A modified cryopreservation method increases the survival of human biopsied cleavage stage embryos

BACKGROUND: The relatively poor survival rate of human biopsied cleavage stage embryos following cryopreservation is a significant obstacle in the application of preimplantation genetic diagnosis (PGD). We have attempted to improve cryosurvival of biopsied embryos by modifying the standard embryo cryopreservation technique. METHODS: Biopsied embryos were cryopreserved in 1.5 mol/l 1,2-propanediol in the presence of an elevated concentration of sucrose (0.2 mol/l) and human serum albumin was replaced by maternal serum (20% vol:vol). An additional initial thawing step in the presence of 0.3 mol/l sucrose was also included. RESULTS: The proportion of biopsied embryos which survived cryopreservation with > or =50% of their blastomeres intact was significantly higher using the modified method (138/185; 75%) than that observed using the standard propanediol method (20/46; 43%; P = 0.022). Total blastomere survival was also significantly increased as a result of the modifications (1010/1513; 67% versus 177/385; 46%; P < 0.001). Six fetal hearts have been detected to date following replacement of biopsied embryos cryopreserved with the modified method. CONCLUSIONS: Survival of human biopsied cleavage stage embryos can be restored to a level similar to that of non-biopsied controls by modification of the cryopreservation procedure. Embryos which have been cryopreserved using the modified method can implant following replacement in utero.     

II. The cryopreservation of human embryos

The general ethical considerations concerning the cryopreservationand ultimate fate of human embryos produced during IVF treatmentsare discussed. The discussion is centred around two generalquestions: who should decide and what should be done with theembryos? Special attention is given to the necessity of consentof both intended parents and to the practical solutions in caseof disagreement. This problem is linked to the question of thevalidity and revocability of the prior agreement or contractsigned by the intended parents concerning the ultimate fateof these embryos.     

Multinucleation in cleavage stage embryos

BACKGROUND: The aim was to analyse multinucleation in relation to its incidence in time and in the population, and its correlation with clinical variables, with other morphological characteristics and with the implantation rate of cleavage stage embryos. METHODS: Retrospective analysis of 10 388 cleaved embryos from 1395 consecutive IVF/ICSI cycles in 700 patients between January 1, 1999 and June 30, 2002. RESULTS: Multinucleation was observed in 3491 (33.6%) embryos in 1107 cycles (79.4%) of 609 (87%) patients, more frequently on day 2 than on day 3: 2848 (27.4%) versus 1567 (15.1%) [relative risk (RR) = 1.82; 95% confidence interval (CI) = 1.72-1.92]. Its incidence increased with fragmentation: 31.0, 34.4 and 36.5% for fragmentation 相似文献   

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.     

Six year follow-up of cryopreserved human embryos1

In 1987, we became aware of the importance of remaining in contactwith couples whose embryos had been cryopreserved for >1year. As a result, a questionnaire was designed to follow thefate of these embryos. Of 407 couples with cryopreserved embryos,262 couples opted to use them within 1 year with the intentionof fulfilling a parental plan. The remaining 145 couples werequestioned by six successive questionnaires sent out between1987 and 1992.By the end of the study, 336 of the 407 couples(82.5%)had chosen to utilize their embryos in a parental plan.In most cases, the maximum delay of response (5 years accordingto the Council of State) was respected. The remaining 71 couples(17.5%) either abandoned the parental plan or had not givenany information by the end of the study. Initially, anonymousdonation to another couple was chosen in preference to destroyingthe surplus embryos (32 versus 18 couples, P < 0.05). Latterly,however, these differences have balanced out (24 versus 28,not significant). Only those couples who initially opted todonate embryos to another couple changed their attitude in lateryears. In the long run, 62 couples decided not to pursue theirparental plan; of these, 24 couples chose to make a gift toanother couple, 28 couples opted for destruction,and 10 choseto make a gift to research. Nine couples (out of 71) declinedto make a decision, but they had all achieved a pregnancy duringan in-vitro fertilization (IVF) attempt.Three of these werelost to follow-up, i.e. 0.7% of all couples benefiting fromthe freezing technique.     

Ploidy in human cleavage stage embryos after fertilization in vitro

Chromosome preparations were made from 15 cleaved human embryosin the 2- to 12-cell stage after in-vitro fertilization. Allshowed two pronuclei before the first cleavage. Twelve had atleast one diploid metaphase, while three had interphase nucleionly. There was no evidence of partheno-genetic haploid cleavage.As expected, the frequency of metaphases increased with durationof colchicine treatment: 25 and 55% of the cells reached metaphaseafter 6–13 and 16–24 hours' treatment, respectively.Cleaved embryos with ‘ideal’ blastomere numbers(2, 4 or 8) showed a considerably higher metaphase frequencythan others. For a more detailed chromosome analysis the techniquewill have to be further improved. The asynchronous cleavageof blastomeres makes optimal treatment by mitogens difficult.     

Influence of the developmental stage and the equilibration time on the outcome of ultrarapid cryopreservation of mouse embryos

Mouse embryos of different developmental stages from 1-to 8-cellinclusive were ultrarapidly frozen in 0.25 ml French strawsafter various periods of equilibration (1, 3, 5 and 9 min) infreezing-buffer containing 3.5 M dimethyl-sulphoxide (DMSO),0.25 M sucrose, and 20% fetal calfserum (FCS) in phosphate bufferedsaline (PBS). After thawing in a 37°C waterbath and dilutionfor 5 min in 0.25 M sucrose in PBS/FCS the embryos were culturedin Ham's F10 medium with 10% FCS (37°C, 5% CO₂, 95% humidity)for 4–6 days. The rates of expanded and hatching blastocystswere then evaluated and compared to the corresponding ratesof non-frozen controls. Thus, for each cleavage stage, the optimalequilibration time was evaluated. It was 1 min for the 1-celland 8-cell stages (32 and 81% blastocysts, respectively), and3–5 min for the 2-cell (76 and 73%, respectively) andthe 4-cell stage (88 and 87%, respectively). It is concludedthat the ultrarapid freezing method described provides satisfactoryresults for all tested cleavage stages, but not for the 1-cellstage.     

Fertilization and early embryology: The protective action of polyvinylpyrrolidone-Percoll during the cryopreservation of mouse 2-cell embryos and its effect on subsequent developmental potential post-thaw in vitro and in vivo

The effects of cryopreservation, in media containing (FS3 +) or omitting (FS3) polyvinylpyrrolidone (PVP) in the form ofPercoll (PVP-Percoll), on the survival of 2-cell mouse embryoswas studied. Survival and zona pellucida disruption post-thaw,growth (assessed by in-vitro culture until the blastocyst stage)and development in vivo (assessed by implantation and livingfetus rates and the birth of live progeny) were all investigated.Initial post-thaw survival showed no statistically significantdifference (P 0.05) between FS3+ (91.1 ± 9.8%) and FS3(84.5 ± 6.6%). However, there was a statistically significant(P 0.05) reduction in the incidence of zona damage when thefreezing solution contained PVP-Percoll compared to the control(3.6 ± 1.0 and 8.7 ± 0.6% respectively) and astatistically significant (P 0.05) greater number of embryosdeveloping in vitro to the blastocyst stage (84.8 ±7.1and 72.3 ± 6.1% respectively). The rates of implantationwere not significantly different: 72.2 ± 7.0% for FS3+and 51.2 ± 30.7% for the non-frozen control group. Thepercentage of live fetuses was also similar between the experimentaland control groups: 27.4 ± 10.6 and 24.3 ± 113%respectively. We conclude that the presence of polymers canprotect embryos against cryoinjury and that PVP in the formof PVP-Percoll provides a non-toxic alternative to PVP in itsnative form, during the cryopreservation of mouse 2-cell embryos.     

Normal survival and in-vitro development after cryopreservation of zona-drilled embryos in mice

The Importance of the zona pellucida on survival after freezingand thawing was Invesligated. Zona-drilled and zona Intact mouseembryos were fertilized in vitro, cultured to the 2, 4 and 8-cellstages and frozen using conventional methods. Zona drillingdid not affect the survival or development of frozen embryosto the blastocyst stage in vitro. We conclude that partial damageto the zona pellucida during micromanipulation procedures Lscompatible with rates of survival and development which arenot different to those observed In zona-Intact control embryos.     

When to avoid creating surplus human embryos

The advice that should be given to a couple considering assisted reproductive technologies for the treatment of their infertility, when they are completely opposed to the destruction of surplus embryos, is discussed. It is urged that they do not use treatments that generate surplus embryos. They should be given the options of declining IVF and considering adoption, or less efficient treatments, namely limited ovarian stimulation, limited insemination of available ova or natural cycle IVF where no surplus embryos are generated.     

Parameters guiding selection of best embryos for transfer after cryopreservation: a reappraisal

BACKGROUND: The purpose of this study was to evaluate the respective influences of blastomere survival and resumption of mitosis on the outcome of frozen-thawed embryos. METHODS: A retrospective analysis was performed in our centre on 363 thawing cycles, involving 4-cell day 2 grade 1 embryos with <10% fragmentation. RESULTS: A higher implantation rate per transferred embryo was observed when all transferred embryos were characterized by fully intact blastomeres (100% blastomere survival) as compared with damaged embryos (50 or 75% blastomere survival) (22.0 versus 7.2%; P < 0.0001). Moreover, the implantation rate per transferred embryo was significantly higher for cleaved embryos compared with uncleaved embryos (19.7 versus 3%; P < 0.0001). Transfer of fully intact, cleaved embryos resulted in the highest implantation rates compared with transfer of damaged and uncleaved embryos (27.4 versus 0%; P < 0.0001). Intermediate implantation rates were observed when only one of the two criteria was fulfilled (13 versus 11% respectively; P > 0.05). Multivariate analysis showed that the clinical pregnancy rate was influenced by both criteria (odds ratio = 3.4 for transfer of embryos with six or more cells versus embryos with less than six cells. CONCLUSION: The results of our study suggest that the most important factor to predict further embryo development is the total number of blastomeres in transferred embryos, however they are obtained (good survival and/or resumption of mitosis).     

Calculating the implantation potential of day 3 embryos in women younger than 38 years of age: a new model

For optimal embryo selection in IVF/intracytoplasmic sperm injection (ICSI), knowledge of the implantation potential is essential. This is a retrospective analysis of morphological characteristics and cleavage kinetics of day 3 embryos resulting in an objective assessment of the relative implantation potential of each distinct type of embryo. Therefore transferred embryos were sampled according to their documented implantation behaviour: all embryos without any implantation on the one hand and all those with 100% ongoing implantation on the other. There were 213 such embryos in the latter group of which only seven (3%) had >20% fragmentation and only one embryo (0.5%) showed multinucleation (an embryo containing >20% fragmentation). For this reason, only embryos with < or =20% fragmentation and without multinucleation were analysed. They were split up according to the amount of fragmentation and the number of blastomeres on day 2 and on day 3. For each type, the implanted fraction was calculated, i.e. the number certainly implanted divided by the sum of the number certainly implanted and the number certainly not implanted, thus describing its relative implantation potential. By extrapolation to the entire population it was possible to establish the implantation potential for each type of embryo. Optimal day 3 embryos were calculated to reach a mean of 47% ongoing implantation. By establishing the implantation potential for most embryos, this model also provides useful information about which embryos are worth freezing in a cost-effective cryopreservation policy.     

Fertilization and early embryology: Improved cleavage rate of human embryos cultured in antibiotic-free medium

Retarded development and blastomere fragmentation of human prelimplantationembryos represent a common phenomenon in in-vitro culture systems.Even though media composition is generally formulated to meetembryo nutritional requirements, the influence of antibioticsupplementation has not been investigated thoroughly. The presentstudy was performed to evaluate the effects of antibiotics onembryo morphology and growth in modified culture media. A totalof 196 zygotes from 18 couples was cultured in three differentmedia: (i) conventional medium (n = 99, control group); (ii)medium modified with half the standard antibiotic concentration(n = 54); and (iii) antibiotic-free medium (n = 43); 49 embryosfrom the control group were selected at the zygote stage andtransferred to the patients on day 2. The remaining 147 zygoteswere cultured to the blastocyst stage for cryopreservatlon;their morphology and cell number were assessed daily at 40,64, 88, and 112 h post-insemination. Overall cleavage rate was95% and embryo scoring revealed 91% grade 1 embryos throughoutthe culture period in the three media. Significantly highercleavage rates were obtained in the antibiotic-free medium ateach observation, including the blastocyst stage, when comparedto the other two groups. In addition, no notable improvementwas observed in the embryos cultured in a reduced concentrationof antibiotics. In conclusion, antibiotic supplementation ofmedia has an adverse effect on the growth rate of preimplantationembryos, even in reduced concentrations, suggesting that antimicrobialdrugs may interfere with the timing of cleavage events eitherby delaying or blocking embryo development.     

Proliferation of blastomeres from biopsied cleavage stage human embryos in vitro: an alternative to blastocyst biopsy for preimplantation diagnosis

Normally fertilized human embryos were biopsied at cleavagestages on the third day after in-vitro fertilization (IVF).One or two blastomeres at the 8-cell stage were removed andco-cultured with the biopsied embryos. Embryos and blastomereswere assessed daily for morphological development until day6, when the number of cells were counted by labelling the nuclei.In all, 53% of the biopsied embryos (25 out of 47) reached theblastocyst stage between day 5 and 6 and the proportion wasthe same irrespective of the number of cells removed. Therewas no significant difference between biopsied embryos fromwhich one or two blastomeres respectively had been removed withregard to total cell numbers at the blastocyst stage (56.2 ±3.0 and 64.7 ± 5.5), number of trophectoderm (45.4 ±3.5 and 44.0 ± 5.7) and inner cell mass cells (14.0 ±1.2 and 16.6 ± 1.8). Overall, 72% of the isolated blastomeresdivided at least once over 3 days in culture and 50% dividedmore than once. The mean overall cell number after 3 days inculture was 3.7 ± 0.48 per blastomere (range 1–8cells) if one cell was removed and 6.9 ± 1.0 if two cellswere removed. If the undivided blastomeres are excluded, themean cell number was 4.8 ± 0.51 and 8.3 ± 1.0respectively. Over this period, 55% of the blastomeres cavitated.Of the blastomeres taken from embryos that developed to theblastocyst stage, 92% divided and 76% cavitated. In those fromarrested embryos, 50% divided (P < 0.002) and 32% cavitated(P < 0.003). From the first group 8% of blastomeres and fromthe second group 41% of blastomeres neither divided nor cavitated(P < 0.008). We conclude that as a more consistent alternativeto blastocyst biopsy, cryopreserving biopsied cleavage stageembryos and culturing blastomeres would increase the numberof cells available for genetic analysis. This could facilitatepreimplantation diagnosis of inherited disease by improvingreliability and possibly allowing combined detection of chromosomaland single cell defects. Further studies will investigate thegenetics of proliferated blastomeres.     

Combined impact of the number of pre-ovulatory oocytes and cryopreservation on IVF outcome

Three hundred and twenty-seven consecutive in-vitro fertilization (IVF) cycles in which luteal-phase leuprolide had been given were ranked according to the number of pre-ovulatory oocytes obtained (1-5, 6-10, greater than 10). Excess pre-embryos were cryopreserved at the pronuclear stage and later transferred into monitored natural cycles on the day after ovulation. The results indicate that the retrieval of large numbers of pre-ovulatory oocytes (greater than 10) has a small negative impact on oocyte quality as judged by fertilization rates (4% lower). However, implantation was not impaired compared to lower levels of retrieval in either the original IVF or subsequent cryo-thaw cycles. Overall, despite the small reduction in fertilization rate, the retrieval of many pre-ovulatory oocytes has produced a 'take-home baby rate' per stimulation cycle of 28.3% when 6-10 pre-ovulatory oocytes were retrieved and 41.5% when greater than 10 were retrieved: even higher rates are anticipated when the remaining cryopreserved pre-embryos are ultimately thawed.     

Chromosome studies on early human embryos fertilized in vitro

The majority of early spontaneous abortions carry a lethal chromosomalanomaly. While it is recognized that several factors would beresponsible for some IVF failures, it is important to determinethe contribution of chromosomal aberrations to the preimplantationloss of embryos produced in vitro. Chromosome analysis of embryosnot destined for replacement in the uterus could help to elucidatethis phenomenon of early embryonic loss. Fifty-five out of 239embryos fertilized in vitro were successfully karyotyped andamongst these the overall rate of diploidy was 25.5% in thisstudy, which mainly comprised rejected embryos. Embryos withoutcleavage had mostly a chromosomal defect (20/38) and only aminority (9/38) were unfertilized. Numerical abnormalities werefound in a total of 33/46 (71.7%) morphologically normal embryos.In contrast a diploid chromosomal complement was found in only11.1% (1/9) of morphologically abnormal embryos.     

Morphology and biochemistry of in-vitro produced bovine embryos: implications for their cryopreservation

Examination of some ultrastructural and physiological characteristicsof in-vitro produced bovine embryos may help to explain whysuch embryos are more sensitive to freezing than their in-vivoderived counterparts. Improvement of embryo survival after freezingcan be achieved by changing the conditions of their culture,selection of embryos based on the kinetics of their development,and changing ‘standard’ freezing procedures. Cryopreservationof embryos by vitrification, in particular, seems to yield highersurvival than conventional slow freezing. Further developmentof protocols requires additional embryo transfer studies toensure that the ability of thawed embryos to develop normallyin vivo correlates strongly with in-vitro survival assays.     

Full term delivery following cryopreservation of human embryos for 7. 5 years.

Successful pregnancy in a 44 year old woman is described following the transfer of embryos which were cryopreserved for 7.5 years. The embryos were obtained during a gamete intra-Fallopian transfer (GIFT) procedure in 1989. To our knowledge this is one of the longest published periods of cryopreservation of embryos which has resulted in a healthy baby. This report illustrates the previously presumed viability and normality of human embryos undergoing long-term cryopreservation. Additionally, it emphasizes the importance for advanced reproductive technique programmes and patients to review and update their embryo status.     

Cryopreservation of human embryos obtained after gamete intra-Fallopian transfer and/or in-vitro fertilization

During a one-year period 636 excess embryos obtained after in-vitrofertilization and gamete intra-Fallopian transfer combined within-vitro fertilization were cryopreserved using two differentprotocols. For early stage embryos including the pronucleatestage, 1,2-propanediol was used as cryoprotectant (procedureA, adapted from Renard) and for later stage embryos dimethylsulphoxidewas used in protocol B, adapted from Trounson and Mohr. Afterthawing 288 embryos, half of them were of sufficient qualityto be replaced. After cryopreservation, procedure A gave thebest survival in embryos having 2 blastomeres; for later stageembryos best survival was obtained using the dimethylsulphoxideprotocol. Survival after cryopreservation was also clearly relatedto the quality of the embryos prior to freezing. Embryos werereplaced during endocrinologically monitored natural cyclesand were transferred in synchrony between endornetrial and embryonicage. After replacement of 126 embryos in 110 patients, 20 pregnanciesoccurred. So far six healthy children have been born, two patientsaborted and 12 pregnancies are ongoing. In this series no statisticaldifference was observed between the implantation rate of embryoscryopreserved by procedure A or B. Six pregnancies occurredin patients from the oocyte and embryo donation programme. Anadequate cryopreservation programme circumvents the difficultproblem of synchronizing the ovarian cycles of donor and acceptorpatients.     

Comparison of the effects of controlled-rate cryopreservation and vitrification on 2-cell mouse embryos and their subsequent development.

Effects of two cryopreservation procedures (conventional slow controlled-rate freezing using a programmable freezer and vitrification by direct plunging into liquid nitrogen) were compared on 2-cell embryos and their subsequent development to blastocysts, fresh or cryopreserved 2-cell mouse embryos were developed into blastocysts in vitro. The percentage of vitrified embryos which developed into blastocysts was significantly lower than that of fresh and slow controlled-rate frozen embryos. Although blastocysts from each cryopreservation procedure appeared morphologically normal and neither number of cells in the blastocysts nor in-vitro trophoblast spreading differed significantly, there were significant differences in their functional viability. First, the glucose incorporation activity in terms of [(3)H]2-deoxyglucose (2-DG) uptake in vitrified and thawed 2-cell embryos significantly decreased compared with fresh or slow controlled-rate frozen and thawed 2-cell embryos. Second, 2-DG uptake by blastocysts developed in vitro from fresh 2-cell embryos and from slow controlled-rate frozen or vitrified 2-cell embryos was 105 +/- 75, 43.0 +/- 28.3 and 22.0 +/- 11.4 fmol/embryo/h respectively. Third, the implantation rate of blastocysts developed in vitro from vitrified 2-cell embryos (10.2%) was significantly lower than that from fresh 2-cell embryos (30.8%) or slow controlled-rate frozen 2-cell embryos (22.1%). Since these data suggest that cryopreservation may have ulterior consequences on the functional development of embryos and that vitrification may exert a more harmful effect than slow controlled-rate freezing, more attention should be paid to its safety before vitrification is used routinely in a clinical programme.