G. Coticchio

Meiotic spindle dynamics in human oocytes following slow-cooling cryopreservation

BACKGROUNDnThe 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.nnnMETHODSnUnfrozen 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.nnnRESULTSnFreezing 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.nnnCONCLUSIONSnSpindle 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.

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.

Ultrastructural markers of quality in human mature oocytes vitrified using cryoleaf and cryoloop

This study describes and compares the possible effects of vitrification on the ultrastructural morphology of 20 human mature oocytes vitrified using two different supports, cryoleaf (n = 10) and cryoloop (n = 10). Fresh human mature oocytes (n = 15) were used as controls. Fresh and vitrified-warmed oocytes appeared rounded, with a homogeneous cytoplasm, an intact oolemma and a continuous zona pellucida. Sparse microvacuolization was only occasionally detected in fresh and vitrified-warmed oocytes, to the same extent. About 50% of the vitrified oocytes contained atypical, small and slender mitochondria-smooth endoplasmic reticulum aggregates, whereas a non-homogeneous microvillar pattern was observable in only 30% of the oocytes subjected to vitrification, regardless of the support utilized. Cortical granule content appeared generally reduced after vitrification, but cryoleaf-supported oocytes contained more cortical granules than cryoloop-supported oocytes (P < 0.05). Thus good overall preservation and virtual absence of cytoplasmic vacuolization seem to be the most relevant markers of quality in vitrified-warmed oocytes, using either support. In addition, cryoleaf-supported oocytes retained a higher number of cortical granules than cryoloop-supported oocytes. The variety of ultrastructural alterations recorded emphasizes the need for further studies aimed at assessing the actual tolerance of human oocytes to vitrification.

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

OBJECTIVEnTo determine whether Polscope analysis can predict different spindle and chromosome configurations of the oocyte metaphase II (MII) spindle.nnnDESIGNnComparison of Polscope and confocal microscopy analysis of the MII spindle.nnnSETTINGnPrivate IVF unit.nnnPATIENT(S)nWomen undergoing IVF treatment for male or unexplained infertility.nnnINTERVENTION(S)nFresh and frozen-thawed mature oocytes were analyzed through the Polscope and, immediately afterward, fixed for confocal microscopy assessment.nnnMAIN OUTCOME MEASURE(S)nComparison 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.nnnRESULT(S)nThe 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.nnnCONCLUSION(S)nRetardance 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.

Natriuretic Peptide Precursor C Delays Meiotic Resumption and Sustains Gap Junction Mediated Communication in Bovine Cumulus Enclosed Oocytes

ABSTRACT Oocyte in vitro maturation (IVM) has become a valuable technological tool for animal breeding and cloning and the treatment of human infertility because it does not require the administration of exogenous gonadotropin to obtain fertilizable oocytes. However, embryo development after IVM is lower compared to in vivo maturation, most likely because oocytes collected for IVM are heterogeneous with respect to their developmental competencies. Attempts to improve IVM outcome have relied upon either prematuration culture (PMC) or two-step maturation strategies in the hope of normalizing variations in developmental competence. Such culture systems invoke the pharmacological arrest of meiosis, in theory providing oocytes sufficient time to complete the acquisition of developmental competence after cumulus-enclosed oocytes isolation from the follicle. The present study was designed to test the efficiency of natriuretic peptide precursor C (NPPC) as a nonpharmacologic meiosis-arresting agent during IVM in a monoovulatory species. NPPC has been shown to maintain meiotic arrest in vivo and in vitro in mice and pigs; however, the use of this molecule for PMC has yet to have been explored. Toward this end, meiotic cell cycle reentry, gap-junction functionality, and chromatin configuration changes were investigated in bovine cumulus-enclosed oocytes cultured in the presence of NPPC. Moreover, oocyte developmental competence was investigated after IVM, in vitro fertilization, and embryo culture and compared to standard IVM-in vitro fertilization protocol without PMC. Our results suggest that NPPC can be used to delay meiotic resumption and increase the developmental competence of bovine oocytes when used in PMC protocols.

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.

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.

Characterization of the human cumulus cell transcriptome during final follicular maturation and ovulation

Cumulus expansion and oocyte maturation are central processes in ovulation. Knowledge gained from rodent and other mammalian models has revealed some of the molecular pathways associated with these processes. However, the equivalent pathways in humans have not been thoroughly studied and remain unidentified. Compact cumulus cells (CCs) from germinal vesicle cumulus oocyte complexes (COCs) were obtained from patients undergoing in vitro maturation (IVM) procedures. Expanded CCs from metaphase 2 COC were obtained from patients undergoing IVF/ICSI. Global transcriptome profiles of the samples were obtained using state-of-the-art RNA sequencing techniques. We identified 1746 differentially expressed (DE) genes between compact and expanded CCs. Most of these genes were involved in cellular growth and proliferation, cellular movement, cell cycle, cell-to-cell signaling and interaction, extracellular matrix and steroidogenesis. Out of the DE genes, we found 89 long noncoding RNAs, of which 12 are encoded within introns of genes known to be involved in granulosa cell processes. This suggests that unique noncoding RNA transcripts may contribute to the regulation of cumulus expansion and oocyte maturation. Using global transcriptome sequencing, we were able to generate a library of genes regulated during cumulus expansion and oocyte maturation processes. Analysis of these genes allowed us to identify important new genes and noncoding RNAs potentially involved in COC maturation and cumulus expansion. These results may increase our understanding of the process of oocyte maturation and could ultimately improve the efficacy of IVM treatment.

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.