Cryopreserved oocytes of infertile couples undergoing assisted reproductive technology could be an important source of oocyte donation: a clinical report of successful pregnancies

BACKGROUND: Surplus oocytes in assisted reproduction treatment cycles could be saved and donated to other couples. ICSI is usually performed for oocytes that have been stored frozen, considering possible exocytosis of cortical granules (CG). The unavoidability of ICSI merits further study. METHODS: We used a slow method to freeze excess oocytes from infertile couples. After thawing, oocytes were fertilized by either IVF or ICSI according to semen parameters. Some oocytes were examined for CG. RESULTS: Twenty-eight infertile couples cryopreserved a proportion of their oocytes and 12 thawed their oocytes. Three couples used their own oocytes, whereas nine donated their oocytes to nine other couples for 12 cycles. The survival rate from thawing was 90% (73/81). The fertilization rate using IVF (83%) was similar to ICSI (82%). Seven pregnancies (47% per cycle) were achieved; one used her own oocytes and six received donated oocytes. Five women delivered six babies including one set of twins. Two pregnancies aborted. The frozen-thawed oocytes (15/15) revealed no exocytosis of CG. CONCLUSIONS: To freeze oocytes of infertile couples undergoing assisted reproduction treatment may help other couples. Our successful experience may facilitate oocyte banks to become a reality. Both IVF and ICSI are valuable for frozen oocytes.     

Observational clinical follow-up of oocyte cryopreservation using a slow-freezing method with 1,2-propanediol plus sucrose followed by ICSI

BACKGROUND: The value of oocyte cryopreservation remains controversial. Two major problems exist: poor survival and injury to the oocyte meiotic spindle after freezing and thawing. METHODS: For slow oocyte cryopreservation, we used 1.5 mol/l 1,2-propanediol and 0.3 mol/l sucrose. We waited 3 h after thawing for possible recovery of the meiotic spindles before performing ICSI. RESULTS: Forty-three women undergoing IVF or ICSI cycles cryopreserved some or all of their harvested oocytes; of these, 20 thawed their cryopreserved oocytes for personal use and one for donation. The survival rate of oocytes after thawing was 75%, with 67% of oocytes fertilizing normally after ICSI. All 21 cycles (100%) resulted in fertilization and embryo transfers. Seven pregnancies (33%) resulted. Four women delivered five babies with normal karyotypes. Three conceptions are ongoing. Compared to 38 cycles of frozen-thawed embryos at the pronuclear stage in the same period, the percentages of survival, pregnancy and implantation were similar. Additionally, four unmarried women with white blood cell diseases underwent oocyte freezing before preconditioning treatment for haematopoietic stem cell transplantation. CONCLUSIONS: This protocol achieved reproducible success of survival, fertilization and pregnancy for freezing and thawing of human oocytes. The 3 h post-thaw incubation could permit restoration of the meiotic spindles, thus facilitating normal fertilization.     

Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures

BACKGROUND: The clinical efficacy of the current methods used for cryopreservation of metaphase II human oocytes is low. Meiotic spindle disorders are thought to be largely responsible for this situation. METHODS: Supernumerary fresh metaphase II human oocytes were cryopreserved in 1,2-propanediol with 0.1 M sucrose using a slow freezing/rapid thawing programme. Meiotic spindles were analysed in these living metaphase II oocytes at sequential steps of the freezing and thawing procedures with the use of a computer-assisted polarization microscopy system (Polscope). RESULTS: The meiotic spindle was detected in all 56 oocytes (from 16 patients) before freezing and remained visible in all these oocytes throughout the preparation for freezing up to the time that they were loaded into cryopreservation straws. Immediately after thawing, the spindle was visible in 35.7% of oocytes, but it disappeared in all of the thawed oocytes during the subsequent washing steps. However, the spindle reappeared in all surviving thawed oocytes after washing (57.4%), by 3 h of incubation at 37 degrees C in culture medium. CONCLUSIONS: The current techniques of oocyte freezing and thawing inevitably cause meiotic spindle destruction. All spindles observed in thawed oocytes result from post-thaw reconstruction.     

Ultrastructure of human mature oocytes after slow cooling cryopreservation using different sucrose concentrations

BACKGROUND: We studied the ultrastructural characteristics of human mature oocytes frozen/thawed (F/T) using different concentrations of sucrose. Fresh human mature oocytes were used as controls. METHODS: The oocytes (n = 48) were fixed in 1.5% glutaraldehyde at sampling (n = 16) or after freeze/thawing performed using a slow cooling method with propane-1,2-diol 1.5 mol/l and sucrose at either 0.1 mol/l (n = 16) or 0.3 mol/l (n = 16) in the freezing solution. The oocytes were then processed for electron microscopy observations. RESULTS: Fresh and F/T oocytes belonging to both study groups were regularly rounded in sections, with a homogeneous cytoplasm and an intact zona pellucida (ZP). Organelles (mainly mitochondria-smooth endoplasmic reticulum aggregates and mitochondria-vesicle complexes) were abundant and uniformly dispersed in the ooplasm. The amount and density of cortical granules appeared to be abnormally reduced in some F/T samples, independently of the sucrose concentration in the freezing solution: this feature was frequently associated with an increased density of the inner ZP, possibly related to the occurrence of zona 'hardening'. Furthermore, slight to moderate microvacuolization was revealed in the ooplasm of some F/T oocytes, particularly in those treated with sucrose 0.3 mol/l. CONCLUSIONS: Freeze/thawing procedures are associated with ultrastructural alterations in specific oocyte microdomains, presumably linked to the reduced developmental potential of mature cryopreserved oocytes. Further work is needed to determine whether or not a high concentration of sucrose plays a role, at least in part, in producing the above alterations.     

常规IVF受精失败补救ICSI后行冻融胚胎移植的临床价值

目的探讨冻融胚胎移植在常规体外受精(IVF)失败后补救卵胞浆内单精子注射(L-ICSI)中的应用价值。方法在12个常规体外受精失败周期中应用ICSI对未受精的MⅡ期卵子进行显微授精,将获得的优质胚胎进行冷冻,再择期行冻融胚胎移植。结果对93个未受精的MⅡ卵子接受L-ICSI,受精63枚,受精率为67.7%(63/93),异常受精3枚(2枚1PN,1枚3PN),57个正常受精卵发生卵裂,卵裂率为95.0%(57/60),优质胚胎率为43.9%(25/57),10例患者冷冻胚胎25枚,其中4例采用程序化冷冻,6例采用玻璃化冷冻。9个患者行冻融胚胎移植,共移植胚胎18枚(其中解冻后胚胎碎裂死亡5枚),其中1个周期因冻融后2个胚胎碎裂放弃移植,2例获得临床妊娠,1例分娩出正常婴儿,1例正在妊娠中,临床妊娠率为22.2%。结论 ICSI可使常规体外受精失败的卵子再受精,冻融胚胎移植可以解决胚胎与子宫内膜不同步的问题,获得相对满意的临床结局,具有一定的应用价值。     

Human oocyte cryopreservation: new perspectives regarding oocyte survival

The success of human oocyte cryopreservation depends on morphological and biophysical factors that could influence oocyte survival after thawing. Various attempts to cryopreserve human oocytes have been performed with contrasting results. Therefore the effect of some factors, such as the presence or absence of the cumulus oophorus, the sucrose concentration in the freezing solution and the exposure time to cryoprotectants, on human oocyte survival after thawing were investigated. The oocytes were cryopreserved in 1,2-propanediol added with sucrose, using a slow-freezing-rapid-thawing programme. After thawing, the oocytes were inseminated by intracytoplasmic sperm injection (ICSI) and the outcomes of insemination and subsequent embryo development were also recorded. The post-thaw cryosurvival rate was not different for the oocytes cryopreserved with their cumuli partially removed mechanically (56%) when compared with those cryopreserved with their cumuli totally removed enzymatically (53%). On the contrary, a significantly higher survival rate was obtained when the oocytes were cryopreserved in the presence of a doubled sucrose concentration (0.2 mol/l) in the freezing solution and the survival rate was even higher when the sucrose concentration was tripled (0.3 mol/l) (60 versus 82% P < 0.001). Furthermore, a longer exposure time (from 10.5 to 15 min) to cryoprotectants, before lowering the temperature, significantly increased the oocyte survival rate (P < 0.005). Intracytoplasmic sperm injection produced a good fertilization rate (57%) of thawed oocytes and a high embryo cleavage rate (91%) and a satisfactory embryo morphology was observed (14 and 34% for grade I and grade II embryos respectively).     

Fertilization and early embryology: Preliminary experience with human oocyte cryopreservation using 1,2-propanediol and sucrose

Feasibility of cryopreservalion of mature human oocytes using1,2-propanediol and sucrose was studied initially utilizrng1 and 2 day old unfertilized oocytes. Of these 285 aged oocytes55% survived thawing, and 41% of 128 oocytes inseminated bysingle sperm intracytoplasmlc injection (ICSI) fertilized normally.Limited embryonic development occurred in 51% of these embryos(n = 27) observed for the next 4 days. Cryosurvlval of freshdonated oocytes (n = 81) was poorer (n = 20; 24.7%), while fertilization(n = 13; 65%) and embryo development (100%) was good prior uterinetransfer on day 3. Eight oocyte recipient cycles were undertaken,in which cryopreserved donated oocytes were thawed and inseminatedby ICSI. Five of these cycles reached embryo transfer, and threepregnancies were initiated though none went successfully toterm. Oocyte cryopreservatlon will ultimately facilitate oocytedonation procedures; however, cryosurvival of fresh frozen oocytesmust be improved to at least the degree observed with aged unfertilizedoocytes.     

Cryopreservation of biopsied embryos at the blastocyst stage

BACKGROUND: The availability of an efficient cryopreservation program is especially important in the case of embryos that have undergone blastomere biopsy for PGD. Unfortunately, the freezing/thawing of biopsied embryos has given disappointing results when performed at the cleavage stage. In this study, embryos diagnosed as normal after PGD were grown to the blastocyst stage, frozen and thawed for successive frozen embryo transfer. METHODS: A total of 34 patients performed a thawing cycle in which 47 blastocysts were thawed. The cryopreservation solutions were based on HEPES-buffered medium supplemented with human serum albumin (HSA), sucrose and 1,2-propanediol. The same protocol was applied to embryos from 88 IVF/ICSI patients, which underwent 92 thawing cycles with 150 thawed blastocysts. RESULTS: The survival rate was similar in the two groups (53% after PGD and 58% in IVF/ICSI cycles), as well as the cumulative pregnancy rate per patient (59% after PGD versus 47% in IVF/ICSI cycles), despite a higher maternal age and a lower proportion of embryos available for transfer or cryopreservation in the PGD group. CONCLUSIONS: Neither the survival rate nor the subsequent development and chances of implantation, differed between embryos frozen at the blastocyst stage following biopsy and those frozen intact.     

Germinal vesicle transfer between fresh and cryopreserved immature mouse oocytes.

BACKGROUND: We assessed the maturational competence and the chromosomal pattern of mouse oocytes reconstructed by germinal vesicle (GV) transfer technique using nuclear and/or cytoplasmic components from cryopreserved GV stage oocytes. METHODS: From 657 GV oocytes (326 fresh and 331 frozen/thawed), four groups of reconstructed oocytes were obtained by micromanipulation and electrofusion: fresh GV-fresh cytoplast (FF), thawed GV-thawed cytoplast (TT), fresh GV-thawed cytoplast (FT), thawed GV-fresh cytoplast (TF). All reconstructed oocytes were cultured in vitro to metaphase II. RESULTS: Survival rate after manipulation and electrofusion, as well as progression to metaphase II, did not differ significantly among the four groups. Comparing reconstructed oocytes with fresh and thawed control pools, the only difference was a slightly but significantly higher maturation rate in the TT pool versus matched controls (P < 0.01). Cytogenetic analysis of 25 reconstructed oocytes showed the expected number of 20 chromosomes in 88% of them. CONCLUSIONS: We conclude that both nuclear and cytoplasmic components derived from cryopreserved immature oocytes are suitable for GV transfer procedure, and generate chromosomally normal oocytes able to progress to metaphase II in vitro. The possibility of using cryostored immature oocytes as a source of nuclei and cytoplasm could help in applying GV transfer procedure, both in research and clinical settings.     

Assessment of the developmental potential of frozen-thawed mouse oocytes

Mouse oocytes were cryopreserved by a protocol shown previouslytominimize damage tot the zona peelucida and cytoskeletal system.After thawing, the incidence of fertilization didnot differfrom that in control groups of oocytes, and after fertilizaiton,the ability of the fertilized frozen–thawed oocytes todevelop tothe balstocyst stage in vitro was only slightly less(77%) than that of the controls (87 and 89%). Transfer of frozen–thawedand fertilized oocytes after their culture to the balstocyststage in vitro resulted in a lower implantation rate (46%) thanfor the controls (68–73%), but of the implanting embryosthe same proportions in experimental and control groups survivedtoyield vaiable fetuses. In contrast, transfer after culturein vitro to the 2-to 4-cell stage rsulted in similar implantationrates for control and frozen–thawed fertilized oocytes(70–84%), but the spontaneous abortion rate was higherfor the embryos derived from frozen–thawed oocytes. Overallthe cumulative survival rate for frozen oocytes transferrd atthe 2-cell stage (36%) was better than after transfer at theblastocyst stage (30%), but both were less than for the transferat any stage of the control oocytes (47–55%). The cumulativesurvival of cryopreserved oocytes to viable fetuses was 30–40%less than that of the control oocytes. These results are comparedwith those form previous studies and the main remaining obstaclesto completely successful cryopreservation are identified     

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.     

Sucrose concentration influences the rate of human oocytes with normal spindle and chromosome configurations after slow-cooling cryopreservation

BACKGROUND: Recently described slow-cooling cryopreservation protocols involving elevated sucrose concentration have improved survival frequencies of human oocytes, potentially overcoming a major hurdle that has limited the adoption of oocyte storage. Because implantation rates of embryos from frozen oocytes remain generally low, it is still debated whether, irrespective of survival rates, this form of cryopreservation leads inevitably to the disruption or complete loss of the metaphase II (MII) spindle. METHODS: Human oocytes with an extruded polar body I (PBI) were cryopreserved using a slow-cooling method including 1.5 mol/l propane-1,2-diol (PrOH) and alternative sucrose concentrations (either 0.1 or 0.3 mol/l) in the freezing solution. Fresh control and frozen-thawed survived oocytes were analysed by confocal microscopy to evaluate MII spindle and chromosome organizations. RESULTS: Of the 104 oocytes included in the unfrozen group, 76 (73.1%) displayed normal bipolar spindles with equatorially aligned chromosomes. Spindle and chromatin organizations were significantly affected (50.8%) after cryopreservation involving lower sucrose concentration (61 oocytes), whereas these parameters were unchanged (69.7%) using the 0.3 mol/l sucrose protocol (152 oocytes). CONCLUSIONS: Partial disruption of the MII spindle and associated chromosomes accompanies inadequate cryopreservation during slow cooling. However, protocols adopting higher sucrose concentration in the freezing solution promote the retention of an intact chromosome segregation apparatus comparable in incidence to freshly collected oocytes.     

Human oocyte cryopreservation as an adjunct to IVF-embryo transfer cycles

BACKGROUND: The purpose of this work was to develop methods for successful cryopreservation of human oocytes. METHODS: Two cryopreservation procedures were used. Method 1 involved use of 1.5 mol/l propanediol (PrOH)-0.1 mol/l sucrose with medium containing sodium (Na) as cryoprotectant medium, seeding at -7 degrees C, and stepwise dilution of cryoprotectant post-thaw. Method 2 used Na-depleted media with 1.5 mol/l PrOH-0.2 mol/l sucrose for freezing, seeding at -6 degrees C, and use of high sucrose (0.5 and 0.2 mol/l) for cryoprotectant removal. RESULTS: The first method was used in seven patients, and gave poor (12.3%) survival results and no pregnancies. The second method was used in 15 patients (16 cycles), and yielded good survival and fertilization rates (74.4 and 59% respectively), with four pregnancies and five healthy infants born to 11 women receiving an embryo transfer. CONCLUSIONS: Using Na-depleted media along with other alterations in freezing and thawing procedures, human oocyte cryopreservation can provide excellent survival and pregnancy rates.     

Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection1

The aim of this study was to determine why oocytes remain unfertilizedor develop three pronuclei after intracytoplasmic sperm injection(ICSI). Unfertilized and abnormally fertilized oocytes werefixed in glutaraldehyde,stained with Hoechst 33342 and examinedby fluorescence microscopy to identify oocyte, sperm and polarbody DNA.One-pronuclear oocytes were considered to be unfertilized.Atotal of 285 unfertilized oocytes were examined (104 ICSI cycles).Overall, 83% of these oocytes were not activated (still at metaphaseII) while 17% had activated and formed a single (female) pronucleus.About 66% of the unfertilized, metaphase II oocytes containeda swollen sperm head, indicating that the oocyte was correctlyinjected but had failed to activate and complete the secondmeiotic division. Premature chromosome condensation of the spermDNA was evident in 6% of these metaphase II oocytes (4% of theunfertilized oocytes). The swollen sperm head was located amongthe oocyte chromosomes in 5%of the metaphase II oocytes. Othercauses of failed fertilization in the metaphase II oocytes werethe failure of sperm head decondensation (11%) and ejectionof the spermatozoon from the oocyte (23%). A similar patternwas observed in one-pronuclear oocytes (52%, swollen sperm head;28%, intact, undecondensed sperm head; 20%, ejection of thespermatozoon), which indicates that asynchronous pronucleardevelopment does not explain the presence of one-pronuclearoocytes. A total of 41 threepronuclear oocytes were examinedand all had a single polar body, which indicates that the retentionof the second polar body leads to the formation of the thirdpronucleus.In conclusion, this study demonstrates that: (i)the major cause of fertilization failure after ICSI is failureof oocyte activation; (ii) ejection of the spermatozoon intothe perivitelline space is not a major cause of fertilizationfailure;and (iii) sperm head decondensation and oocyte activationafter ICSI can occur independently.     

Volume changes of mature human oocytes on exposure to cryoprotectant solutions used in slow cooling procedures

BACKGROUND: Despite the recent increase in pregnancies from cryopreserved human oocytes, success in terms of births per thawed oocyte is still poor. Modifications to cryopreservation protocols have not been based on measurement of the osmotic response of oocytes, and methodologies are often poorly described or protocols not strictly adhered to, inevitably resulting in variability. METHODS: Volume change of mature human oocytes was measured on exposure to cryoprotectant. Oocytes were exposed to either 0.75 mol/l propane-1,2-diol (PrOH) for 10 min; 1.5 mol/l PrOH for 10 min, having been exposed to 0.75 mol/l PrOH for 7.5 min; or 1.5 mol/l PrOH plus 0.2 or 0.3 mol/l sucrose for 10 min, having been exposed to 1.5 mol/l PrOH for 10 min. RESULTS: On exposure to PrOH alone, oocytes shrank and then re-expanded, having reached 75 and 84% of their starting volume in 0.75 and 1.5 mol/l, respectively. Oocytes shrank continuously in PrOH plus sucrose, reaching 67 or 55% of their initial volume in 0.2 or 0.3 mol/l sucrose, respectively. CONCLUSIONS: To improve consistency following cryopreservation, protocols must be strictly adhered to; small changes in duration of exposure to cryoprotectant can result in drastic changes in cellular hydration and thus the fate of the cell during freezing/thawing.     

Fertilization and early embryology: Intracytoplasmic single sperm injection of 1-day-old unfertilized human oocytes

Although the average fertilization rate in most in-vitro fertilization(IVF) centres is 60–70%, there are cases of complete orvirtually complete fertilization failures. The aim of our workwas to study the fertilization and the subsequent cleavage characteristicsof 1-day-old human oocytes treated by intracytoplasmic singlesperm injection (ICSI) after failing to fertilize during thestandard IVF procedure. A total of 115 metaphase II 1-day-oldunfertilized oocytes were collected from 23 patients. No additionaltreatment was applied to the oocytes or to the semen sample.A single spermatozoon from the patient's husband was injectedinto the cytoplasm of each of these oocytes 21–33 h afterovum retrieval. Injected oocytes were observed at 16–18h and again 42–44 h after the ICSI procedure. Of the injectedoocytes, 92% (n = 106) were intact after ICSI, 38% (n = 44)had two distinct pronuclei and there was no difference in thefertilization rate of oocytes when andrological and non-andrologicalpatients were compared. Similarly, there was no difference inthe fertilization rate after ICSI where patients with acceptableor good (> 15%) fertilization after standard IVF were comparedto patients who had poor (<15%) fertilization after IVF.There was no significant difference in the sperm concentrationor in the progressive forward motility (a + b motility) in thesegroups except where a + b motility of andrological and non-adrologicalpatients was compared. The majority (84%) of the normally fertilizedoocytes cleaved and most (77%) of these embryos showed <20%fragmentation 2 days after the ICSI procedure. From this studyit can be concluded that 1-day-old metaphase II oocytes whichhave failed to be fertilized after standard IVF procedure canbe fertilized and cleave when ICSI is performed on them theday after oocyte retrieval.     

Comparison between intracytoplasmic sperm injection and in-vitro fertilization (IVF) with high insemination concentration after total fertilization failure in a previous IVF attempt.

The aim of this prospective study was to evaluate whether couples with total fertilization failure in a previous in-vitro fertilization (IVF) attempt should be offered an additional IVF treatment with elevated insemination concentration or should be treated with intracytoplasmic sperm injection (ICSI). In 23 cycles 228 sibling metaphase II (MII) oocytes were randomly divided: 143 and 85 oocytes were utilized for ICSI and IVF respectively. Of the 143 injected (ICSI) oocytes, 90 (62.9%) were normally fertilized (two pronuclei), whereas 21 (14.7%) oocytes were damaged by the ICSI procedure. Of the fertilized oocytes 72 (80%) developed into transferable embryos. No fertilization at all was observed in the 85 sibling MII oocytes which were inseminated (P < 0.001). In all 23 cycles at least one embryo, obtained by ICSI, could be replaced. Eight pregnancies were achieved of which six resulted in the delivery of nine healthy children. In conclusion, for couples with no or almost no fertilization of oocytes in previous IVF attempts, ICSI appeared to be far superior to an additional IVF attempt with further elevated insemination concentrations.     

Cryopreservation of supernumerary oocytes in IVF/ICSI cycles

BACKGROUND: The aim of the present study is to investigate cryopreservation of oocytes in patients refusing embryo cryopreservation for ethical reasons, patients from whom no sperm could be retrieved and patients with enough oocytes to yield a number of fresh and cryopreserved embryos to transfer. METHODS: A total of 2900 oocytes out of 6216 retrieved were cryopreserved in 286 patients undergoing 303 cycles. The reasons for cryopreservation were because no sperm was found in 16 cycles, for ethical or personal reasons in 80, and in 207 only supernumerary oocytes were frozen. In 159 cycles, the oocytes were thawed and the surviving metaphase II oocytes microinjected. RESULTS: A total of 1087 oocytes were thawed, 760 (69.9%) survived and 687 were microinjected. We obtained 368 (53.5%) normally cleaved embryos, 331 were transferred and 37 were cryopreserved. One hundred and forty-five transfers (range 1-3 embryos/patient) were performed and 18 (12.4%) pregnancies were obtained. Twelve patients delivered 13 healthy children, and six first trimester abortions were observed (33.3%). CONCLUSION: Although a low implantation rate was observed and a higher abortion rate than in fresh cycles, our results show that in sibling oocytes, the process of cryopreservation apparently does not affect the fertilization and cleavage rate. In this group of patients, producing a large number of mature gametes, oocyte cryopreservation gives the couple extra chances to achieve a pregnancy within a single retrieval and is a good effort towards reducing the number of embryos cryopreserved and enhancing our experience in this new technology.     

A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage

BACKGROUND: Routine oocyte cryopreservation remains an elusive technique in the wide range of assisted reproductive technologies available. This study examines the effect of a cryopreservation protocol on the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle (GV) and metaphase II (MII) stage. METHODS: GV oocytes were randomly assigned to one of three groups: (i) control oocytes matured in vitro to MII stage (n = 156); (ii) oocytes cryopreserved at the GV stage and then matured in vitro (n = 90); (iii) oocytes cryopreserved at the MII stage (n = 147). Following cryopreservation and in-vitro maturation, immunostaining of tubulin and chromatin was performed, before visualization using confocal microscopy. RESULTS: A statistically significant increase was observed in the survival rate in group 2 (73.3%, 66/90) compared to group 3 (55.7%, 82/147) (P < 0.007). Exposure of oocytes to the cryoprotective solutions without freezing had no effect on the structure of their second meiotic spindle. However, statistically significant differences were observed on both spindle and chromosome configurations of oocytes from group 2 (5.2 and 5.2% respectively) and group 3 (16.2 and 18.8% respectively) compared with group 1 oocytes (71.6 and 82.0% respectively) (P < 0.001 in all cases). CONCLUSIONS: The protocol followed results in high rates of survival and potential for in-vitro maturation, but has a deleterious effect on the organization of the meiotic spindle of human oocytes cryopreserved at both the GV and MII stages.     

Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium

BACKGROUND: Oocyte cryopreservation may have significant potential for assisted reproductive technology. However, to date, successful results have been limited. We report a preliminary series of IVF outcomes after fertilization of oocytes that were frozen in a low-sodium medium. METHODS: In this retrospective analysis, 12 patients (21-41 years old), who underwent IVF in a fertility clinic affiliated to the University of Buenos Aires, had oocytes cryopreserved in a modified phosphate buffered saline medium, in which sodium chloride was replaced by choline chloride. A slow-freezing, rapid-thawing protocol was used and oocytes were inseminated by ICSI. Outcome measures included oocyte survival, fertilization, implantation and pregnancy rates. RESULTS: Median oocyte survival was 63%. Median fertilization rate was 59%. Overall implantation rate was 25%. Six clinical pregnancies were achieved; two of these pregnancies went to term resulting in the birth of two babies. CONCLUSIONS: To the best of our knowledge, these are the first pregnancies and normal births using oocytes that were cryopreserved in a choline-based medium. The small sample size prevents us from concluding that freezing in a low-sodium medium is superior to using a conventional one.