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.     

How to facilitate decisions about surplus embryos: patients' views

BACKGROUND: In Victoria, Australia, legislation governing fertility treatment provides that surplus human embryos must not be stored for longer than 5 years. Couples must then choose one of three options: discard, donate to research or donate to another infertile couple. Previous research suggests that many people find these decisions difficult and emotionally distressing. This study aims to elucidate the nature of these difficulties and to identify ways in which the decision-making process could be facilitated. METHODS: This project used a combination of qualitative research methods. In total, 42 people agreed to participate in either a structured interview or a group discussion. All participants had completed IVF treatment and had surplus embryos in storage. The aim of the interviews was to discuss participants' decision making regarding their surplus embryos. Data were thematically analysed. RESULTS: Most participants described the decision-making process as difficult and emotional. Findings indicate that participants could be assisted by more information about each of their current options, and opportunities to talk to others in similar situations. Many responded positively to the idea of having more options, including choice about which research projects to donate to (directed research), and about the recipients of their donated embryos (directed donation). CONCLUSIONS: This study suggests that there are practical ways to assist people in making decisions about their surplus embryos, which could be easily implemented. In addition, the study demonstrated interest in the possibility of directed donation to other couples.     

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.     

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.     

Patients' attitudes towards donation of surplus cryopreserved embryos for treatment or research

BACKGROUND: The aim of the study was 2-fold: first, to investigate couples' reasons for not using cryopreserved embryos within the maximum storage period; second, to study their attitudes towards potential embryo donation for specific purposes. METHODS: A questionnaire was sent to 284 IVF/ICSI couples who experienced destruction of their cryopreserved embryos (n=1180) because the cryopreservation period exceeded the Danish legislative limit of 24 months. RESULTS: Seventy-four per cent of the couples responded. The main reasons for not utilizing surplus embryos was 'successful delivery' (85%), 'consider family completed' (61%) and 'too short legislative limit for cryopreservation' (59%). Sixty per cent of the couples agreed to the concept of donation of cryopreserved embryos for infertility research, 57% responded affirmatively to donation for stem cell research and 49% for stem cell treatment, but only 29% agreed to the concept of donation to infertile couples. Multiple logistic regression analysis showed that delivery of a child after IVF treatment (OR 3.8, 95% CI 1.4-10.2) and female age <35 years (OR 2.2, 95% CI 1.3-6.0) were predictive of agreement to the idea of donation for stem cell research and stem cell treatment respectively; however, male age, duration of infertility, mode of conception (IVF or ICSI) and having IVF children were not significant predictors. The following predictive variables were entered into the analysis: female and male age, duration of infertility, IVF versus ICSI, donor semen and +/- IVF children. CONCLUSIONS: This study shows that 23% of all couples having cryopreserved embryos do not utilize them for further treatment within the legislative storage period of 2 years. A major reason is successful delivery. More than half of these patients agreed to the concept of donation of surplus outdated embryos for research, whereas less than one-third agreed to donation to other infertile couples. Based on these figures, an alternative utilization of surplus embryos for stem cell research would require a 100-fold larger pool of available embryos to provide a realistic basis for this purpose.     

The fate of cryopreserved human embryos approaching their legal limit of storage within a West Australian in-vitro fertilization clinic.

Human embryos can only be stored in the first instance for 3 years in Western Australia, according to the West Australian Reproductive Technology Act. Thereafter, an application must be made to the local regulatory body, the Reproductive Technology Council, for an extension. Of the 650 batches of embryos frozen between 8 April 1993 and 31 October 1995, 170 (26.2%) batches were still in storage after 2.5 years. A reminding letter was sent at this time to the couples to whom the embryos belonged, i.e. 6 months before the expiry of the initial storage period, asking for clarification of what was to be done with the embryos. A large proportion of patients (64.7%) chose to either extend the storage period or thaw and transfer the embryos. Curiously, more batches of embryos were discarded (18.8%) than donated to other couples (5.9%). Contact with the patients was lost in a small but significant proportion of cases; more of these had been unsuccessful in their treatment (20.4%) than had achieved a pregnancy (4.3%).     

Non-invasive measurement of pyruvate and glucose uptake and lactate production by single human preimplantation embryos

The consumption of pyruvate and glucose and the production of lactate by 40 single human preimplantation embryos has been measured using a non-invasive technique. Twelve of the embryos showed abnormal fertilization. Of the 28 normally fertilized embryos, nine (32%) developed to the blastocyst stage in culture while the remainder degenerated or arrested during cleavage. In the normal embryos, pyruvate uptake exceeded that of glucose in the early developmental stages (days 2-5 post-insemination) before glucose became the predominant substrate in the blastocyst (day 6). Considerable quantities of lactate were formed throughout development, rising from a value of 43.6 pmol/embryo/h on day 2.5 to 95.4 pmol/embryo/h on day 5.5. The values of pyruvate and glucose uptake and lactate production of those embryos which arrested were below those which developed normally. On the basis that one mole of glucose can give rise to two moles of lactate, only 50% of the lactate produced could be accounted for in terms of glucose uptake from the medium. This figure rose to 90% in the blastocyst. The remaining lactate must be derived from endogenous sources, most probably glycogen. It is proposed that the high production of lactate by human preimplantation embryos in vitro is an adaptation to the conditions of culture.     

Improvement of the culture conditions for the development of human preimplantation embryos

The culture of human preimplantation embryos from the 1-cell to the morula/blastocyst stage of development is not satisfactory at present. The success of various IVF laboratories ranges from 18 to 23%, therefore there is a requirement for improvement in the standard conditions used to culture the embryo. Using a limited number of 'spare' human embryos which were donated for research, in-vitro studies have been undertaken using various culture media. The results show that a significant improvement in viability is achieved using Ham's F-12 medium compared with other media presently used for culturing embryos.     

Transfers of frozen-thawed human embryos in cycles stimulated by HMG

A total of 130 transfers of frozen-thawed (F-T) human embryos was carried out after moderate ovarian stimulation with human menopausal gonadotrophin (HMG). Embryos were replaced 3 days after the spontaneous luteinizing hormone (LH) surge or 4 days if ovulation was induced by human chorionic gonadotrophin (HCG). Embryos were thawed a few hours prior to transfer. One-hundred-and-twenty-three transfers were effective and 23 pregnancies were achieved. The rate of ongoing pregnancies per transfer was 17.9% (22/123). The survival rate of embryos originating from cycles stimulated by a combination of an LHRH analogue and HMG in a long protocol (LA-HMG protocol) was significantly lower when compared with the rate of embryos retrieved from clomiphene citrate-HMG (CC-HMG protocol) stimulated cycles (52 versus 67%, P less than 0.05). When fresh embryos originated from cycles stimulated with an LHRH analogue and HMG in a short protocol (SA-HMG protocol), the survival rate was not affected (59 versus 67%, NS). Although the difference was not significant, the ongoing pregnancy rate per transfer according to the three protocols from which the embryos originated seemed to be better with the SA-HMG protocol: 16% with the CC-HMG protocol, 14.5% with the LA-HMG protocol versus 27.6% with the SA-HMG protocol. The success rate was independent of the number of F-T transferred embryos if at least one embryo with 100% intact blastomeres was replaced.     

Non-invasive measurement of glucose and pyrovate uptake by individual human oocytes and preimplantation embryos

Pyruvate and glucose uptake by 73 individual human oocytes andpreimplantation embryos was measured non-invasively, using anultramicrofluorescence assay to analyse changes in substratelevels in mkrodroplets of culture medium. The uptake of bothsubstrates was measured over successive daily incubations betweendays 1 (unfertilized oocytes) or 2 (‘spare’ embryoswhich were not transferred) and day 6 (day 0 = day of insemination).Under these conditions, 58% (25/43) of fertilized embryos withtwo pronuclei on day 1 developed to the blastocyst stage byday 6. The pyruvate uptake of these embryos increased from –28to a maximum of 40 pmol/ embryo/h between days 2.5 and 4.5.Similarly, glucose uptake increased from 8 to 14 pmol/embryo/hbetween days 2.5 and 4.5, but then increased further to 24 pmol/embryo/hon day 5 at the blastocyst stage. The pyruvate uptake of fertilizedembryos which arrested at cleavage stages was significantlylower than for those which developed to the blastocyst stage.Polyspermk and parthenogenetic embryos, and unfertilized oocytesalso had lower pyruvate uptakes at later stages. The glucoseuptake of unfertilized oocytes and abnormal embryos never reachedthe level of fertilized embryos at the blastocyst stage on day5.5. Non-invasive measurement of pyruvate uptake before embryotransfer may provide a valuable functional criterion for theselection of viable embryos capable of developing to the blastocyststage.     

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.     

Anaphase lagging mainly explains chromosomal mosaicism in human preimplantation embryos

BACKGROUND: Cleavage stage embryos as well as postimplantation embryos have been studied extensively over the years. However, our knowledge with respect to the chromosomal constitution of human embryos at the blastocyst stage is still rudimentary. METHODS: In the present paper, a large series of human blastocysts was examined by means of fluorescent in situ hybridization (FISH). RESULTS: It was found that only one in four blastocysts (25%) displayed a normal chromosomal pattern. We defined a group of blastocysts (26%) displaying a simple mosaic chromosome pattern (different cell lines resulting from one chromosomal error), an about equally large group of blastocysts (31%) displaying a complex mosaic chromosome pattern, and a smaller group of blastocysts (11%) showing a chaotic chromosome distribution pattern. Six per cent of all blastocysts analysed could not be assigned one of the previously mentioned chromosomal patterns. CONCLUSION: Anaphase lagging appeared to be the major mechanism through which human embryos acquire a mosaic chromosome pattern during preimplantation development to the blastocyst stage.     

A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos

The impact of cryopreservation on the implantation potential of early cleavage stage (day 2) embryos was assessed by analysing the outcome from > 5000 thawed embryos in relation to the outcome from a similar number of fresh embryos. Analysis of procedures in which all transferred embryos fulfilled equivalent defined criteria revealed no significant difference in the implantation rates (fetal hearts/100 embryos transferred) of fresh 4-cell embryos (16.6%) and fully intact thawed 4-cell embryos (16.9%). Although 2-cell embryos implanted at significantly lower rates, there was again no significant difference between fresh (6.5%) and fully intact thawed (7.2%) embryos. Similar analysis of all embryos (irrespective of cell number on day 2) demonstrated that the implantation potential of partially intact thawed embryos was related to the extent of blastomere loss with the implantation rate of embryos with 50% cell survival (5.4%) being approximately half the rate of fully intact embryos (11.3%). Combining the values obtained from 'pure' data for the implantation rates of embryos with defined levels of survival with their relative prevalence in the total population of thawed embryos gave a predicted number of implantations (441) which was similar to the observed outcome (463). This number was approximately 30% less than the number expected had the same embryos been transferred fresh (635). The results suggest that intact thawed embryos have the same implantation potential as equivalent fresh embryos and that the impact of cryopreservation is limited to blastomere loss which is directly related to loss of implantation potential. The observed frequency of blastomere loss results in a reduction of approximately 30% in the implantation potential of a population of embryos following cryopreservation.     

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.     

Clinical experience with ultrarapid cryopreservation of human embryos resulting from intracytoplasmic sperm injection: brief communication.

A total of 41 patients requested thawing of supernumerary embryos in an intracytoplasmic sperm injection (ICSI) programme. Mean patient age was 30.8 +/- 3.8 years. Embryo freezing by the ultrarapid method was performed at room temperature in 3 mol/l DMSO and 0.25 mol/l sucrose. Total freezing time was 2.5 min including filling of the straw. In the thawing process, the embryos were removed from liquid N(2), left at room temperature for 30 s, immersed for 40 s at 30 degrees C, and then successively transferred at room temperature for 10 min to each of three sucrose solutions of decreasing concentration. The embryos were kept in culture and only those that presented cleavage after 24 h were transferred. Embryos from 42 cycles were thawed and a total of 24 transfers was performed. The mean number of thawed embryos was 5.0 +/- 3.2 per cycle and the mean number of transferred embryos was 2.83 +/- 1.3. The clinical pregnancy rate per cycle obtained after the thawing process was 16. 6%. The clinical pregnancy rate per transfer was 29.2% and the implantation rate was 13.2%. The abortion rate was 14.3%. Six deliveries have been performed, with the birth of seven infants.     

Cryopreservation of human and rabbit oocytes and one-cell embryos: a comparison of DMSO and propanediol

The aim of this study was to improve the cryopreservation ofhuman oocytes and pronuclear embryos. One-step and multiple-stepaddition of dimethyl sulphoxide (DMSO) and 1, 2-propanediol(PROH) and three different freezing protocols with intermediatetemperatures of –35, –70 and –110°C wereinvestigated. This work was performed using rabbit oocytes aswell as human oocytes and one-cell embryos from the routineIVF programme. Also, human polyploid pronucleate oocytes wereused in controlled prospective studies of morphological intactnessand development in vitro. Rabbit oocytes survived best (113/126)when PROH was added in one step and controlled freezing stoppedat –110°C. But the development was better (141/187)if DMSO was added in multiple steps and the oocytes were cooledto –70°C before being plunged into liquid nitrogen.The mode of addition of the cryoprotectant influenced developmentonly if slow freezing was stopped at –35°C (51 versus34%). Using PROH, the development after thawing was also betterif cooling was stopped at –35°C (51 versus 37%) andDMSO was superior to PROH when the oocytes were cooled slowlyto –110°C (66 versus 37%). In the human, significantlymore pronucleated than unfertilized oocytes developed afterfreezing (92 versus 50%). The best results were achieved withpronuclear embryos using 1.5 M PROH and cooling to –110°C,when 91.7% of the surviving oocytes developed further. Thisis a marked improvement of the development rate and comparableto embryo freezing