Raffaella Sciajno

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.

Meiotic spindle dynamics in human oocytes following slow-cooling cryopreservation

BACKGROUND The demand for cryopreservation of human oocytes is increasing in assisted reproduction clinics and yet remains an experimental procedure. Surprisingly, little is known about the effects of cryopreservation on spindle-chromosome interactions and the recovery of meiotic spindle functionality. The goal of these studies was to evaluate the process of meiotic spindle reassembly and chromosome alignment in cryopreserved human metaphase II oocytes. METHODS Unfrozen control oocytes were compared with frozen oocytes fixed at 0, 1, 2 and 3 h after thawing. Oocytes were analysed by confocal microscopy and subjected to 3-dimensional image analysis to evaluate spindle integrity. RESULTS Freezing resulted in a loss of spindle bipolarity and chromosome alignment. One hour following thawing, most oocytes recovered spindle bipolarity and equatorial chromosomal alignment. However, between 2 and 3 h, a progressive loss of chromosome alignment was observed. Further analysis revealed a positive correlation between spindle length and number of displaced chromosomes following freezing. This time-dependent redistribution of chromosomes involved outward displacement from the equatorial plate and retention at the surface of the meiotic spindle. CONCLUSIONS Spindle disassembly incurred by cryopreservation is rapidly reversed and is coordinated with chromosome alignment within 1 h but is not sustained at later times.

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.

Endometrial preparation for frozen-thawed embryo transfer with or without pretreatment with gonadotropin-releasing hormone agonist

OBJECTIVE To test the efficacy of endometrial preparation with exogenous steroids, without pretreatment with gonadotropin-releasing hormone (GnRH) agonist, in women with normal ovarian function. DESIGN Prospective randomized study. SETTING Private outpatient infertility clinic. PATIENT(S) Two hundred ninety-six women undergoing frozen-thawed embryo transfer. INTERVENTION(S) In group 1 (146 patients), depot GnRH agonist was administered in the luteal phase; treatment with 17beta-estradiol transdermal patches at steadily increasing dosage from 100 to 300 microg was then given for at least 12 days. In group 2 (150 patients), endometrial preparation began on day 1 of menstrual cycle. The starting dose was 200 microg; this was increased to 300 microg after 7 days. MAIN OUTCOME MEASURE(S) Pregnancy, abortion, implantation and cancellation rates. RESULT(S) In group 2, six cycles (4%) were cancelled due to evidence of ovulation. Groups were similar in the percentage of embryos that survived freezing-thawing (77.1% in group 1 and 76.6% in group 2) and in the number of embryos transferred per patient (2.1 +/- 0.6 and 2.1 +/- 0.7, respectively). Groups 1 and 2 did not differ significantly in rates of pregnancy (19.7% and 24.1%), abortion (17.8% and 11.7%), and implantation (10.4% and 11.9%). CONCLUSION(S) Endometrial preparation for frozen-thawed embryo transfer based exclusively on steroid administration appears to be as effective as the more conventional protocol involving preliminary desensitization with a GnRH agonist. This simplified protocol reduces costs, minimizes pharmacologic treatment, and increases patient compliance.

Vitrification may increase the rate of chromosome misalignment in the metaphase II spindle of human mature oocytes

The metaphase II (MII) spindle of the human oocyte may be damaged by cryopreservation. High performance confocal microscopy was used to assess meiotic spindle and chromosome organization in oocytes after vitrification by the cryoleaf system. Three hours after retrieval, donor mature oocytes were fixed or vitrified. Vitrification was performed by equilibration in 7.5% ethylene glycol (EG) and 7.5% dimethylsulphoxide (DMSO), transfer to 15% EG, 15% DMSO and 0.5 mol/l sucrose, and loading onto cryoleaf strips. Tubulin staining was found in all survived vitrified-warmed oocytes, the majority (62.8%) of which displayed a bipolar spindle. A normal bipolar spindle configuration and equatorial chromosome alignment was observed only in a part of vitrified-warmed oocytes (32.6%). This frequency was significantly lower in comparison to fresh oocytes (59.1%). In another fraction of vitrified-warmed oocytes (30.2%), spindle bipolarity was associated to one or more non-aligned scattered chromosomes that often appeared tenuously associated with the lateral microtubules of the spindle. Furthermore, in cryopreserved oocytes with a bipolar spindle, a significantly increased pole-to-pole distance (14.9 +/- 2.3 microm) was found in comparison to the fresh control (12.4 +/- 2.6 microm) (P = 0.001). Therefore, under the conditions tested, vitrified-warmed oocytes maintain a MII spindle with a bipolar organization. However, chromosome alignment appears to be partly compromised.

Comparative analysis of the metaphase II spindle of human oocytes through polarized light and high-performance confocal microscopy

OBJECTIVE To determine whether Polscope analysis can predict different spindle and chromosome configurations of the oocyte metaphase II (MII) spindle. DESIGN Comparison of Polscope and confocal microscopy analysis of the MII spindle. SETTING Private IVF unit. PATIENT(S) Women undergoing IVF treatment for male or unexplained infertility. INTERVENTION(S) Fresh and frozen-thawed mature oocytes were analyzed through the Polscope and, immediately afterward, fixed for confocal microscopy assessment. MAIN OUTCOME MEASURE(S) Comparison of retardance values, derived from Polscope analysis, between spindles with different microtubule and chromosome configurations, defined by confocal microscopy evaluation. Measurements of spindle longitudinal axis through the Polscope and confocal microscopy. RESULT(S) The mean retardance values of different categories of spindle configuration were not statistically significant in almost all cases, allowing only the identification of spindles with highly disorganized microtubules and chromosomes in frozen-thawed oocytes. In spindles with bipolar organization, the Polscope produced measurements of the spindle main axis which were in all cases statistically smaller compared with confocal microscopy evaluation. CONCLUSION(S) Retardance measurements have limited predictive value of the degree of spindle fiber order and chromosome position in routine clinical settings. Also, under the conditions tested, morphometric evaluation of the spindle through the Polscope is not consistent with confocal analysis. This suggests that the Polscope may still be a rather inefficient method for assessing the metaphase II spindle and, as a result, for noninvasive oocyte selection.

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 oocytes 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%).

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.