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.     

Fertilization and early embryology: Factors affecting activation and fertilization of human oocytes following intracytoplasmic injection

Intracytoplasmic sperm injection (ICSI) has dramatically alteredthe treatment of severe male factor infertility, resulting inimproved fertilization and pregnancy rates. The purpose of thisstudy was to investigate oocyte activation and fertilizationin aged human oocytes following ICSI. Non-viable spermatozoawere injected into 24 h old human oocytes in the presence andabsence of calcium and were assessed for evidence of activationand fertilization 16–19 h after the injection procedure.Sham injections were also carried out to assess the effect ofthe injection procedure itself and the presence of calcium inthe injection medium on oocyte activation. Non-viable spermatozoainjected in the presence of 1.78 mM calcium were capable ofnormally fertilizing aged human oocytes and the resulting zygotesunderwent cleavage. None of the oocytes injected with non-viablespermatozoa in the absence of calcium were fertilized normally,although the rates of activation following all treatments weresimilar.     

Fertilization and early embryology: The quarantine of fertilized donated oocytes

The fertilization and subsequent cryopreservation of donatedoocytes have enabled the resulting embryos to be quarantinedfor a minimum of 6 months, and to be thawed and replaced onlyafter the donor has had a second negative human immunodeficiencyvirus (HIV) test. In the study described here, a total of 39women had 56 embryo transfers, and 12 pregnancies (21% per transfer)were achieved. The logic of the protocol for minimizing therisk of infection of the recipient, which is in line with thatof semen donation, is presented, together with an argument forthe feasibility of such an approach.     

Fertilization and early embryology: Osmotic and physiological responses of mouse zygotes and human oocytes to mono- and disaccharides

To survive cryopreservation, oocytes, zygotes and embryos musttolerate a sequence of volumetric contractions and expansions.These result as an egg or an embryo is exposed to a permeatingcryoprotective additive, then to an increase followed by a decreasein the osmolality of its extracellular milieu as water freezesduring cooling and then melts during warming, and finally tothe dilution of the cryoprotective additive solution. Measurementsof the extent to which mouse zygotes and human oocytes undergoosmotic contraction have been made by exposing them to solutionsof monosaccharides (fructose, galactose, glucose) or disaccharides(maltose, sucrose, trehalose), ranging in concentration from0.25 to 1.50 M. Mouse zygotes and human oocytes exhibit verysimilar responses to these solutions. Their volumes contractlinearly as a function of 1/(osmolality) of the solutions, yieldingestimates of non-osmotic volumes of 13–23%. Mouse zygotesexposed to 1.5 M concentrations of these solutions for 10 minlose 85% of their cell water. Yet >75% of treated zygotesdevelop into hatching blastocysts. Human oocytes also appearto survive such extreme dehydration, based on a vital dye assay.These results suggest that solutions of various non-permeatingsaccharides can serve as osmotic buffers for the recovery ofcryopreserved oocytes, zygotes and embryos.     

Cryopreservation of mouse and human oocytes using 1, 2-propanediol and the configuration of the meiotic spindle

Human and mouse oocytes were cryopreserved by a slow freeze,rapid thaw method, using propanediol (PROH) as the cryoprotectant.A simulated cryopreservation was also included in the studyto detect the level of damage attributable to the PROH alone.Comparison of the mouse and human oocytes cryopreserved by thesame method showed opposing results, with a poor morphologicalsurvival rate of 4% observed for mouse oocytes and a subsequentnormal fertilization rate of 0%. In 171 cryopreserved humanoocytes a higher survival rate of 64% was achieved, and thisshowed more similarity to the mouse pronuclear oocytes survivalof 53%. A comparison of human oocytes, cryopreserved withinthe cumulus and denuded of cumulus and corona prior to cryopreservation,demonstrated a higher survival rate in the denuded oocytes of69% compared to 48%. A delay prior to cryopreservation of 1or 2 days had no effect on the immediate survival of oocytes,but culture for a further 24 h after thawing reduced survival,with the day 1 oocytes exhibiting the most dramatic reductionin survival (28%). Using a lectin binding method, abundant corticalgranules were observed in all cryopreserved oocytes analysed.The meiotic spindle and chromosomes were examined in cryopreservedoocytes using fluorescence microscopy and 60% of the survivingoocytes had a normal spindle and chromosome configuration.     

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.     

Fertilization and development of the human oocyte following exposure to cryoprotectants, low temperatures and cryopreservation: a comparison of two techniques.

Both glycerol and dimethyl sulphoxide (DMSO) equilibration prior to cryopreservation produced adequate rates of survival and development to the hatching blastocyst stage for mouse oocytes using the two chosen cooling and warming regimes. Successful fertilization of human oocytes and further development of the embryos produced, was achieved following equilibration with DMSO at 0 degrees C. Although fertilization of fresh human ova previously exposed to glycerol was recorded, all oocytes with two pronuclei failed to continue to undergo cell division by 48 h in culture. Following cryopreservation, human oocytes were successfully inseminated after equilibration with both glycerol and DMSO. However, cell division to the 2-cell stage was recorded in only one oocyte which had undergone exposure to DMSO, freezing and thawing. No cell divisions were recorded after glycerol cryopreservation, or even after simple exposure to glycerol. Therefore it appears that DMSO and slow cooling may be the best protocol, although further evaluation will be necessary.     

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.     

Fertilization and embryonic development of human oocytes after cooling.

Injury to living cells resulting from rapid cooling to temperatures at or near 0 degrees C has long been recognized, and the phenomenon, which is termed 'cold shock', has been known to occur in some mammalian gametes. Although human embryos have been successfully stored at low temperatures, cryopreservation of the human oocyte is proving to be more difficult. Whether or not this lack of success is a direct result of cellular injury brought about by 'cold shock' is the purpose of the current investigation. Human oocytes were cooled, in the absence of cryoprotectants, at two different cooling rates (-3 degrees C/min and -1000+ degrees C/min) to a temperature of 0 degrees C and rewarmed prior to insemination. In both cases fertilization after cooling was similar to the rates achieved in a routine in-vitro fertilization and embryo transfer procedure. After cooling at -3 degrees C/min, the rate of fertilization was 19/22 (86%) and after cooling at -1000+ degrees C/min, 9/9 (100%), with non-cooled control rates of 62/87 (71%) and 35/50 (70%) respectively. Fertilized oocytes from both groups were successfully cultured for a further 24 h before termination of the experiment.     

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.     

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.     

Fertilization and early embryology: Oscillations in intracellular free calcium induced by spermatozoa in human oocytes at fertilization

Calcium has an important role in the events of egg activationand early preimplantation development. We investigated changesin intracellular calcium concentration in human oocytes at fertilizationusing the calcium-sensitive photoprotein aequorin. Oocytes weredonated for research by patients undergoing in-vitro fertilizationtreatment in the Department of Obstetrics and Gynaecology. Cumuluscells, and in some cases zonae pellucidae, were removed by appropriateenzyme treatment. Single oocytes were micro-injected with aequorinand incubated in a chamber perfused with pre-equilibrated culturemedium in a photomultiplier system. Eleven zona-intact and 15zona-free oocytes were incubated with sperm, and oocytes fromeach group were incubated without sperm as controls. Dramatictransient increases in intracellular free calcium concentrationwere recorded in three zona-intact and seven zona-free oocytes,thought to be the first direct measurements of intracellularchanges in human oocytes at fertilization. The amplitude (upto 2.5 µM), duration (120 s) and frequency (every 10–35min) of these transients were similar in zona-intact and zona-freeoocytes. They resemble those recorded in mouse oocytes, whichmay therefore be a suitable model for biochemical events athuman fertilization.     

Fertilization and early embryology: Cryopreservation of immature mouse oocytes

Mouse oocytes enclosed in cumulus cells were isolated from antralfollicles at the germinal vesicle (GV) stage. They were storedin straws at – 196°C by a conventional mouse embryofreezing method using dimethylsulphoxide (1.5 M) as the cryoprotectant.Overall survival assessed after removal of the cumulus cellswas 93% (299/320). A significantly greater proportion of freshoocytes remained arrested at the GV stage during culture (11versus 1%), but the rate of maturation to metaphase II was notsignificantly different between frozen and fresh oocytes (83versus 74%). The rate of fertilization in vitro was similarfor frozen and fresh oocytes matured in vitro (70 versus 81%)but significantly less than with mature ovulated oocytes (96%).Fertilization of frozen and fresh oocytes arrested after germinalvesicle breakdown was similar (77 versus 95%. No evidence ofparthenogenetic activation was found in the different groupsafter overnight incubation of metaphase II oocytes. Implantationwas similar for embryos derived from fresh and frozen GV-stageoocytes matured in vitro and mature ovulated oocytes, but theloss of embryos after implantation was significantly higherin the in-vitro matured groups (frozen, 40% and fresh, 46% versus24%). The overall survival of oocytes frozen at the GV stagewas 27%. This compares favourably with the estimated overallsurvival of mature oocytes cryopreserved by a similar procedure.We conclude that the increased post-implantation loss is dueto suboptimal conditions for maturation in vitro rather thanfreezing injury.     

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.     

Fertilization and early embryology: Behaviour of spermatozoa in human oocytes displaying no or one pronucleus after intracytoplasmic sperm injection

The behaviour of sperm cells after intracytoplasmic sperm injection(ICSI) was investigated by analysing 192 unfertilized and 37one-pronuclear (1PN) oocytes following ICSI. Eighty-two unfertilizedoocytes were directly fixed whereas 110 were first parthenogeneticallyactivated by puromycin. In contrast to the findings in unfertilizedoocytes after in-vitro fertilization, most unfertilized oocytesafter ICSI (n = 76) contained evidence of the presence of spermatozoain the cytoplasm. Few oocytes (n = 6) contained prematurelycondensed sperm chromosomes (PCC), whereas the majority containedeither intact sperm heads (n = 31) or swollen sperm nuclei (n= 39) along with metaphase II chromosomes of the oocyte. Followingactivation by puromycin, swollen sperm nuclei and PCC were nolonger observed, whereas unchanged sperm heads persisted in12 oocytes displaying a single pronucleus. A non-decondensedsperm nucleus along with decondensed maternal chromatin werealso discovered in 32 out of 37 oocytes displaying a singlepronucleus after ICSI. The findings in unfertilized and 1PNoocytes after ICSI indicate that successful sperm injection,even followed by oocyte activation, is not sufficient to guaranteenormal fertilization. It seems that partial sperm membrane damageprior to injection is also required to ensure normal sperm decondensation.     

Resistance of human follicular oocytes to parthenogenetic activation: DNA distribution and content in oocytes maintained in vitro

Sixty-nine human oocytes were subjected to various parthenogenetic stimuli but no activation was observed. Analysis of Feulgen-stained DNA (F-DNA) distribution and content showed anomalies of chromosomal material in 36% of oocytes at 24-75 h after retrieval. A significant loss of F-DNA was noticed in apparently normal metaphase II oocytes remaining in culture for 2-3 days.     

Fertilization and early embryology: Polypeptide profiles of human oocytes and preimplantation embryos

The polypeptides that direct fertilization and early developmentuntil activation of the embryonic genome occurs, at the 4–8cell stage in the human, are exclusively maternal in origin,and are either synthesized during oogenesis or translated laterfrom maternal mRNA. Using sodium dodecyl sulphate—polyacrylamidegel electrophoresis and silver stain, we have visualized andcompared the polypeptides present in different populations ofhuman oocytes and cleavage stage embryos obtained after superovulationand insemination in vitro. Two polypeptide patterns were resolved,differing in the region of mol. wt 69 kDa. The distributionof these patterns showed no correlation with the ability ofindividual oocytes to achieve fertilization and develop normallyto the 8-cell stage.     

Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1, 2-propanediol.

Thin slices of human ovarian cortex were evaluated following cryopreservation in 1,2-propanediol (PROH)/sucrose under various conditions. Following rapid thawing, 1 microm sections were assessed by light microscopy and oocyte abnormalities were further examined by electron microscopy. Follicles (n = 503) were predominantly primordial (91%), with no follicles larger than the proliferating primary stage. Proportions of intact pre-granulosa cells and oocytes (expressed as percentages of the total numbers observed) were significantly reduced following cooling at three different rates with the highest levels of intactness (55 and 85% respectively) being achieved with slow cooling. The frequency of oocyte abnormalities [loss of organelles (mitochondria), organelle-free areas, and/or cytoplasmic vacuolation] was significantly increased at all cooling rates with slow cooling resulting in the highest proportion (56%) of normal oocytes. With slow cooling, increasing dehydration time increased the proportions of intact pre-granulosa cells and oocytes (maximum 74 and 91% respectively after 90 min dehydration). Under these conditions, the highest proportion of follicles with all pre-granulosa cells intact (44%) was observed, as was the highest proportion of 'normal' oocytes (85%). In this study, single step dehydration in PROH/sucrose for 90 min and slow cooling/rapid thawing results in the highest proportion of intact human primordial and primary follicles.     

The effect of osmotic stress on the metaphase II spindle of human oocytes, and the relevance to cryopreservation

BACKGROUND: Knowing osmotic tolerance limits is important in the design of optimal cryopreservation procedures for cells. METHODS: Mature human oocytes were exposed to anisosmotic sucrose solutions at concentrations of 35, 75, 150, 600, 1200, or 2400 (+/-5) milliosmolal (mOsm) at 37 degrees C. A control treatment at 290 mOsm was also utilized. Oocytes were randomly allocated to each experimental treatment. After the treatment, the oocytes were cultured for 1 h, then fixed in cold methanol. Immunocytochemical staining and fluorescence microscopy were used to assess the morphology of the metaphase II (MII) spindle. Logistic regression was used to determine if media osmolality had a significant effect on spindle structure. RESULTS: Osmolality was a significant predictor of spindle morphology. Hyposmotic effects at 35, 75, and 150 mOsm resulted in 100, 67, and 56% of oocytes having abnormal spindles, respectively. Hyperosmotic effects at 600, 1200, and 2400 mOsm resulted in 44, 44, and 100% of the spindles with abnormal structure, respectively. CONCLUSIONS: Anisosmotic conditions lead to disruption of the MII spindle in human oocytes. Applying this fundamental knowledge to human oocyte cryopreservation should result in increased numbers of cells maintaining viability.