OBJECTIVETo evaluate the safety and risk of cryopreservation in female fertility preservation.

DATA SOURCESThe data analyzed in this review were the English articles from 1980 to 2013 from journal databases, primarily PubMed and Google scholar. The criteria used in the literature search show as following: (1) human; embryo; cryopreservation/freezing/vitrification, (2) human; oocyte/immature oocyte; cryopreservation/ freezing/vitrification, (3) human; ovarian tissue transplantation; cryopreservation/freezing/vitrification, (4) human; aneuploidy/DNA damage/epigenetic; cryopreservation/freezing/vitrification, and (5) human; fertility preservation; maternal age.

STUDY SELECTIONThe risk ratios based on survival rate, maturation rate, fertilization rate, cleavage rate, implantation rate, pregnancy rate, and clinical risk rate were acquired from relevant meta-analysis studies. These studies included randomized controlled trials or studies with one of the primary outcome measures covering cryopreservation of human mature oocytes, embryos, and ovarian tissues within the last 7 years (from 2006 to 2013, since the pregnancy rates of oocyte vitrification were significantly increased due to the improved techniques). The data involving immature oocyte cryopreservation obtained from individual studies was also reviewed by the authors.

RESULTSVitrifications of mature oocytes and embryos obtained better clinical outcomes and did not increase the risks of DNA damage, spindle configuration, embryonic aneuploidy, and genomic imprinting as compared with fresh and slow-freezing procedures, respectively.

CONCLUSIONSBoth embryo and oocyte vitrifications are safe applications in female fertility preservation.

Cryopreservation ; methods ; Female ; Humans ; Oocytes ; cytology ; Ovary ; cytology ; Pregnancy

To study the effects of mouse cytomegalovirus (MCMV) on the in vitro maturation, fertilization, cleavage and blastula formation of mouse oocytes, the immature oocytes were infected in vitro by MCMVs of different dosages (100 TCID(50), 10 TCID(50) and 1 TCID(50)). The oocytes were then observed for in vitro maturation, fertilization, cleavage and blastula formation and the ultrastructural changes after the culture with the viruses. Our results showed that no significant differences were found in IVM, IVF, cleavage and blastula formation among the groups treated with of virus of various dosages. And ultrastructural abnormality was observed in the oocytes treated by 100 TCID(50) of viruses. It is concluded that MCMV did not have any conspicuous effects on IVM, IVF, cleavage and blastula formation of murine immature oocytes.
Blastocyst ; Cells, Cultured ; Cleavage Stage, Ovum ; Cytomegalovirus Infections ; Fertilization ; Muromegalovirus/*pathogenicity ; Oocytes/cytology ; Oocytes/growth & development ; Oocytes/*virology

Mouse follicular oocytes, denuded and intact, were cultured in pyruvate salt sol and glutamine salt sol supplemented bovine serum albumin to compare the maturation rate. Glutamine has no effect on maturation of the denuded mouse oocyte but has an effect on maturation of the intact oocyte by increasing the maturation rate, depending on the increased concentration of glutamine (0.4 mM to 2 mM). Changes in osmolarity of the operation medium from 280 mOsm to 310 mOsm has no discernible effect on the oocyte maturation. A high frequency of abnormal 1st polar bodies was observed in pyruvate salt sol. and this may be due to the increased energy source in the cytoplasm of the 1st polar body when the po1ar body was extruded into the perivitelline space after the 1st meiosis.
Animal ; Cell Division ; Female ; Glutamine/metabolism ; In Vitro ; Mice ; Oocytes/cytology ; Oocytes/metabolism* ; Ovum/metabolism* ; Pyruvates/metabolism

Mouse follicular oocytes, denuded and intact, were cultured in pyruvate salt sol and glutamine salt sol supplemented bovine serum albumin to compare the maturation rate. Glutamine has no effect on maturation of the denuded mouse oocyte but has an effect on maturation of the intact oocyte by increasing the maturation rate, depending on the increased concentration of glutamine (0.4 mM to 2 mM). Changes in osmolarity of the operation medium from 280 mOsm to 310 mOsm has no discernible effect on the oocyte maturation. A high frequency of abnormal 1st polar bodies was observed in pyruvate salt sol. and this may be due to the increased energy source in the cytoplasm of the 1st polar body when the po1ar body was extruded into the perivitelline space after the 1st meiosis.
Animal ; Cell Division ; Female ; Glutamine/metabolism ; In Vitro ; Mice ; Oocytes/cytology ; Oocytes/metabolism* ; Ovum/metabolism* ; Pyruvates/metabolism

This experiment was undertaken in order to know the effect of leucocytes on the maturation of mouse oocytes in vitro. Leucocytes obtained from heart puncture of mouse (3 X 10(4) cells/mm3) inhibited the maturation of mouse oocytes. The egg toxic activity declined with decreasing leucocyte concentration. It was found that egg toxic effect of leucocytes is not species specific. The activity of intact leucocytes or equal numbers of leucocytes that were destroyed was similar and which seems not to be influenced by the physiological stats of leucocytes.
Animal ; Culture Media ; Female ; Leukocytes* ; Metaphase ; Mice ; Oocytes/cytology* ; Ovum/cytology*

The successful cryopreservation of reproductive cells has important practical significance in many fields. In order to improve the recovery rate and viability of cryopreserved cells, it is necessary to study the permeability characteristics of cell membrane to both water and cryoprotectant. In this paper we review the studies on membrane permeability of animal reproductive cell for the recent years. We firstly list the typical permeability data of spermatozoa and oocyte membrane for water and cryoprotectant. We then analyze the effects of these characteristics on the design of cryopreservation protocol. We also introduce the latest experimental methods to measure the cell membrane permeability.
Animals ; Cell Membrane Permeability ; physiology ; Cryopreservation ; methods ; Female ; Humans ; Male ; Oocytes ; cytology ; Spermatozoa ; cytology

This experiment was undertaken in order to find out if there is any morphological change in oocytes and two-cell embryos whose development have been suppressed by progesterone for six hours in vitro. It can be observed that some part of the outer side of nuclear membrane of the suppressed oocytes was damaged. The number of nuclear pores has decreased in suppressed oocytes and this suggests that progesterone might suppress the transport of intermediary metabolites between cytoplasm and nucleus. Sometimes, closely packed aggregates of parallel or irregular endoplasmic reticula were observed in suppressed oocytes. Microvilli of suppresed oocytes showed signs of degradation and the perivitelline space became apparent. Thus it is presumed that the egg membrane has constricted during cultivation under progesterone in vitro. The other cell organelles such as mitochondria, multivesicular bodies, cortical granules and fibrillar lattices showed no difference in morphology between treated and control (intact) oocytes. In two-cell embryos, there was also no evident morphological change except for the fact that many vacuoles appeared clearly in suppressed embryonal cells. In brief, there was no fundamental morphological change in the oocytes and the embryonal cells exposed to progesterone for six hours even though it inhibits their development. The action of progesterone should be investigated thoroughly.
Animal ; Embryo/cytology* ; Embryo/drug effects ; Female ; In Vitro ; Mice ; Oocytes/drug effects ; Oocytes/ultrastructure* ; Ovum/ultrastructure* ; Progesterone/pharmacology*

Oocyte-like cells (OLC) can be generated by stem cells after the induction and differentiation in vitro, and maturated when transplanted in vivo to improve the development potential. Human amniotic fluid stem cells (hAFSC) were cultured for 10 days in porcine follicle fluid (pFF) that was extracted from the medium follicle with high levels of hormones and Bmp 15 protein. After the induction, the cell aggregates showed the germ cell-like cells and produced the germ cell marker oct4, and triggered epigenetic changes with high expression of methylation transferase gene dnmt3b. The cell aggregates were packaged into porcine theca folliculi to form grafts, which were then transplanted into mouse renal capsule. After one month of transplantation, the morphology of OLC from a graft was not only similar to oocytes, but also expressed the germ cells markers (oct4, nanog, stella, ifitm3, dazl, nanos3, bmp15, and gd9). The results demonstrate that the in vivo differentiation model was useful for OLC development.
Amniotic Fluid ; cytology ; Animals ; Biomarkers ; Cell Differentiation ; Female ; Humans ; Mice ; Oocytes ; cytology ; Ovarian Follicle ; Stem Cells ; cytology ; Swine

Total or near-total fertilization failure after intracytoplasmic sperm injection (ICSI) is a rare event, but it occurs repeatedly because of sperm defects in activating oocyte. The case presents a successful pregnancy and live birth after calcium ionophore A23187 (A23187) activation on one-day-old unfertilized oocytes in a patient whose husband suffered oligoasthenoteratozoospermia, and who had experienced repeated near-total fertilization failure after ICSI. In the second ICSI cycle, only one oocyte was fertilized while nine were unfertilized. Oocyte activation with A23187 were performed on the one-day-old unfertilized oocytes after ICSI and resulted in fertilization and embryo transfer. A clinical pregnancy was achieved and a healthy baby was born. To our knowledge, this is the first reported case of a healthy birth after oocyte activation on the one-day-old unfertilized oocyte. This indicates that "rescue oocyte activation" on one-day-old unfertilized oocytes after ICSI may be helpful for preventing total or near-total fertilization failure after ICSI.
Adult ; Female ; Fertilization in Vitro ; methods ; Humans ; Live Birth ; Male ; Oocytes ; cytology ; Pregnancy ; Sperm Injections, Intracytoplasmic ; methods

BACKGROUNDIt is still unclear whether the vitrification procedure itself is associated with the incidence of abnormal DNA methylation during oocytes vitrification. The purpose of this study was to evaluate the epigenetic profile of mouse oocytes, which went through vitrification either at a mature stage or at an immature stage following in vitro maturation (IVM) by analyzing the global DNA methylation.

METHODSMetaphase II (M II) stage and germinal vesicle (GV) stage oocytes were collected from adult female mice and were vitrified respectively. The M II oocytes were assessed for cryo-survival and global DNA methylation. The GV oocytes were assessed for cryo-survival and only the surviving GV oocytes were cultured in vitro for subsequent assessment of global DNA methylation in mature oocytes. In vivo matured fresh M II oocytes without undergoing vitrification were used as control. The level of global DNA methylation in the M II oocytes was then examined by immunofluorescence using an anti-5-methylcytosine (anti-5-MeC) monoclonal antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG under a laser scanning confocal microscope.

RESULTSIn terms of the effect of vitrification on global DNA methylation status in matured oocytes, in the M II-v group, all the examined oocytes (90/90) were found with hypermethylation, including 63.3% (57/90) of them displaying DNA methylation of a very high level, 25.6% (23/90) with a high level, and 11.1% (10/90) with an intermediate level, whereas in the GV-v group, all the matured oocytes (129/129) were also examined with hypermethylation, including 67.4% (87/129) of them displaying DNA methylation of a very high level, 23.3% (30/129) with a high level, and 9.3% (12/129) with an intermediate level. Statistically, it was similar between both groups, which were similar to the control: 68.6% (83/121) of fresh M II oocytes displayed DNA methylation of a very high level, 21.5% (26/121) with a high level, and 9.9%(12/121) with an intermediate level (P > 0.05). In terms of the effect of IVM on global DNA methylation status in matured oocytes, in the in vivo matured oocytes group, all oocytes examined (94/94) were found with hypermethylation, including 80.9% (76/94) displaying DNA methylation of a very high level and 19.1% (18/94) with a high level, whereas in the in vitro matured oocytes group, all oocytes examined (69/69) were also found with hypermethylation: 85.2% (56/69) of them displayed with DNA methylation of very high level, 11.9% (11/69) with high level, and 2% (2/69) with intermediate level. This result was similar to that in in vivo matured fresh M II oocytes (P > 0.05).

CONCLUSIONThe vitrification procedure at GV stage does not induce widespread alteration of global DNA methylation status of mouse oocytes subsequently matured in vitro.

Animals ; DNA Methylation ; physiology ; Female ; Fertilization in Vitro ; Mice ; Microscopy, Confocal ; Oocytes ; cytology ; metabolism ; Vitrification