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The most important results of Human Clinical Embryology :

From Oocytes to Preimplantation Embryos

 

INTRODUCTION

 
 

The female gamete plays a crucial role in determining embryo competence and therefore in vitro fertilization (IVF) results. Oocyte quality is not only influenced by the nuclear and mitochondrial genome, but also by the microenvironment provided by the ovary and the pre-ovulatory follicle that influences transcription and translation, and as a consequence, cytoplasmic maturity. In contrast to in vivo processes, the application of ovarian hormone stimulation protocols for IVF bypasses the complicated selection procedure that usually occurs during oocyte development and maturation of a single oocyte for ovulation, and allows for the maturation of many oocytes, often with compromised quality.

It has been speculated (Van Blerkom and Henry, 1992) that some morphological irregularities, which can easily be assessed at the light microscopy level, may reflect a compromised developmental ability of the oocytes and could therefore represent a useful tool for selecting competent oocytes prior to fertilization. Oocyte morphological assessment in the laboratory is first based on the presentation of the cumulus–corona cells. For mature oocytes, the cumulus–corona mass should appear as an expanded and mucified layer, due to active secretion of hyaluronic acid. This extracellular matrix molecule interposes between the cumulus cells (CCs), separating them and conferring to the cumulus–corona mass a fluffy ‘cloud-like’ appearance. However, stimulated cycles may be characterized by asynchrony between the nuclear maturation status of the oocyte and the expansion of the cumulus–corona cell mass. This has been suggested to be caused by a different sensitivity of the oocyte and the cumulus–corona mass to the stimulants (Laufer et al., 1984).

Following the removal of the cumulus–corona cells in preparation for intracytoplasmic sperm injection (ICSI), oocyte evaluation is more accurate and is based on the nuclear maturation status, the morphology of the cytoplasm and on the appearance of the extracytoplasmic structures. The presence of the first polar body (PBI) is normally considered to be a marker of oocyte nuclear maturity. However, recent studies using polarized light microscopy have shown that oocytes displaying a polar body may still be immature (Rienzi et al., 2005). Only those displaying a meiotic spindle (MS) can in fact be considered as true, mature, Metaphase II (MII) stage oocytes. The presence, position and retardance of the MS have been suggested to be related to developmental competence. In accordance with a recent meta-analysis (Petersen et al., 2009), however, only in vitro development can be related to the morphology of the MS. Analyses of in vivo development are relatively rare in the literature and the meta-analysis failed to show significant differences in implantation rates between embryos derived from oocytes displaying a detectable MS and those without.

Nuclear maturity alone is, in fact, not enough to determine the quality of an oocyte. Nuclear and cytoplasmic maturation should be completed in a coordinated manner to ensure optimal conditions for subsequent fertilization. An ideal mature human oocyte, based on morphological characteristics, should have a ‘normal-looking’ cytoplasm, a single polar body, an appropriate zona pellucida (ZP) thickness and proper perivitelline space (PVS; Swain and Pool, 2008). However, the majority of the oocytes retrieved after ovarian hyperstimulation exhibit one or more variations in the described ‘ideal’ morphological criteria (De Sutter et al., 1996Xia, 1997Balaban et al., 1998Mikkelsen and Lindenberg, 2001; Balaban and Urman, 2006Ebner et al., 2006Rienzi et al., 2008). This is also true for oocytes obtained from proven fertile donors (Ten et al., 2007). Morphology, moreover, often fails to predict fertilizing ability and developmental competence (Rienzi et al., 2011). Only a few morphologically detectable features of the Metaphase II oocyte indicate compromised developmental ability. According to the Istanbul consensus workshop on embryo assessment (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011) extracytoplasmic anomalies (PBI morphology, PVS size, the appearance of the ZP) are simply phenotypic variations often related to in vitro culture and/or oocyte aging. On the other hand, a special deviation in the cytoplasmic texture, namely the presence of aggregations of smooth endoplasmic reticulum (SER) is potentially lethal and developmental competence of these oocytes should be interpreted with caution. Oocyte morphology may also reflect genetic abnormalities. This is the case for giant oocytes that contain one additional set of chromosomes. These oocytes, when observed with polarized light microscopy, display two distinct MS. Although, the occurrence of giant oocytes is relatively rare after ovarian hyperstimulation, the use of these cells for IVF is dangerous.

Owing to the complex mechanisms related to oocyte maturation and acquisition of competence, it is unlikely that a single characteristic (with the exception of oocyte size and the presence of SER aggregates) can adequately reflect the quality of the cell. Accordingly, to obtain information about the competence of the oocyte, morphological assessment should be combined with other approaches (i.e. cumulus–corona cell gene expression, metabolomics and oxygen consumption). Further predictive value could be obtained by combining the oocyte evaluation with evaluations of preimplantation development (pronuclear stage, cleavage stage and blastocyst stage).

A. CUMULUS-ENCLOSED OOCYTES

During follicular antrum formation, granulosa cells (GCs) differentiate into mural GCs, lining the follicular wall, and CCs, surrounding the oocyte. Within the cumulus mass, CCs in close contact with the oocyte (corona cells) develop cytoplasmic projections which cross the ZP and form gap junctions with the oolemma. This organized structure is called the cumulus–oocyte complex (COC; Fig. 1; Albertini et al., 2001). In natural spontaneous cycles, oocyte nuclear maturation runs parallel to the gradual FSH-dependent expansion of the cumulus and corona cells, whereas this synchrony may be disturbed in stimulated cycles (Laufer et al., 1984). Immature COCs (Fig. 2), commonly retrieved from small follicles during in vitro maturation (IVM) cycles, show a typically unexpanded cumulus with multilayers of compact GCs adhering to the ZP of an immature oocyte at prophase I [germinal-vesicle stage (GV); Figs 3 and 4]. IVM of such immature COCs aims for expansion of CCs and oocyte nuclear maturation.

In stimulated cycles, 34–38 h after triggering ovulation, a typical mature pre-ovulatory COC displays radiating corona cells surrounded by the expanded, loose mass of CCs (Fig. 5). In the majority of expanded COCs, oocytes are mature at the MII stage, although it is possible after gonadotrophin stimulation to find in a mucified cumulus and radiating corona cells an immature oocyte at the GV or metaphase I (MI) stage (Fig. 6). It is common, in stimulated cycles, to recover COCs with an expanded cumulus cell mass but compact, non-radiating corona cells (Figs 7a, 8a and 9a). Indeed, at recovery, the presence of the surrounding cumulus and corona cells usually prevents identification of the PBI in the PVS, an indicator of successful completion of meiosis I with arrest at the MII stage of development (Fig. 1). In preparation for ICSI, oocyte denudation is performed via enzymatic action of hyaluronidase (Figs 7b , 8b and 9b) and mechanical pipetting, allowing the accurate determination of the oocyte nuclear status (Figs 7c , 8c and 9c).

The Alpha-ESHRE consensus document states that, although there is little corroborated evidence to support a correlation between the appearance of the COC and embryo developmental competence, a binary score (0 or 1) with a ‘good’ COC (score 1) defined as having an expanded cumulus and a radiating corona should be documented (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011).

B. OOCYTE MATURATION STAGE 

The removal of the cumulus–corona cell mass gives the unique opportunity to evaluate oocyte morphology prior to fertilization, and in particular, the nuclear maturation stage. Oocyte nuclear maturity, as assessed by light microscopy, is assumed to be at the MII stage when the PBI is visible in the PVS (Figs 10 and 11). The MII stage is characterized by the alignment of the homologous chromosomes on the spindle equatorial plate during metaphase of the second meiotic division. It is generally recognized that 85% of the retrieved oocytes following ovarian hyperstimulation display the PBI and are classified as MII, whereas 10% present an intracytoplasmic nucleus called the ‘germinal vesicle’ (GV; Figs 12–14), characteristic of prophase I of the first meiotic division. Approximately 5% of the oocytes have neither a visible GV nor PBI and these oocytes are generally classified as MI oocytes (Figs 15–17; Rienzi and Ubaldi, 2009). These oocytes may, however, be at the GV breakdown stage where the nuclear envelope has broken down but has not as yet progressed to true MI where the chromosomes are aligned on the metaphase plate in preparation for the completion of the first meiotic division.

Additional information on oocyte nuclear status can be obtained with the use of polarized light microscopy combined with software for image processing for the non-invasive visualization of the MS and other oocyte birefringent structures. The MS is a microtubular structure involved in chromosome segregation, and therefore is crucial in the sequence of events leading to the correct completion of meiosis and subsequent fertilization. Parallel-aligned MS microtubules are birefringent and able to shift the plane of polarized light inducing a retardance; these properties enable the system to generate contrast and image the MS structure (Oldenbourg, 1999; Fig. 18). The presence of the MS gives more accurate information about the nuclear stage of the oocyte. In particular, some oocytes can be immature (at the stage of early telophase I) when observed with polarized light microscopy, despite the presence of PBI in the PVS. At this stage, in fact, there is continuity between the ooplasm of the oocyte and the forming PBI and the MS is interposed between the two separating cells (Figs 19–22). This condition normally has a duration of 75–90 min. The MS has been found to disappear in late telophase I (Fig. 23), reforming only 40–60 min later (Montag et al., 2011). However, it must be underlined that other factors, such as sub-optimal culture conditions, temperature fluctuations and chemical stress during manipulation, can contribute to MS disassembly (Rienzi and Ubaldi, 2009). Finally, the percentage of oocytes with detectable MS is also related to the time elapsed from HCG administration and is higher after 38 h (Cohen et al., 2004). In general, it is expected that at least 80% of oocytes recovered following ovarian hyperstimulation are MS positive when viewed by polarized light microscopy.

 

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The bidirectional communication between the oocyte and CCs, crucial for the acquisition of oocyte competence (Gilchrist et al., 2008), might perhaps be investigated in the future, through non-invasive analysis of CCs (pattern of gene expression or protein synthesis), offering new biomarkers of oocyte quality, compensating for the inadequacy of the COC morphological assessment (Feuerstein et al., 2007Ouandaogo et al., 2011).

 

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Post Author: IRIFIV AISRG