisted reproductive technology (ART) usually employs fresh ejaculate, it is essential to synchronize ejaculation and ovulation. However, if the sperm is efficiently cryo-accumulated, synchronization would not be necessary and much sperm could be provided for fertilization or insemination. In recent years, survival of young males suffering from some cancers has improved due to advanced treatments including high-dose chemotherapy and radiotherapy. However, testicular functions, especially spermatogenesis, are usually sacrificed temporarily or permanently by these treatments. Sperm cryopreservation liberates these patients from iatrogenic infertility and allows them to retain reproductive capability.

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The conditions suitable for cryopreservation differ among oocytes and embryos at different stages even in the same species. Differences in cryobiological properties would affect these conditions. Permeability to water and cryoprotectants is important because it modulates major forms of cell injury from cryopreservation. In the mouse, morulae and blastocysts tolerate cryopreservation better than oocytes or early stage embryos, because they are highly permeable to water and cryoprotectants. The permeability appears to stem from a substantial expression of water/cryoprotectant-permeable channels, such as aquaporin-3. Such facilitated diffusion by channel proteins is less dependent on temperature than the process of simple diffusion via the lipid bilayer. Therefore, it is important to know the expression level and permeation properties of water/cryoprotectant channels for efficient cryopreservation of oocytes and embryos.

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Human blastocysts have been successfully cryopreserved and many live births occur each year using blastocysts that have been cryopreserved once. However, there is a paucity of information on re-cryopreservation of human blastocysts developed from frozen embryos. Therefore, we document the data for 27 blastocysts from 15 cycles (12 patients) of warming to review the efficacy of re-cryopreservation by vitrification of human blastocysts developed from frozen-cleaved embryos. A total of 27 surplus human blastocysts developed from frozen-cleaved embryos, obtained from 12 healthy infertile women undergoing IVF treatment between February 2004 and December 2006, were re-cryopreserved by vitrification. Of these, 26 (96%) re-expanded after warming and were transferred into 12 patients (15 cycles). Following transfer, the implantation rate was 35% (9/26) and the pregnancy rate was 47% (7/15). Two pregnancies ended in miscarriage, 5 healthy babies were born in 4 deliveries, and 1 pregnancy is ongoing. Our results suggest that re-cryopreservation by vitrification of human blastocysts developed from frozen-cleaved embryos can be performed without impairing their implantation ability after warming.

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Rat oocytes are known to be activated in vitro spontaneously. In the present study, we examined the effect of oocyte preincubation on the survival and development of oocytes after intracytoplasmic sperm injection (ICSI) in rats. Some presumptive oocytes produced by ICSI were transferred into the oviduct or ovarian bursa of recipient females to observe the development to term. When ICSI was performed without oocyte preincubation, the rate of oocyte survival was 90.5%, normally fertilized, 82.5%, and cleaved, 58.7%. These rates were reduced by oocyte preincubation for 3 or 5 h and the reduction could be related to the incomplete spontaneous activation of oocytes, which is known to result in no pronucleus formation and no cleavage. Both oviduct and ovarian bursa transfer allowed us to produce live offspring after ICSI (19.5 and 21.7% of transferred oocytes, respectively). Rat offspring could be produced by the ICSI protocol commonly used in mice. In rats, shortening of the period from oocyte recovery to sperm injection might be an important factor in the production of live offspring after ICSI. Intra-ovarian bursa transfer is technically easy and has been successfully applied to the production of rat offspring after various manipulations of oocytes, including ICSI.

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Bisphenol A (BPA) and nonylphenol (NP) are endocrine disrupting chemicals (EDCs) which induce reproductive abnormalities. While single exposure studies have been reported, few investigations of the simultaneous administration of several chemicals to animals have been conducted. The purpose of this study was to estimate the effect of gestational exposure to BPA and NP simultaneously on reproductive function. Pregnant female ICR mice were given BPA, NP or BPA plus NP by subcutaneous injection from gestational day (GD) 5 to delivery. The daily doses of BPA and NP were 1/1,000 or 1/100 the median lethal doses (LD₅₀; BPA: 2,500 mg/kg, NP: 1,231 mg/kg). On postnatal day (PND) 1, the pups (F1) were thinned out to 8. On PND 42, the body weights of some F1 were recorded and they were sacrificed; the livers, testes, epididymes, ovaries, and uteri were then weighed. The remaining F1 mice were mated with non-treated heterosexual mice. On GD 17, female mice were dissected to count the total number of fetuses and dead fetuses. All NP-treated mice exhibited decreased body and testis weight on PND 42. The pregnancy rate was 100% in all treated female groups, although it declined in untreated female mice mated with male mice from some treatment groups. The average dead fetus rate changed significantly according to the dosage combination. This study shows that BPA and NP can enhance, suppress or be neutral for the effect of the other in combined exposure to BPA and NP.

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