Survival and developmental potential of stored human early cleavage stage embryos.

Human early cleavage stage embryos which survive cryopreservation and thawing fully intact demonstrate similar developmental potential to equivalent non frozen embryos when returned to the in vivo environment, whereas blastomere loss is directly related to the loss of potential for subsequent implantation in thawed embryos. This suggests that blastomere lysis during freezing and thawing does not occur preferentially in non viable blastomeres. Prefreeze growth rate rather than prefreeze blastomere number per se correlates with the developmental potential of stored embryos. When blastomere loss occurs as a consequence of cryopreservation, development of thawed early cleavage stage embryos to the blastocyst stage in vitro is impaired and the resultant blastocysts have a reduced total cell content. Blastomere loss is more prevalent in embryos which have been biopsied for preimplantation genetic diagnosis but this increased sensitivity can be circumvented by modification of the standard cryopreservation protocol.

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.

The effects of cryopreservation regimens on the morphology of human ovarian tissue.

Human ovarian tissue consisting of stroma, pre-granulosa cells and oocytes, has been frozen using a variety of cooling rates and dehydration regimens. The differential survival of the various cell types under these conditions highlights the difficulty in defining optimum protocols for the cryopreservation of multicellular tissue.

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.

Skewed segregation of the mtDNA nt 8993 (T-->G) mutation in human oocytes.

Rapid changes in mtDNA variants between generations have led to the bottleneck theory, which proposes a dramatic reduction in mtDNA numbers during early oogenesis. We studied oocytes from a woman with heteroplasmic expression of the mtDNA nt 8993 (T-->G) mutation. Of seven oocytes analyzed, one showed no evidence of the mutation, and the remaining six had a mutant load > 95%. This skewed expression of the mutation in oocytes is not compatible with the conventional bottleneck theory. A possible explanation is that, during amplification of mtDNA in the developing oocyte, mtDNA from one mitochondrion is preferentially amplified. Thus, subsequent mature oocytes may contain predominantly wild-type or mutant mitochondrial genomes.

Intracytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2-propanediol.

This study reports the subsequent embryo development of cryopreserved mature human oocytes following insemination or intracytoplasmic sperm injection (ICSI). Metaphase II oocytes were cryopreserved using a slow freezing-rapid thawing procedure employing the cryoprotectant 1,2-propanediol. The study was conducted at two centres. The normal insemination of cryopreserved oocytes was undertaken in one centre, and ICSI of cryopreserved oocytes in the other. Both methods resulted in a 50% normal fertilization rate. A low rate of abnormal fertilization was observed in the inseminated group of oocytes (5%) compared with 21% for the ICSI oocytes; this was not significantly different. Embryo development was assessed daily for 7 days. All normal fertilized cryopreserved oocytes in both groups cleaved on day 2, with a similar appearance to in-vitro fertilization and ICSI embryos. In the normal inseminated oocytes, there was a significant decrease in the number of embryos cleaving on day 3 (33%) compared with the development of ICSI oocytes, with a subsequent gradual reduction over days 4 and 5 (22 and 11% respectively) resulting in one early blastocyst on day 7 (11%). In contrast, all ICSI-generated embryos continued to cleave on day 3, with a gradual reduction over subsequent days (day 4, 86%; day 5, 57%; day 6, 43%; day 7, 29%). By day 7, two of the blastocysts had started to hatch, resulting in a 66% hatching rate of blastocysts formed from ICSI of cryopreserved oocytes. This is the first study to show normal development to the hatching blastocyst stage following ICSI of cryopreserved human oocytes.

Parthenogenetic activation of human oocytes following cryopreservation using 1,2-propanediol.

Fresh and aged human oocytes were cryopreserved using 1,2-propanediol (PROH). After thawing, the oocytes were cultured for 20 h and examined for parthenogenetic activation using light microscopy and an ultraviolet DNA stain. Control fresh or aged oocytes and oocytes exposed to PROH without cryoperservation were also examined for activation. No control oocytes were observed to activate spontaneously (n = 43) and parthenogenetic activation was not induced by exposure to PROH alone (n = 26). In both fresh and aged cryopreserved oocytes, 27 and 29% of the oocytes respectively were activated, and these proportions were significantly elevated compared with the controls (P < 0.01). Although a similar rate of activation was observed for the cryopreserved fresh and aged oocytes, the form of parthenogenetic activation varied between these two types of oocyte. A single pronucleus was observed in 18% of the fresh and 5% of the aged cryopreserved oocytes. In contrast, the presence of two or more pronuclei was observed in 0% of the fresh and 19% of the aged cryopreserved oocytes.

Fertilization of human oocytes following cryopreservation; normal karyotypes and absence of stray chromosomes.

Survival following cryopreservation of fresh and aged human oocytes by the propanediol (PROH) procedure was observed in 51 and 73% of oocytes respectively, immediately after thawing. This survival was reduced in both types of oocytes at the time of insemination (3-4 h) to 41% in fresh and 61% in aged oocytes. Insemination of the cryopreserved and control oocytes with spermatozoa from one donor resulted in total fertilization rates similar to our in-vitro fertilization (IVF) rate for non-male factor patients. The normal fertilization rate for fresh cryopreserved oocytes was slightly lower (46%) than the rate for IVF oocytes (59%) (P < 0.05), while the abnormal fertilization rates were not significantly different (16 and 15% respectively). In contrast, a reduction in the normal fertilization rate was observed for the aged cryopreserved oocytes (13%) compared to the IVF rate (P < 0.001). Associated with this was an increase in the abnormal fertilization rate for the aged cryopreserved oocytes, which was significantly higher (47%) than the IVF rate (15%) (P < 0.001). Significant differences in the total and normal fertilization rates were observed between cryopreserved oocytes obtained from cohorts with < or = 27 (total: 84%, normal: 68%) and > 27 oocytes (total: 55%, normal: 33%) (P < 0.05). Fertilized oocytes and oocytes with abnormal or absent spindles were examined for chromosomal loss and no stray chromosomes were observed in any of these cryopreserved oocytes (n = 137). In the cryopreserved oocytes which had undergone normal fertilization, four scorable karyotypes were achieved and in all of these two sets of 23 chromosomes were observed.

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 study to detect the level of damage attributable to the PROH alone. Comparison of the mouse and human oocytes cryopreserved by the same method showed opposing results, with a poor morphological survival rate of 4% observed for mouse oocytes and a subsequent normal fertilization rate of 0%. In 171 cryopreserved human oocytes a higher survival rate of 64% was achieved, and this showed more similarity to the mouse pronuclear oocytes survival of 53%. A comparison of human oocytes, cryopreserved within the cumulus and denuded of cumulus and corona prior to cryopreservation, demonstrated a higher survival rate in the denuded oocytes of 69% compared to 48%. A delay prior to cryopreservation of 1 or > or = 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 reduction in survival (28%). Using a lectin binding method, abundant cortical granules were observed in all cryopreserved oocytes analysed. The meiotic spindle and chromosomes were examined in cryopreserved oocytes using fluorescence microscopy and 60% of the surviving oocytes had a normal spindle and chromosome configuration.

An analysis of human oocytes and follicles from stimulated cycles: oocyte morphology and associated follicular fluid characteristics.

The aim of this study was to determine whether certain characteristics of the follicular biochemistry, previously shown by us to be associated with oocyte developmental capacity, also reflected differences in oocyte appearance, and to determine the range of oocyte characteristics induced by ovarian stimulation. A representative sample of 33 human oocytes and associated follicular fluids was obtained after a follicular growth period considered suitable for IVF. Individual follicular fluid protein and proteoglycan levels, and follicular volume were compared with the morphological characteristics of each oocyte. Oocytes which retained the germinal vesicle nuclear status after exposure to human chorionic gonadotrophin tended to occur in small follicles (less than or equal to 2 ml) and to be highly vacuolated and with characteristic predicted a low potential for cleavage. Among those oocytes which had progressed to MII nuclear maturity [in the medium (2.5-6.5 ml) and large (greater than or equal to 7 ml) volume follicles] the degree of oocyte vacuolation was negatively correlated with alpha 1-antitrypsin level, while the degree of organelle clumping was correlated with proteoglycan and immunoglobulin levels. Only five of the oocytes (15%) in this sample had follicular characteristics consistent with a normal potential for pregnancy. These MII oocytes occurred within the medium volume range, had low vacuolation levels, and a low degree of organelle clumping. In contrast, those oocytes with a low potential for cleavage based on their follicular biochemistry, showed high cytoplasmic vacuolation levels.

Prediction of outcome in human in vitro fertilization based on follicular and stimulation response variables.

Since in vitro fertilization (IVF) pregnancy rates have reached a plateau in recent years, there is need for a system of assessment, which could provide a guide for improvements. The follicular characteristics, the response to stimulation, the quality of sperm used for insemination, and the embryonic human chorionic gonadotropin production of 222 women who had undergone routine IVF treatment have been analyzed. Models, predictive of IVF outcome, have been developed using these parameters in various combinations. The results have shown that follicular health and maturity are critical to IVF outcome and that certain patterns of response to ovarian stimulation are associated with the more frequent occurrence of oocytes capable of normal embryonic development after fertilization.

Follicular characteristics associated with viable pregnancy after in vitro fertilization in humans.

The aim of this study has been the development of a noninvasive method of predicting the pregnancy potential of human oocytes and embryos intended for in vitro fertilization and embryo replacement. A multifactorial system which distinguishes, with a high degree of accuracy, between normal pregnancy, abnormal pregnancy, and non-pregnancy-producing embryos is reported. The variables included are (1) follicular fluid proteins alpha 1-antitrypsin, complement C3, immunoglobulin IgG2, and total protein, and total proteoglycan level separated by isoelectric focusing; (2) follicular volume; and (3) an embryo appearance rating. The study group consisted of (1) follicles which produced embryos of known performance after transfer (a) when the number of embryos transferred = the number of implantations and, (b) where one embryo transferred = no pregnancy; (2) follicles which produced oocytes which did not cleave after insemination; and (3) follicles from which no oocyte was aspirated. Canonical discriminant analysis of follicular fluid variables and follicular volume has been used to characterize the oocyte performance groups. Correct classification was achieved in 69% of normal pregnancy, 70% of abnormal pregnancy, 33% of no pregnancy, and 47% of no cleavage oocytes. An embryo appearance rating was included with the above variables for a separate discriminant analysis of only those oocytes which had formed embryos after insemination. Correct classification was achieved in 81% of normal-pregnancy, 70% of abnormal-pregnancy, and 70% of no-pregnancy embryos.