E Sereni

Predictive factors for embryo implantation potential

In spite of recent improvements in IVF, pregnancy rates have not increased significantly and one of the major problems remains the high multiple pregnancy rate. Better criteria are therefore necessary to establish the viability of a transferable embryo. Early prognosis of the developmental fate of the oocyte would help in selecting the best embryos to transfer, but non-invasive selection at the oocyte stage (extracytoplasmic and intracytoplasmic morphology) has proved to be of little prognostic value. Recently, it has been shown that follicular vascularization appears to be predictive of oocyte developmental fate, making it a good first-step approach for selection. Observation of pronuclei patterns at the zygote stage appears to offer an additional prognostic tool, correlating well with IVF outcome. Morphological evaluation of the embryo at days 2-3 remains the most used and valid method of selection, even though it is not sufficient to select embryos with the higher implantation potential. Blastocyst culture is another possible strategy for selecting the best embryos with reduced risk of aneuploidies, though not all major chromosomal aberrations are excluded by prolonged in-vitro culture. In summary, selecting the best embryo for transfer is a decision that should be based on choices made during the different stages of assisted reproductive technologies.

Evidence-based clinical outcome of oocyte slow cooling

In the last few years, there has been a significant improvement in oocyte cryopreservation techniques. To investigate the clinical significance of oocyte freezing, an assessment of the cumulative pregnancy rate per started cycle derived from the use of fresh and frozen-thawed oocytes was performed. Between 2004 and 2006, 749 cycles were carried out, in which no more than three fresh oocytes were inseminated either by standard IVF or microinjection. Supernumerary mature oocytes were cryopreserved by slow cooling. Cryopreservation of fresh embryos was performed in rare cases to prevent the risk of ovarian hyperstimulation syndrome using a standard embryo freezing protocol. Fresh embryo transfer cycles totalled 680, 257 of which resulted in pregnancy. The pregnancy rates per patient and per transfer were 34.3% and 37.8% respectively. When frozen-thawed oocytes were used, following 660 thawing cycles, 590 embryo transfers were performed in 510 patients. Eighty-eight pregnancies were achieved with embryos from frozen oocytes, with a success rate of 17.2% per cycle. When fresh and frozen-thawed cycles were combined, the number of pregnancies was 355, giving a cumulative pregnancy rate of 47.4%. Oocyte cryopreservation can contribute considerably to the overall clinical success, ensuring a cumulative rate approaching that achievable with embryo storage.

Truths and myths of oocyte sensitivity to controlled rate freezing

The mammalian oocyte is especially sensitive to cryopreservation. Because of its size and physiology, it can easily undergo cell death or sub-lethal damage as a consequence of intracellular ice formation, increase in the concentration of solutes and other undesired effects during the conversion of extracellular water into ice. This has generated the belief that oocyte storage cannot be achieved with the necessary efficiency and safety. However, many concerns raised by oocyte freezing are the result of unproven hypotheses or observations conducted under sometimes inappropriate conditions. For instance, spindle organization can undergo damage under certain freezing conditions but not with other protocols. The controversial suggestion that cryopreservation induces cortical granule discharge and zona pellucida hardening somehow questions the routine use of sperm microinjection. Damage to mouse oocytes caused by solute concentration is well documented but, in the human, there is no solid evidence that modifications of freezing mixtures, to prevent this problem, provide an actual advantage. The hope of developing oocyte cryopreservation as a major IVF option is becoming increasingly realistic, but major efforts are still required to clarify the authentic implications of oocyte cryopreservation at the cellular level and identify freezing conditions compatible with the preservation of viability and developmental ability.

Freezing spermatozoa obtained by testicular fine needle aspiration: a new technique

Testicular fine needle aspiration (TEFNA) of spermatozoa in azoospermic patients in advance of intracytoplasmic sperm injection could be useful to avoid the possibility of no recovery of spermatozoa on the day of oocyte retrieval. The conventional freezing procedure for these spermatozoa is not appropriate because of their very low number and poor in-situ motility. This article presents a new procedure for the freezing of TEFNA-recovered spermatozoa. A total of 1063 spermatozoa (10-340 cells/sample) were frozen by this method for research purposes. Before freezing, 13.7% were motile. The recovery rate after thawing was 100%. After thawing, 3.6% motility was observed. In a separate study group, the total number of frozen spermatozoa was 431 (2-300 cells/sample). Before freezing, the sperm motility rate was 3.5%. After thawing, 100% of the spermatozoa were retrieved with a motility rate of 2.3%. One biochemical pregnancy was obtained. The procedure yielded excellent recovery and good motility rates after thawing. However, because of the low number of cases, any conclusion about the efficiency of the technique is premature.

A PolScope evaluation of meiotic spindle dynamics in frozen–thawed oocytes

In mature human oocytes, the metaphase II (MII) spindle presence and birefringence signal detected through the PolScope may vary before and after freezing. In particular, spindle dynamics during the first few hours after thawing is still under study. In this study, oocytes from stimulated ovaries were cryopreserved in 1.5 mol/l 1,2-propanediol with 0.3 mol/l sucrose using a slow freezing-rapid thawing method. Oocytes were examined with the PolScope for the presence, intensity of signal birefringence and size of the meiotic spindle before freezing and at 0, 1 and 2 h post-thaw (where 0 h = the time of the end of the thawing procedure). Of the 173 surviving oocytes exhibiting a spindle before freezing, 82.7% (143/173) showed spindle birefringence within 1 h of thawing. However, at the end of the thawing procedure the intensity of spindle birefringence (retardance) and the spindle length were smaller in comparison to the pre-freezing condition. These parameters increased after 1 h, although were not restored to the value observed before freezing. No significant changes were observed by extending the culture to 2 h.

The effect of leuprorelin on steroidogenesis of human preovulatory granulosa cells in vitro

AbstractPurpose: LHRH analogues are commonly used in in vitro fertilization protocols to induce hypogonadotropic hypogonadism. The aim of our study was to evaluate the action of the LHRH agonist leuprorelin on the E2 steroidogenesis of human preovulatory granulosa cells. Results: FSH causes a significant increase in E2 production which is double that of the basal condition (P<0.01). At concentrations of 1, 10, and 100 ng/ml, leuprorelin does not produce any modification with respect to the basal condition during the 24- or 72-hr culture period. The FSH, added at different analogue concentrations, produces a significant increase in E2 production as compared to the basal condition (P<0.05) and the E2 production percentage is similar to the values obtained with FSH alone during the 24- or 72-hr culture period. Conclusions: Leuprorelin has no effect on the in vitro E2 production at any concentration. The treatment with different doses of leuprorelin does not suppress FSH-stimulated E2 production. Our findings suggest that human granulosa cells are not acutely sensitive to a direct action on E2 steroidogenesis by LHRH analogues.