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.     

Perinatal outcome and follow-up of 82 children aged 1-9 years old conceived from cryopreserved embryos

Embryo cryopreservation is routinely used in in-vitro fertilization(IVF)—embryo transfer programmes. Yet very few studieshave reported the follow-up of children conceived with frozen/thawedembryos. This study was designed to follow up the total cohortof children conceived with cryopreserved embryos in A. BéclèreHospital in Clamart, France between 1986 and 1994. We were ableto study 89 children, aged 1–9 years old, out of the 93conceived during this period (lost to follow-up: 4.3%). Theprematurity rate was 14.7% for the singleton and 85.7% for thetwins. Half of these premature deliveries occurred during the36th week of amenorrhea. In all, 8% of the singleton and 28.6%of the twins were small for gestational age. At the time ofthe study, only three children aged 1 and 2 years old were belowthe 10th percentile. The total malformation rate was 3.4% whentwo abortions performed during the study period were added tothe one (short ureter) found in our study group. The medicaland surgical illness as well as principal acquisitions for children<5 years old and scholastic performance for older childrendid not show pathological features.     

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.     

Changes in patient preferences in the disposal of cryopreserved embryos

BACKGROUND: The disposal of unused cryopreserved embryos can be a difficult decision for patients and the existence of unclaimed embryos raises ethical concerns for clinics. This study examined changes in patients' preferences for disposition of unused embryos and the relevance of a two-stage process for obtaining consent. METHODS: Patients who had not returned for cryopreserved embryos for over 5 years were contacted and asked to specify their current preferences for embryo disposition. These preferences were compared with dispositional choices made at the time of embryo freezing. RESULTS: Over one-third of patients had not returned for cryopreserved embryos within 5 years, and 31% of these declined to provide an updated directive. Those with a live birth through treatment were more likely to provide a new directive and more likely to choose to discard rather than donate embryos for research. Prior to IVF, the majority of non-returnees had elected to donate unused embryos for research, but 59% of all couples changed their minds after treatment. CONCLUSIONS: Changes in preferences for embryo disposition was linked to treatment outcome and highlighted the need for a two-stage process to obtain fully informed consent. In this Canadian sample, patients' affinity for research declined after treatment.     

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.     

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.     

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.     

Development of antral follicles in human cryopreserved ovarian tissue following xenografting

This study reports the first gross morphological and histological evidence of antral follicle development in human ovarian tissue following cryopreservation. Human ovarian tissue was cryopreserved using propanediol and sucrose and grafted under the renal capsule of bilaterally oophorectomized severe combined immunodeficient (SCID) mice. Follicles at all stages of development were observed in the grafted tissue whereas, prior to grafting, only primary and primordial follicles were present. Antral follicles were rarely observed on grafts examined <20 weeks after grafting either non-frozen tissue (fresh, 1/7) or cryopreserved tissue (0/11). In contrast, development of at least one antral follicle was evident on the majority of sites > or = 20 weeks after grafting (fresh 7/9, cryopreserved 18/24). Antral follicle diameters ranged from 0.1 to 5.0 mm. Histological examination of these antral follicles indicated normal follicular morphology, i.e. antral cavities encapsulated by concentric layers of theca and granulosa cells. Pedicles containing germinal vesicle oocytes were observed protruding from the granulosa cell layers. The development and morphology of the cryopreserved and fresh tissue following grafting was similar.     

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.     

Development of mouse embryos cryopreserved by an ultra-rapid method of freezing

High concentrations of cryoprotectant combined with sucrosewere utilized in an ultra-rapid freezing protocol for mousepreimplantation embryos. Dimethylsulphoxide (DMSO, 1.5 or 3.5M) or propanediol (PROH, 1.5 or 3.0 M) combined with 0.25 Msucrose were used as freezing solutions. One–,2- or 8-cellembryos were placed directly into these solutions at room temperature,loaded into straws and plunged into liquid nitrogen within 2–3min. The straws were rapidly thawed and the embryos expelledinto the solution in which they were frozen for 10 min. Thecryoprotectants were then removed by single- or multi-step dilution.Survival and development of the embryos in vitro and in vivowere assessed. DMSO (1.5 M) and both concentrations of PROHwere totally inadequate as a cryoprotectant in this freezingprotocol. A concentration of 3.5 M DMSO gave high survival anddevelopment rates when a multi-step dilution procedure was used,but not with a single-step dilution. One-cell embryos gave 71%survival, 35% in-vitro development and 10% in-vivo viability;2-cell embryos showed 87% survival, 77% in-vitro developmentand 66% in-vivo viability; and 8-cell embryos showed 97% survival,87% in-vitro development and 62% in-vivo viability. The resultsfor the 2- and 8-cell stages compared favourably with non-frozencontrols, which had 71% in-vivo viability. This method of cryopreservationis therefore fast and viable     

Chromosome analysis of human oocytes and embryos: does delayed fertilization increase chromosome imbalance?

Thirty per cent of a sample of 120 unfertilized human oocytescarried chromosome abnormalities highly correlated with maternalage (38% in patients >35, as compared with 24% in youngerpatients). Fertilized eggs, when observed 17 h after insemination,showed in 1.6% a single pronudeus suggesting parthenogeitelicactivation. In 92% of the cases two pronuclei were observedand the rate of chromosome anomalies depended on the morphologicalaspect of the embryos. Triploidy was also encountered in 6.4%of the eggs leading to an overall rate of chromosome aberrationsreaching 29.2%. Delayed fertilization drastically increasedthe rate of chromosome anomalIes (87%) as well as the rate ofmosaicism: 30% versus 10.6% in timely fertilized eggs. The highrate of chromosome disorders in early life after in-vitro fertilization(IVF) raises the ethical question of the opportunity of carryingout a genetic control of normality in human embryos at the preimplantationstage.     

Ultrastructure of preimplantation human embryos co-cultured with human ampullary cells

Ova with two pronuclei were co-cultured with established human ampullary cell lines and various stages of preimplantation embryonic development were monitored by Nomarski optics and then assessed by transmission electron microscopy (TEM). Fifteen embryos ranging from the 2-cell stage to blastocyst hatching were examined for normal and abnormal features. Their ultrastructure was similar to that of embryos cultured in Whittingham's T6 medium, reported previously. Seven embryos were evidently morphologically normal and showed good organization of fine structure. Most cellular organelles underwent progressive changes during early development. There was evidence of enhanced embryonic genome activation at the 8-cell stage. Invariably, all embryos had few too many fragments, some internalized, which were later segregated into the blastocoele or found outside the trophoblast of the late morula and blastocysts. Six grossly 'normal' embryos assessed by Nomarski had multiple nuclei of various dimensions, which highlights the subjectivity of embryo assessment in the IVF laboratory. Incomplete incorporation of chromatin into nuclei and formation of micronuclei were evident in some blastomeres. The results are discussed in relation to early embryonic loss, prevalent in IVF. Significant events reported include the detection of centrioles at the 8-cell stage, cavitation of the early blastocyst and the initiation of blastocyst hatching visualized by TEM.     

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.     

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.     

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%).     

Diagnostic assessment of the developmental potential of human cryopreserved ovarian tissue from multiple patients using xenografting

BACKGROUND: Although ovarian tissue cryopreservation for women at risk of losing ovarian function is offered by many clinics, there is a lack of evidence relating to the developmental potential of the stored tissue and, therefore, its clinical potential. This study was designed to use xenografting of cryopreserved tissue from multiple patients to assess the reproducibility of preservating developmental potential, the variation in developing follicle profile and the relationship between pre-freeze histology and post-thaw development. METHODS: Using previously published methods, cryopreserved ovarian cortex from nine patients was thawed and grafted under the kidney capsules of immunodeficient mice. Development of follicles was assessed after 26 weeks and compared to histology prior to freezing. RESULTS: Multiple growing follicles including antral stages were observed in multiple grafts of tissue from all patients. Metaphase II oocytes (n=9) were observed in follicles in grafts from five patients. There was no relationship between pre-freeze histology and developing follicle profile in xenografts. CONCLUSIONS: The propanediol freezing method used in this study is capable of reproducibly preserving the developmental potential of human ovarian follicles. The developing follicle profile after cryopreservation cannot be accurately predicted from pre-freeze histology. Xenografting provides a powerful tool for assessing the potential of human cryopreserved ovarian tissue.     

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.     

Oocyte maturation,follicle rupture and luteinization in human cryopreserved ovarian tissue following xenografting

BACKGROUND: Previous studies have demonstrated development of antral follicles in cryopreserved human ovarian tissue after autografting and xenografting, thus indicating successful preservation of follicular function. The study aim was to assess whether these follicles could also undergo periovulatory changes in response to hCG. METHODS: Ovarian tissue from three patients were dehydrated in propanediol (PROH)/sucrose and cryopreserved using the slow cooling/rapid thaw procedure. Thawed tissue was placed under the kidney capsule in immunodeficient mice. Following growth (>20 weeks) in the presence of gonadotrophin, hCG was administered and ovarian tissue examined histologically. RESULTS: Thirty-two antral follicles (diameter range 0.6 to 5 mm) were examined. Histological evidence of a response to hCG was evident in all follicles. Disruption of the concentric layers of mural granulosa and theca cells was apparent in all antral cavities. In 17 (53%) follicles the exterior follicular wall had reduced to a few cells thick, and in eight (25%) the wall had ruptured. Mucified oocyte-cumulus cell complexes were present in 32 follicles, 17 of which had begun to detach from the pedicle. Resumption of meiosis had occurred in over half the oocytes (five metaphase II and seven metaphase I oocytes, eight germinal vesicle breakdown). Two corpora lutea were also detected. CONCLUSIONS: Follicles cryopreserved within human ovarian tissue using the PROH procedure, can develop to the antral stage and undergo periovulatory changes following xenografting and exposure to a luteinizing stimulus.     

Birth characteristics and perinatal outcome of babies conceived from cryopreserved embryos

The objective of this retrospective study was to compare thebirth characteristics and perinatal mortality of babies conceivedfrom the use of cryopreserved embryos with those resulting fromin-vitro fertilization (IVF) and fresh embryo transfer. A totalof 232 consecutive births, one pregnancy termination and a totalof 283 babies in the cryopreserved group were studied. The IVFdata included 763 births, three terminations and 961 babies,based on a previous analysis. There was no difference in theincidence of twin (18 versus 19%) and triplet births (2 versus3%) in the cryopreserved and IVF groups respectively. The meangestational age and birthweight of singleton, twin and tripletbirths were not significantly different between the groups.No difference was found in the perinatal mortality rates. Theincidence of major congenital malformations in the cryopreservedgroup (1%) was significantly lower than that in the IVF group(3%, P < 0.05). It is concluded that the birth characteristicsof babies conceived from cryopreserved/thawed embryos are similarto those from fresh embryos. There are fewer congenital malformationsin the cryopreserved group.     

Fertilization and early embryology: Nuclei number in human embryos co-cultured with human ampullary cells

A study was undertaken to evaluate the effect on nuclei numberin human embryos cultured in vitro with primary cell lines ofhuman Fallopian tube epithelium. The development of 203 surplushuman embryos, cultured in standard culture medium (Earle'sbalanced salt solution+15% A5) with or without ampullary cells,was observed from day 2 to day 5.5 post-insemination. The expandedblastocysts in both culture conditions were analysed for nucleinumbers per blastocyst. Embryos transferred to co-culture atthe 2-cell stage had an average of 120.7 nuclei per blastocyst,which was significantly higher than the average of 62.9 nucleiper blastocyst (P=0.023) for the embryos transferred to co-cultureat the 4-cell stage. The embryos cultured in the control mediumhad an average of 42.1 nuclei per blastocyst, which was significantlyless than co cultured embryos (P=0.04). Severely fragmentedembryos (grades 3 and 4) did not show recovery in co-culture.Our results show that when human embryos are transferred toco-cultures before the 4-cell stage, the blastulation rate andthe cell number per embryo increase significantly compared tothe embryos cultured in standard culture medium. The possibleeffect of co-culture on embryonic gene expression is discussed.