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.

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.

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.

Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART.

BACKGROUND: The relationship between early embryo post-implantation development in couples undergoing assisted reproductive techniques (ARTs) and sperm chromatin alterations has not been satisfactorily explained. The aim of this study was to assess the relationship between sperm DNA fragmentation in IVF/ICSI patients, sperm parameters (concentration, motility and morphology) and ART outcome, especially with regard to clinical pregnancy and pregnancy loss (spontaneous miscarriage or biochemical pregnancy). METHODS: DNA fragmentation was evaluated by TUNEL assay, performed on sperm suspensions after density gradient separation, in 132 men undergoing an ART cycle (82 IVF and 50 ICSI) and correlated with sperm parameters and ART outcome. RESULTS: A highly significant negative correlation was found between DNA fragmentation and sperm parameters. There was a close relationship between DNA fragmentation and post-implantation development in ICSI patients: the clinical pregnancy and pregnancy loss rates significantly differed between patients with high and low sperm DNA fragmentation (P = 0.007 and P = 0.009, respectively). CONCLUSIONS: Sperm DNA fragmentation seems to affect embryo post-implantation development in ICSI procedures: high sperm DNA fragmentation can compromise 'embryo viability', resulting in pregnancy loss.

Strategies and clinical outcome of 250 cycles of Preimplantation Genetic Diagnosis for single gene disorders.

BACKGROUND: We report on our experience with preimplantation genetic diagnosis (PGD) for single gene disorders (SGDs), from 1999 to 2004, describing strategies and overall clinical outcome of 250 cycles in 174 couples for 23 different genetic conditions. METHODS: PGD cycles included 15 for autosomal dominant, 148 for autosomal recessive and 19 for X-linked SGDs. In addition, 68 cycles of PGD for SGDs were performed in combination with HLA matching. The strategy in each case used an initial multiplex PCR, followed by minisequencing to identify the mutation(s) combined with multiplex PCR for closely linked informative markers to increase accuracy. Linkage analysis, using intragenic and/or extragenic polymorphic microsatellite markers, was performed in cases where the disease-causing mutation(s) was unknown or undetectable. RESULTS: In 250 PGD cycles, a total of 1961 cleavage stage embryos were biopsied. PCR was successful in 3409 out of 3149 (92.4%) biopsied blastomeres and a diagnosis was possible in 1849 (94.3%) embryos. Four hundred and twenty-seven embryos were transferred in 211 cycles, resulting in 71 pregnancies (33.6% per embryo transfer), including 15 biochemical pregnancies, six spontaneous miscarriages, two ectopic pregnancies, which were terminated, and nine pregnancies which are still ongoing. The remaining pregnancies were confirmed to be unaffected and went to term without complications, resulting in the birth of 35 healthy babies. CONCLUSIONS: Minisequencing for mutation detection combined with multiplex fluorescence PCR for linkage analysis is an efficient, accurate and widely applicable strategy for PGD of SGDs. Our experience provides a further demonstration that PGD is an effective clinical tool and a useful option for many couples with a high risk of transmitting a genetic disease.

Criteria to assess human oocyte quality after cryopreservation.

Oocyte cryopreservation certainly represents one of the most attractive developments in the field of assisted reproduction, with the aim of preserving female fertility and circumventing the ethical and legal drawbacks associated with embryo freezing. Despite the achievement of the first pregnancy from frozen oocytes dating back as early as 1987, since then fewer than 150 pregnancies have been reported. Over a long period of time, application of oocyte storage on a large scale has been prevented by various factors, namely poor post-thaw survival. Fertilization rates remained low even after the introduction of intracytoplasmic sperm injection. Modifications of slow-freezing protocols, mainly based on the increase of the concentration of sucrose used as non-penetrating cryoprotectant (CPA) and the replacement of sodium with choline, appear to have decisively improved survival rates to over 80%. Investigations at the cellular level on thawed oocytes are largely lacking. Fertilization rates have also benefited from protocol modifications, reaching values indistinguishable from those normally obtained with fresh material. Vitrification protocols have also been tested, giving rise to improvements whose reproducibility is still uncertain. Data on the dynamics of fertilization and preimplantation development of embryos derived from frozen oocytes are extremely scarce. At the moment, clinical efficiency of oocyte cryopreservation cannot be precisely assessed because of the lack of controlled studies, although it appears to be considerably lower than that achieved with embryo freezing. In summary, encouraging advances have been made in the field of oocyte cryopreservation, but presently no protocol can ensure standards of success and safety comparable to those guaranteed by embryo storage.

Oocyte cryopreservation: a biological perspective.

Oocyte cryopreservation would amount to a major breakthrough in reproductive medicine. Diverse strategies have been tested to minimise cooling-induced cell injury. Nevertheless, oocytes from various species have shown a particular sensitivity to freezing, due to their unique biological characteristics. Storage of human mature oocytes with slow freezing has resulted in low survival rates, although recent studies based on modified methods have reported higher success. Survival after thawing is not necessarily a guarantee of unaltered viability. Developmental failure at pre- or postimplantation stages may originate from critical perturbations of various cell components, such as the chromosome segregation apparatus, the intracellular calcium signalling system, and the cytoskeleton. Germinal vesicle (GV)-stage oocytes have been suggested to be more amenable to freezing. But their use would require efficient in vitro maturation systems, which are not presently available. Oocyte cryopreservation remains a realistic objective, provided that more systematic approaches are applied, such as thorough analysis of the plasmalemma permeability to water and the diverse cryoprotectants.

Fate of stored embryos: our 10 years experience.

Couples accepting embryo cryopreservation signed an informed consent in which directives were given in case of death, divorce or absence. In this study, we tried to analyse our experience in terms of benefits achieved and to understand the feelings of couples about their embryos. (1) The majority of couples decided, or simply let, their embryos be discarded. In fact, a considerable proportion of them (25.1%) did not want to assume the responsibility of signing the disposal of their embryos. (2) Embryo donation is the most uncommon and difficult choice to make (6.0%) and this ought to be an important point to think over while dealing with law design. (3) An unexpected proportion of couples, who did not conceive, still have their embryos stored and this forces us to reflect on the difficulty of getting over the treatment failure.

GnRH analogs: depot versus short formulations.

Gonadotropin releasing hormone agonists (GnRH-a) are widely used in controlled ovarian hyperstimulation (COH) for assisted reproduction (ART). Two different formulations are now available: short formulations and depot formulation. Some authors have suggested that depot GnRH-a induce a too high pituitary suppression and have put forward protocols using reduced GnRH-a doses. A reduced dose of daily triptorelin is enough for pituitary suppression during ovarian stimulation but provides no significant improvement in IVF cycle outcome when compared with depot formulation in normally responding women. However, it seems to improve ovarian response and overall results in poor responding patients. Low doses of short GnRH-a allow shorter treatment, requiring lower amounts of gonadotropins. This possibility should be considered in view of its economic advantage.

Perifollicular vascularity and its relationship with oocyte maturity and IVF outcome.

New markers of embryo ability to implant are pursued continuously. Understanding whether an oocyte is really "mature," that is, ready to be fertilized, would be of great help in choosing an embryo that will implant. It is usual to pay attention to the phase of meiosis, considering the extrusion of the polar body (metaphase II) to be the only sign of the maturity of the oocytes. Nevertheless, understanding more about how the cytoplasm contributes to an oocyte's competency also shows promise as a method of predicting which embryos will implant. Some studies about perifollicular vascularity have demonstrated that embryos originating from oocytes developed in well-vascularized follicles have a higher implantation rate than those originating from oocytes developed in follicles with poor vascularization. Here, we report our results from a preliminary study in which embryos were transferred according to the degree of vascularization of the follicle. Women who received embryos originating from oocytes developed in well-vascularized follicles had a statistically higher pregnancy rate than women who received embryos deriving from oocytes grown in more poorly vascularized follicles (34% vs. 13.7%).

Cryopreservation of human oocytes.

Cryopreservation is a well established technique by which preimplantation stage embryos can be stored for later use. However, it would be preferable to cryopreserve unfertilized oocytes to overcome the problems related to the ethical and legal status of the embryo. This procedure has been successful in mice, but attempts to extend this approach to other species have been less successful. In human in vitro fertilization (IVF) in particular, very few live births have been reported after the cryopreservation of oocytes. The reason for this lack of success is unclear. The widespread contention that compromised oocyte viability originates principally from damage to the meiotic spindle induced by cryopreservation conditions is unproven. The treatment of large groups of women has shown that pregnancies derived from stored oocytes can be achieved in a reproducible fashion, although success rates are still lower than those derived from frozen embryos. To date, human oocytes have been stored largely by applying methods originally designed for cleaving embryos. Therefore, it is reasonable to envisage that appropriate modification of current freezing techniques will increase survival rates and make oocyte storage an important option in IVF treatment.

Effect of reduced dose of triptorelin at the start of ovarian stimulation on the outcome of IVF: a randomized study.

BACKGROUND: Partial pituitary desensitization using gonadotrophin-releasing hormone (GnRH) agonists may be sufficient in women undergoing controlled ovarian hyperstimulation for assisted reproduction. However, the minimal effective agonist dose remains to be determined. The aim of the study was to investigate the effect of a reduced daily dose of triptorelin, administered at the start of ovarian stimulation, on the results of IVF and intracytoplasmic sperm injection. METHODS: A total of 132 patients was randomized in two groups. Pituitary desensitization was obtained in group 1 (66 patients) with a single 3.75 mg injection (i.m.) of triptorelin. In group 2, 66 patients received 100 microg triptorelin daily, which was then reduced to 50 microg at the start of follicle-stimulating hormone (FSH) stimulation. RESULTS: No significant differences were found in terms of pregnancy rate per transfer (38% in group 1 versus 34.9% in group 2), implantation rate (20.2 versus 18%) and abortion rate (8.3 versus 9.1%). The number of FSH ampoules used, as well as the number of days stimulation required, was significantly reduced in group 2 (41 +/- 26 versus 46.6 +/- 25.3, P < 0.03 and 11 +/- 1.3 versus 11.8 +/- 1.5, P < 0.002 respectively). No significant differences were seen in oestradiol concentrations and in follicle number, in the quantity of oocytes collected and fertilized, or in the number of embryos obtained or transferred. CONCLUSION: A reduced dose of triptorelin is enough for pituitary suppression during ovarian stimulation but provides no significant improvement in IVF cycle outcome when compared with depot formulation. The possibility of a shorter treatment protocol requiring lower amounts of gonadotrophins should be considered in view of its economic advantage.

Oocyte donation programme: results obtained with intracytoplasmic sperm injection in cases of severe male factor infertility or previous failed fertilization.

Intracytoplasmic sperm injection was carried out in 15 oocyte donation cycles of 15 infertile couples where oocytes had failed to fertilize after in-vitro fertilization (IVF) procedures or where the male partner had severe male factor infertility. A total of 62 oocytes were donated, but only 46 of these, in metaphase II, were injected. Of the injected oocytes, 31 (67.3%) had two pronuclei the morning after the injection procedure. On the following day, 29 embryos were obtained (93% of the fertilized oocytes) and 25 were transferred. Two patients were not successful and consequently did not undergo embryo transfer. A total of five clinical pregnancies were obtained, giving pregnancy rates of 33.3 and 38.4% per started cycle and embryo transfer respectively.