Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step-wise versus direct rehydration

BACKGROUND: This study compared the viability of human pronuclear oocytes subjected to vitrification followed by post-thaw step-wise removal of cryoprotectants versus direct rehydration, in terms of their subsequent in vitro survival and ultrastructural features. METHODS: A total of 115 three-pronuclei stage oocytes were cryopreserved in super-open-pulled straws by vitrification in 40% ethylene glycol + 0.75 mol/l sucrose for either 1 min or 10 s at 38 degrees C, followed by direct plunging into liquid nitrogen. After thawing, oocytes vitrified for 1 min (group 1) or 10 s (group 2) were expelled into a graded series of sucrose solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) for removal of the cryoprotectant in five 2.5 min steps. A second batch of oocytes vitrified for either 1 min (group 3) or 10 s (group 4) were directly expelled into culture medium at 38 degrees C after thawing. Finally, the ultrastructural changes occurring in oocytes in each of the treatment groups were evaluated. RESULTS: Oocyte development (division to two-blastomere stage) rates after in vitro culture were 82, 83, 0 and 0% for groups 1, 2, 3 and 4, respectively. The harsh osmotic process involved in direct rehydration provoked ultrastructural changes, including the disruption of cytoplasmic and pronuclear membranes as well as intracellular organelles. CONCLUSION: The direct post-thaw rehydration of human pronuclear oocytes has lethal osmotic effects, such that protocols for vitrifying human pronuclear oocytes should include the step-wise removal of the cryoprotectant.     

Vitrification of mouse oocytes using closed pulled straws (CPS) achieves a high survival and preserves good patterns of meiotic spindles, compared with conventional straws, open pulled straws (OPS) and grids.

BACKGROUND: We modified the loading of pulled straws into a new closed system, called closed pulled straws (CPS) for holding oocytes for vitrification. The morphological survival, dynamics of meiotic spindles, and fertilization in vitro of vitrified oocytes using CPS were compared with conventional straws, open pulled straws (OPS), and grids. METHODS: Surviving oocytes were stained for spindles and chromosomes after 1, 2 and 3 h incubations, and compared with controls. The capacity of fertilization and embryonic cleavage were examined in vitro. RESULTS: The survival rates of the CPS (79%) and straw (77%) groups were significantly higher (P < 0.05) than the OPS (63%) and grid (39%) groups. At a 1h incubation, vitrified oocytes of four groups had significantly fewer normal spindles than controls (P < 0.05). The straw group was inferior to the others in spindle morphology (P < 0.05). After a 3 h incubation, recovery of vitrified oocytes with normal spindles was significantly improved in all groups (P < 0.05). The percentages of fertilization and blastocyst formation of vitrified oocytes after a 1 h incubation was significantly lower than controls (P < 0.05), but they were improved after 2 or 3 h incubations (P < 0.05). CONCLUSIONS: Oocytes vitrified using CPS, OPS or grids could lessen spindle injuries and expedite recuperation. The survival using OPS or grids is lower. Sufficient culture time for recovery of meiotic spindle would be imperative for fertilization events of vitrified oocytes. CPS has the advantages of achieving a high survival and preserving good spindles.     

Open pulled straws for vitrification of mature mouse oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws

Vitrification of oocytes has been applied recently for humans, but remains elusive. The microtubules of oocytes are vulnerable to cryoprotectants and thermal changes. Using mouse oocytes, the effects of vitrification in open pulled straws (OPS) were investigated on survival, the meiotic spindle, and chromosomes and compared with conventional straws. Mature oocytes were allocated to four groups for exposure to cryoprotectants, vitrification in conventional straws, or vitrification in OPS. They were diluted in stepwise sucrose solutions. Oocytes without treatments were used as controls. The surviving oocytes were stained for meiotic spindles and chromosomes. After dilution, all of the oocytes exposed to cryoprotectants survived. Vitrification sometimes resulted in lysis so that survival using OPS (62%) was significantly (P < 0.05) smaller than that using conventional straws (81%). Oocytes exposed to cryoprotectants or vitrified exhibited serious disturbances of microtubules immediately post-dilution. After 1 h incubation, the microtubules could repolymerize so that the OPS group had significantly (P < 0.05) more normal spindles (78%) than did the conventional straw group (21%). The former also tended to have more compact chromosomes (87%) than did the latter (78%). OPS for vitrification of oocytes achieve more rapid cooling, warming, and dilution and so reduce spindle injury. However, the lower survival rate in OPS needs improvement.     

Cryoloop vitrification of rabbit oocytes

BACKGROUND: Vitrification is assumed to be a promising method to cryopreserve human oocytes but still needs optimization. In this study, rabbit oocytes (fertilized by ICSI) were vitrified with cryoloops, and the effect of three different cryopreservation protocols on spindle configuration and embryo quality was assessed. METHODS: Metaphase II rabbit oocytes were randomly assigned to one of four groups: (i) control; (ii) E40 [40% ethylene glycol (EG)]; (iii) ED20 [20% EG + 20% dimethylsulphoxide (DMSO)]; and (iv) ED20 + M (20% EG + 20% DMSO + vitrification machine). After warming, one part of each group was fertilized by ICSI to examine the fertilization and embryo cleavage ability, and the others were immunostained for tubulin and chromatin before visualization using confocal microscopy. RESULTS: The survival rates after warming were 79.1, 83.1 and 82.3%, respectively. In protocols E40 and ED20, the spindles were severely injured and the embryo quality not good compared with those in the ED20 + M group. CONCLUSIONS: The fastest cooling rate in combination with EG and DMSO as cryoprotectants had the fewest adverse effects on the spindle configuration of rabbit oocytes and embryo development.     

Ongoing twin pregnancy after vitrification of blastocysts produced by in-vitro matured oocytes retrieved from a woman with polycystic ovary syndrome: Case report

This case report describes an ongoing pregnancy after cryopreservation of blastocysts produced by in-vitro matured oocytes retrieved from a woman with polycystic ovary syndrome (PCOS). Oocyte retrieval was performed on day 18. The patient was administered 10 000 IU of hCG s.c. 36 h prior to oocyte collection. A total of 61 immature oocytes was obtained. Following incubation for 24-72 h in the YS maturation medium supplemented with 30% follicular fluid (hFF), 1 IU/ml FSH, 10 IU/ml hCG and 10 ng/ml rhEGF, 65.6% (40/61) of the oocytes were at the metaphase II stage. Thirty-eight oocytes (38/40, 95.0%) were fertilized after ICSI with the patient's husband's sperm and the 2PN oocytes were co-cultured with cumulus cells in YS medium supplemented with 10% hFF. Four embryos were transferred into the uterus on day 4 following oocyte retrieval but this failed to result in pregnancy. Eight embryos were developed to expanded blastocyst stage. The blastocysts were vitrified on electron microscope grids. Two years after cryopreservation, four blastocysts were thawed, three re-expanded and these frozen-thawed blastocysts were transferred to the uterus. A viable twin pregnancy was confirmed by ultrasound scan.     

应用玻璃化冷冻技术保存人未成熟卵母细胞的研究

目的利用玻璃化冷冻保存技术合理利用常规促排卵周期获得的未成熟卵母细胞。方法1．对注射hCG后38h、43h、60h获得GV期卵母细胞进行玻璃化冷冻保存,冻融后的未成熟卵母细胞行体外成熟培养（IVM）,用ICSI技术对获得的成熟卵母细胞完成授精,并体外胚胎培养至囊胚期。比较各组间的冻融存活率、体外成熟率、受精率、卵裂率及囊胚形成率。2．对GV卵分别行先玻璃化冻融后IVM和先IVM后玻璃化冻融,比较它们的冻融存活率、体外成熟率、受精率、卵裂率及囊胚形成率。结果1．38h与43h卵龄GV卵在各项指标比较上没有差异,60h卵与前2组有相似的冻融存活率,但在体外成熟率、受精率和卵裂率上较前2组出现明显下降趋势。3组均无囊胚形成。2．先玻璃化冻融后IVM和先IVM后玻璃化冻融在未成熟卵母细胞的利用率上没有差异。结论1．冷冻卵龄较短的GV期卵母细胞能获得更好的冷冻保存效果。2．玻璃化冻融和IVM先后不影响未成熟卵母细胞的利用。     

Birth following vitrification of a small number of human oocytes: case report

We report the birth of a healthy baby girl at 37 weeks gestation to a 47 year old recipient, after vitrification of mature oocytes from four in-vitro fertilization (IVF) patients. A total of 17 oocytes was vitrified in 1-2 microl of ethylene glycol (40%) and 0.6 mol/l sucrose (20.54%) in open pulled straws. Eleven oocytes survived after vitrification and five pronuclear zygotes were obtained after intracytoplasmic sperm injection (ICSI). Three embryos were transferred to three patients, two of whom were the original oocyte donors and pregnancy was not established. The third embryo was donated to a 47 year old infertile woman after preimplantation diagnosis had confirmed euploidy for chromosomes X, 13, 14, 15, 16, 18, 21 and 22. The successfully completed pregnancy is encouraging for further research to explore the potential benefits of vitrification for the cryopreservation of human oocytes, given the relatively low success of conventional freezing of human oocytes by slow cooling methods.     

Characterization of abnormal one pronuclear human oocytes by morphology, cytogenetics and in-situ hybridization

The morphology, chromosomal constitution and developmental capabilityof abnormal human oocytes (94/3500 oocytes; 2.7%) which afterinsemination exhibited only one pronucleus were examined. Themajority of one pronuclear oocytes exhibited two or more distinctpolar bodies. Dividing oocytes showed irregular chromosome distributionfrom haploid to diploid. Embryos resulting from abnormal oocytesdisplayed limited developmental potential. Many of them underwentfragmentation or were arrested at the 2- or 8-cell stage ofdevelopment, and only some reached the morula or blastocyststate (11/35 oocytes). In 45% (15/33) of examined oocytes,decondensed sperm heads or tiny nucleus-like structures werefound in addition to a single nucleus. Chromosome Y was alsodetected in chromosomal preparations in 10% of the oocytesby in-situ hybridization utilizing the human Y chromosome-specificDNA probe (DYZ3). These observations provided strong evidencethat many of these oocytes originated from fertilized oocytes.The origin of the other one pronuclear oocytes could not bedetermined. Parthenogenetic activation of some oocytes cannotbe excluded and other explanations concerning the origin ofabnormal oocytes are discussed.     

Time-dependent capability of human oocytes for activation and pronuclear formation during metaphase II arrest

BACKGROUND: The purpose of this study was to investigate the fertilization rate and developmental potential of human oocytes in relation to the duration of their metaphase II (MII) arrest stage following the extrusion of the first polar body (1PB). METHODS: Immature metaphase I oocytes (MI; study oocytes, n = 468) that underwent meiotic maturation during brief in vitro culture and their matured in vivo, MII siblings (control oocytes, n = 3293) were subjected to ICSI. Fertilization and early cleavage were evaluated in both study and control groups. RESULTS: The overall fertilization rate was significantly lower in the oocytes matured in vitro than in those matured in vivo (42 versus 77%, P < 0.0001). A significant relationship was observed between oocyte activation potential and the length of MII arrest. The majority of study oocytes injected soon after PB extrusion remained unfertilized (64%; 98/154 oocytes). The proportion of normally activated oocytes that contained two pronuclei and two PBs gradually increased with prolonged time of MII arrest (43 and 61% at 2 and 3-6 h after 1PB extrusion). Significantly more embryos originating from the study than control oocytes were arrested soon after the first two cleavage divisions (39 and 17%; P < 0.0001) and exhibited multinucleated blastomeres (23 and 13%; P < 0.0001), which suggests the existence of chromosomal abnormalities. CONCLUSIONS: Human oocytes progressively develop the ability for full activation and normal development during the MII arrest stage.     

Simple, efficient and successful vitrification of bovine blastocysts using electron microscope grids.

This study demonstrates that higher survival of vitrified-thawed bovine blastocysts can be obtained using electron microscope (EM) grids as embryo containers at freezing, rather than plastic straws. In-vitro produced day 7 bovine blastocysts after in-vitro fertilization (IVF) were vitrified on grids or in straws with EFS40 freezing solution and their survival after thawing was compared. Embryo survival was assessed as re-expanded and hatched rates at 24 and 48 h after thawing respectively. When the effects of exposure to vitrification solution and chilling injury from the freezing procedure were examined, embryo survival in the exposure group (24 h: 100, 48 h: 73.3%) was not different compared with that in the control group (100, 84.4%). After vitrification, the hatched rate of the EM grid group 48 h after thawing (67.8%) was significantly higher than that of the straw group (53.3%) (P < 0.05). Fast developing embryos (expanded blastocyst and early hatching blastocyst stage) showed better resistance to freezing than delayed ones (early blastocyst stage), irrespective of embryo containers (early: 24 h, 57.1 and 48 h, 24.4%; expanded: 84.7 and 60.6%; early hatching: 91.7 and 80.0%) (P < 0.001). When using expanded and early hatching blastocysts, embryo survival rates in the vitrification-EM grid group (67.8, 95.0% respectively) were significantly higher than that of the vitrification-straw group (53.0, 65.0%) at 48 h.     

Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes

BACKGROUND: Vitrification of human blastocysts has been successfully applied using grids, straws and cryoloops. We assessed the survival rate of human compacted morulae and early blastocysts vitrified in pipette tips with a smaller inner diameter and solution volume than the previously described open pulled straw (OPS) method. METHODS: Excess day 5 human embryos (n = 63) were experimentally vitrified in vessels. Embryos were incubated at 37 degrees C with sperm preparation medium (SPM) for 1 min, SPM + 7.5% ethylene glycol (EG)/dimethylsulphoxide (DMSO) for 3 min, and SPM + 16.5% EG + 16.5% DMSO + 0.67 mol/l sucrose for 25 s. They were then aspirated (0.5 microl) into a plastic micropipette tip (0.36 mm inner diameter), exposed to liquid nitrogen (LN(2)) vapour for 2 min before being placed into a pre-cooled cryotube, which was then closed and plunged into LN(2). Embryos were warmed and diluted using 0.33 mol/l and 0.2 mol/l sucrose. RESULTS: The survival rate for compacted morulae was 73% (22/30) and 82% (27/33) for early blastocysts. CONCLUSIONS: The survival rates of human compacted morulae and early blastocysts after vitrification with this simple technique are similar to those reported in the literature achieved by slow cooling and other vitrification protocols.     

Fertilization and early embryology: Parthenogenetic activation of human oocytes following cryopreservation using 1, 2-propanediol

Fresh and aged human oocytes were cryopreserved using 1, 2-propanediol(PROH). After thawing, the oocytes were cultured for 20 h andexamined for parthenogenetic activation using light microscopyand an ultraviolet DNA stain. Control fresh or aged oocytesand oocytes exposed to PROH without cryopreservation were alsoexamined for activation. No control oocytes were observed toactivate spontaneously (n = 43) and parthenogenetic activationwas not induced by exposure to PROH alone (n = 26). In bothfresh and aged cryopreserved oocytes, 27 and 29% of the oocytesrespectively were activated, and these proportions were significantlyelevated compared with the controls (P < 0.01). Althougha similar rate of activation was observed for the cryopreservedfresh and aged oocytes, the form of parthenogenetic activationvaried between these two types of oocyte. A single pronucleuswas observed in 18% of the fresh and 5% of the aged cryopreservedoocytes. In contrast, the presence of two or more pronucleiwas observed in 0% of the fresh and 19% of the aged cryopreservedoocytes.     

Cryopreservation of human oocytes

Three different methods were used for freezing human excessoocytes (320 frozen, 205 thawed) in our IYF programmme and theresults of these methods weax compared. A high fertilizationrate (75%) could be achieved after thawing, using 1,2 propanediolas a cryoprotectant. Polyploidy rates of 20% and 40% were observedusing DMSO and l,2-propanediol as cryoprotectants, respectively.Using the ultracooling method, the survival rate was poor (4%).     

Bulk vitrification of human embryonic stem cells

BACKGROUND: The traditional vitrification method cannot keep up with the increased culture and propagation efficiency required to cryopreserve large quantities of vigorously proliferating human embryonic stem (HES) cells. In this study, we describe a newly invented vitrification carrier for cryopreserving large amount of HES cells and evaluate whether this bulk vitrification (BV) method is as effective as the popular open-pulled straw (OPS) vitrification method. METHODS: HES cell clumps were harvested after passage and transferred to a cell strainer; only those clumps with a diameter more than 70 microm were included in the study and randomly selected to be cryopreserved by the BV method, OPS vitrification or slow freezing method. HES cell survival, growth and pluripotency were analyzed after thawing. RESULTS: Bulk vitrification method with cell strainer could cryopreserve 136 +/- 23.4 cell clumps at one time (round), which was 30 times as high as those for OPS method (4 +/- 1.5). After thawing, bulk-vitrified HES cells exhibited high survival rate up to 94.3%, comparable with the OPS method. All surviving cell clumps generated HES cell colonies. Teratomas comprising all three primordial germ layers were formed in severe combined immunodeficient mice after subcutaneous injection of post-thawed, bulk-vitrified HES cell clumps, confirming pluripotency. CONCLUSIONS: This new BV method could cryopreserve a large quantity of HES cell clumps at one time, which not only would satisfy routine cryopreservation of HES cell during daily culture process but also guarantee researchers have large quantity of efficiently cryopreserved HES cells ready for a scheduled study at any time.     

Cytogenetic analysis of unfertilized human oocytes

Cytogenetic studies were carried out on 180 oocytes that appeared unfertilized after in-vitro fertilization. The majority of the 135 that were informative had grossly haploid second meiotic metaphases, two were grossly diploid, and five had a variety of different abnormalities. Twenty-one oocytes were abnormally fertilized and included prematurely condensed sperm chromosomes. The frequency of this phenomenon varied according to the stimulation protocol, those oocytes maturing longer in vivo showing less propensity to abnormal fertilizations. Thirteen per cent of the analysable haploid metaphases were hyperhaploid but none contained extra whole chromosomes. The extra components were a single chromatid (one case), or two single chromatids replacing a whole chromosome (four cases). The data suggest that the chromatids arose as a result of premature centromere division at meiosis I, and that this may be a major mechanism for trisomy formation rather than non-disjunction of whole bivalents at meiosis I, as generally believed.     

Cryopreservation of human oocytes: a review of current problems and perspectives

It is well recognized that the ability to cryopreserve unfertilizedhuman oocytes would make a significant contribution to infertilitytreatment. However, despite considerable interest, very fewsuccessful pregnancies have arisen from cryopreserved oocytesafter thawing, insemination and transfer of the subsequent embryo.The reasons for this lack of progress may well result from adearth of information on how the various biophysical changesduring a cryopreservation regimen affect human oocyte function.Recently, fundamental studies on the effects of cooling, membranepermeability, cryoprotectant addition and ice formation havebeen performed on human oocytes by a number of groups, and theseform the basis of the current review. It is likely that successfulhuman oocyte cryopreservation will only follow once these factorsare fully understood, but the existing base of knowledge shouldprovide a platform for further improvements in the techniquescurrently employed.     

Cryopreservation of human embryos and oocytes

The success rate of human embryo cryopreservatlon depends ontechnical and embryonic parameters. First of all, the cryoprotectantcan affect embryo survival as we found by comparing two freeze-thawprocedures using propanediol (PROH) (1.5 mol) alone or withsucrose (0.1 mol). Embryo survival was significantly enhancedwith sucrose (62 versus 32%). Embryo quality is another majorparameter involved in the success of freezing; the rates ofpositive survival were found to be 67% for morphologically normalembryos versus 49% for embryos with fragments (P < 0.001).The efficiency of embryo cryopreservatlon in an IVF programmecould be estimated in 1986: a woman with extra embryos, storedafter transfer of 3–4 fresh embryos (16% of all cydes),can expect a 22% pregnancy rate per transfer of fresh embryosand a 32% pregnancy rate per collection after transfer of thestored eggs. A comparative study of the cryopreservability ofimmature or mature oocytes was performed in humans. Human oocyteshave a low survival rate (36%) whatever the cryopreservationprotocol or the initial maturation stage. Immature human oocytescould survive freezing and thawing, mature and be fertilizedin vitro, but with a very low efficiency.     

Intracytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2-propanediol

This study reports the subsequent embryo development of cryopreservedmature human oocytes following insemination or intracytoplasmicsperm injection (ICSI). Metaphase II oocytes were cryopreservedusing a slow freezingrapid thawing procedure employing the cryoprotectant1,2- propanediol. The study was conducted at two centres. Thenormal insemination of cryopreserved oocytes was undertakenin one centre, and ICSI of cryopreserved oocytes in the other.Both methods resulted in a 50% normal fertilization rate. Alow rate of abnormal fertilization was observed in the inseminatedgroup of oocytes (5%) compared with 21% for the ICSI oocytes;this was not significantly different. Embryo development wasassessed daily for 7 days. All normal fertilized cryopreservedoocytes in both groups cleaved on day 2, with a similar appearanceto in-vitro fertilization and ICSI embryos. In the normal inseminatedoocytes, there was a significant decrease in the number of embryoscleaving on day 3 (33%) compared with the development of ICSIoocytes, with a subsequent gradual reduction over days 4 and5 (22 and 11% respectively) resulting in one early blastocyston day 7 (11%). In contrast, all ICSI-generated embryos continuedto cleave on day 3, with a gradual reduction over subsequentdays (day 4, 86%; day 5, 57%; day 6, 43%; day 7, 29%). By day7, two of the blastocysts had started to hatch, resulting ina 66% hatching rate of blastocysts formed from ICSI of cryopreservedoocytes. This is the first study to show normal developmentto the hatching blastocyst stage following ICSI of cryopreservedhuman oocytes.     

A novel method for chromosome analysis of human sperm using enucleated mouse oocytes

BACKGROUND: Mouse oocytes can be used in conjunction with intracytoplasmic sperm injection (ICSI) as a technique to permit chromosomal analysis of human sperm. However, chromosomes derived both from the human sperm and the mouse oocyte appear simultaneously following ICSI. The present study focused on evaluating whether or not previously enucleated mouse oocytes are usable for the analysis of human sperm chromosomes. METHODS: The metaphase chromosome-spindle complex was removed from a mouse oocyte. Human sperm from a donor with proven fertility were injected into mouse enucleated oocytes or intact oocytes. The presence of pronuclei in the oocytes was confirmed approximately 7-11 h after ICSI, and the oocytes were then fixed so that the nuclei could be observed as chromosome samples at 15-16 h after ICSI. RESULTS: The formation rate of one pronucleus in enucleated oocytes after ICSI was 93.9% (186/198) while that of two pronuclei in intact oocytes after ICSI was 85.4% (88/103). The appearance rate of metaphase chromosomes of human sperm in the enucleated oocytes, 89.4% (160/179), was significantly higher than that in intact oocytes, 78.7% (74/94) (P = 0.017). CONCLUSIONS: An efficient ICSI method using enucleated mouse oocytes was established to allow the visualization of the human sperm chromosome complement without the risk of confusion with mouse oocyte chromosomes.     

Experimentally induced parthenogenetic activation of human oocytes

A total of 486 unfertilized, aged human oocytes were exposedto ethanol, calcium ionophore A23187, phorbol ester or puromycinand examined for evidence of activation. Five per cent of controloocytes (3/58) were spontaneously activated. Of the two agentswhich cause the release of intracellular Ca²⁺ ions, Ca²⁺ ionophoreinduced activation of only 16% of unfertilized oocytes, whileethanol did not have any effect. Phorbol ester, a stimulatorof protein kinase C, also resulted in limited activation (14%of oocytes). In contrast, puromycin, an inhibitor of proteinsynthesis, resulted in activation of 91% of the exposed oocytes.It is speculated that puromycin probably inhibits a specificcytostatic factor or factors which are responsible for maintenanceof the metaphase II block. Morphologically activated oocytesusually retained the second polar body and formed subnuclei.The developmental potential of activated oocytes appeared tobe reduced, with only some oocytes capable of a single division.