Hum. Reprod. Advance Access originally published online on October 30, 2007
Human Reproduction 2008 23(1):62-66; doi:10.1093/humrep/dem280
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Developmental fate of ovoid oocytes
Landes- Frauen- und Kinderklinik, IVF-Unit, Krankenhausstr. 26-30, A-4020 Linz, Austria
1 Correspondence address. E-mail: thomas.ebner{at}gespag.at
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Abstract |
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BACKGROUND: Irregularities in composition, thickness and/or color of the zona pellucida may impair optimal function and result in reduced outcome. Anomalies of oocyte shape have not been investigated in detail in this respect.
METHODS: Therefore, all patients attending our clinic within a period of 1 year were screened for the presence of ovoid gametes and the corresponding developmental potential was evaluated. For all elongated gametes, a roundness index (RI; length divided by width) was calculated in order to quantify shape.
RESULTS: RI did not affect fertilizability (P > 0.05). The degree of dysmorphism was found to be related to cleavage pattern. The more ovoid a gamete was, the higher was the risk of the corresponding zygote not cleaving like a tetrahedron (P < 0.01). Abnormal cleavage (a rather flat array of blastomeres) was associated with delayed compaction (P < 0.01) and blastocyst formation (P < 0.001). The quality of blastocysts was not affected at any stage in ovoid concepti (P > 0.05).
CONCLUSIONS: Ovoid oocytes with abnormal cleavage pattern show delayed preimplantation development, probably due to a reduced number of cell-to-cell contacts.
Key words: blastocyst formation/compaction/ovoid shape/roundness index/zona pellucida
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Introduction |
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Ovarian endocrinologic milieu under controlled ovarian hyperstimulation differs from that of natural cycle, and may lead to maturation and ovulation of germ cells with reduced developmental potential. Oocyte maturation within the follicle consists of two separate processes, nuclear and cyctoplasmic maturation, both of which should take place in a well-coordinated and synchronized manner in order to ensure adequate oocyte quality. Unfortunately, intrinsic oocyte-specific defects caused by any disturbance of these processes are hardly detectable at a light-microscopic level. Thus, the actual implantation potential may be overestimated as the oocyte morphology, fertilization and cleavage rate may appear inconspicuous at first glance.
Nevertheless, embryologists tend to facilitate selection process in terms of embryo transfer by introducing morphological criteria at different developmental phases (Ebner et al., 2003
; Borini et al., 2005; Rienzi et al., 2005). Defining oocyte characteristics which allow one to predict developmental potential could increase the chances of better embryo selection for transfer (Ebner et al., 2006).
In this respect, numerous morphological anomalies of the gametes are pooled in order to enable adequate analysis. Usually, dysmorphisms are classified as cytoplasmic and extracytoplasmic anomalies (Ebner et al., 2001). The latter group consists of several anomalies of different origin, such as fragmented polar bodies (Ebner et al., 2000), perivitelline space granularity (Hassan-Ali et al., 1998), discoloration of the gamete (Esfandiari et al., 2006) and defects in zona pellucida (ZP) structure (Shen et al., 2005).
It is likely that any irregularity in composition, thickness, colour or shape of the ZP may affect optimal function and result in reduced outcome. In extreme cases, oocytes of certain patients may be characterized by expression of very thin zonae (Stanger et al., 2001) or complete absence of ZP (Veeck, 1998). Such gametes are probably less resistant to manipulation procedures for insemination which might result in a higher degeneration rate. Another negative phenomenon has been documented by Shen et al. (2005), who showed that the thickness (and retardance) of the innermost layer of the ZP was significantly increased in conception cycles when compared with failed cycles. Using polarizing microscopy, these authors (Shen et al., 2005) also occasionally noted splitting of the inner ZP layer which was associated with implantation failure.
One particular dysmorphism definitely affecting ZP structure is a distinct shape anomaly, e.g. an ovoid appearance. Although a case report indicates that oocytes with ovoid ZP may result in successful pregnancy (Esfandiari et al., 2005), the fertilization capacity and further fate of ovoid gametes have not been analyzed to date. In order to check the developmental potential of such dysmorphic ova, we prospectively analyzed all ICSI cases within 1 year for elongated gametes. The preimplantation development of these dysmorphic oocytes was compared with the cleavage behavior of their sibling spheric counterparts.
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Materials and Methods |
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A total of 525 ICSI cycles were involved in this 1 year prospective screening for elongated oocytes. Permission of the Institutional Review Board was not sought since it was an observational study based on routine in vitro culture methods.
More than half of the couples (n = 341) were treated using an antagonist protocol (mean age: 33.1 ± 4.8). In these cases, stimulation was started at the beginning of the cycle (Day 2) using FSH (Puregon®, Organon, Vienna, Austria). After 5–6 days of stimulation, a GnRH-antagonist (Orgalutran®; Organon, Vienna, Austria) was administered when the leading follicle reached 12–13 mm.
In other cases, a long protocol (n = 182; mean age: 33.6 ± 4.9) was used and down-regulation was achieved with the GnRH agonist Suprecur® (Aventis Pharma, Vienna, Austria). After an ultrasound scan as well as a blood analysis, stimulation was initiated with human menopausal gonadotrophin (Menogon®, Ferring, Kiel, Germany). In another two patients, oocytes were collected in a natural cycle.
In all cycles, ovulation was induced with 10 000 IU human chorionic gonadotrophin (hCG, Pregnyl®, Organon). Oocyte retrieval was carried out transvaginally under ultrasound guidance 36 h after hCG driven ovulation induction (based on hormonal parameters and follicular size).
Collected oocyte–cumulus complexes were incubated for 2–3 h (BM1 medium, NMS Bio-Medical, Praroman, Switzerland) prior to ICSI. Brief exposure of oocytes (1 min) to 80 IU/ml hyaluronidase (MediCult, Copenhagen, Denmark) facilitated mechanical removal of the cumulus cells in order to ensure adequate handling and scoring of the gametes (approximately 10–20 cumulus cells were left attached). Special care was taken not to deform oocytes during the denudation process; thus, glass pipettes of a larger diameter were used (approximately 180–200 µm). Consequently, the vast majority of elongated gametes was presumably generated during follicular growth.
Prior to injection, oocytes were screened for morphological anomalies. In the case of larger vacuoles and aggregations of the smooth endoplasmic reticulum, ova were not used for fertilization. Study oocytes (appearing ovoid) were documented appropriately using an imaging and archival software (Octax Eyeware®, MTG, Altdorf, Germany). For all elongated gametes, a roundness Index (RI, length divided by width) was calculated in order to quantify shape (Richter et al., 2001). Two indices were actually estimated to analyze whether the whole oocyte was affected (Fig. 1a), i.e. an ovoid ooplasm and ZP, or whether only the ZP was of ovoid shape (Fig. 1b) with the ooplasm being round (with an RI of 1). Special care was taken to detect split inner layers of the ZP that might keep the ooplasm in a round shape whereas the ZP appeared ovoid (Fig. 1b). In order to account for potential measuring inaccuracies (±1 µm), all oocytes showing an RI of up to 1.03 were considered to be of round shape.
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ICSI was performed as previously described with the first polar body being held at the 6-o' clock position (Ebner et al., 2001
). However, ovoid gametes at metaphase II (MII) could not always be injected at this site since adequate fixation by means of the holding pipette could not be guaranteed if the first polar body was located on the long side (n = 2). From the moment of ICSI, elongated oocytes were kept in individual culture in order to follow their actual cleavage behavior. Round oocytes were cultured in groups (at least 10 µl medium per conceptus) since no difference in outcome was reported for groups when compared with singleton culture (Rijnders and Jansen, 1999). In the morning of Day 1 (16–18 h after insemination), the oocytes were checked for regular fertilization, characterized by two pronuclei and two polar bodies.
The following day (Day 2), the number and size of blastomeres, and degree of fragmentation and multinucleation were checked along with the actual cleavage pattern. In detail, it was determined if the ovoid embryo cleaved as expected (Fig. 2a), ie. giving a crosswise arrangement of four cells with three blastomeres lying side-by-side (Edwards et al., 1970), or if the ovoid ZP failed to exert its shape forming function, resulting in rather flat arrangement of the blastomeres (Fig. 2b). If only one cleavage step had occurred (n = 24; two-cell embryo) on the morning of Day 2, we waited until the four-cell stage was reached in the afternoon, prior to scoring the type of cleavage.
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On Day 3, conventional criteria were applied (number of blastomeres, fragmentation, multinucleation), and on Day 4, we searched for signs of compaction. Blastocyst formation and blastocyst quality according to Gardner and Schoolcraft (1999)
were checked on Days 5 and 6 (Fig. 3), respectively. It has to be mentioned that if not transferred on Day 3 (n = 15), all ovoid embryos were cultured up to the blastocyst stage. If blastocyst quality was promising, these concepti were vitrified for further usage, along with the blastocysts derived from round oocytes.
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Our media of choice (changed daily) were BM1 medium on Day 0, Blastassist System Medium 1 (MediCult) until Day 3 and Blastassist System Medium 2 in the case of extended culture to Day 5.
We tried to not transfer embryos or blastocysts derived from ovoid gametes; however, in cases where no other concepti were available, ovoid embryos were considered for transfer (n = 15).
Statistical comparisons were performed using the 
2- and t-test. Statistical significance was defined as P < 0.05.
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Results |
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A total of 94 cycles, representing 17.9% of all ICSI attempts within the study period (January 2006 to December 2006), showed at least one ovoid MII-gamete (mean ± SD: 1.46 ± 0.73; range 1–4). Within this group of cycles containing ovoid oocytes, 16.6% (137/827) of all mature oocytes showed anomalies in shape. This percentage decreased to 3.9% if MII-gametes of all study patients were considered (137/3524).
Table I shows that the majority of demographic and stimulation data were not different between the cycles with or without elongated oocytes (P > 0.05). Out of the 137 ovoid oocytes, ooplasm deformation was involved in 
60% (n = 82) with a mean RI of 1.16 ± 0.14 µm. The mean RI of the corresponding ZP was 1.29 ± 0.14 µm. There were 55 gametes which were considered round (RI between 1 and 1.03); however, their ZP were found to be ovoid (RI of 1.19 ± 0.09 µm). Splitting of the inner zona layer was observed in 19 cases (13.9% of all ovoid ova).
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In terms of fertilizability, no correlation could be found between degree of shape anomaly, as expressed as RI of ooplasm and/or ZP, and rate of fertilization (P > 0.05). Overall, 102 of 137 ovoid oocytes were fertilized correctly (74.5%), matching well with the fertilization rate (75.9%) of their sibling counterparts of round shape (524/690).
Some 97% of all ovoid fertilized oocytes cleaved on Day 2 (99/102) allowing for proper identification of the cleavage pattern (Table II). There were 60 ovoid embryos (60.6%) which showed regular cleavage (like a tetrahedron), whereas the remaining 39 four-cell embryos showed irregularities in cleavage (four or five contact points). The RI of the ZP (P = 0.00125) but not of the ooplasm (P = 0.24) was associated with cleavage pattern. In detail, oocytes showed comparable RI (1.09 ± 0.11 µm vs. 1.12 ± 0.15 µm) in oocytes that cleaved normally and abnormally, whereas the ZP differed significantly (1.21 ± 0.09 vs. 1.29 ± 0.16 µm).
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Table II indicates that in cycles positive for elongated gametes, sibling oocytes of spherical shape showed more regular cleavage patterns than did the ovoid oocytes (P < 0.001). However, if the number of contact points is ignored, the ovoid gametes showed the same cleavage behavior (P > 0.05) as their round counterparts (Table II).
In terms of blastomere number and degree of fragmentation, no relationship to any RI value was noticed on Days 2 and 3 (P > 0.05). The same was true for embryo quality and cleavage pattern (P > 0.05).
Since 15 ovoid embryos were transferred on day 3, only 84 dysmorphic ones were considered for blastocyst culture. On day 4, a total of 30 embryos had at least started to compact (35.7%). This was slightly, but not significantly, different (P = 0.13) from sibling embryos derived from round oocytes (182/408 or 44.6% with signs of compaction). Compaction on the fourth day of development was not related to any RI value (P > 0.05), but it was highly correlated to cleavage pattern (P = 0.0026).
On Day 5, only 15/84 (17.9%) ovoid embryos reached blastocyst stage when compared with 128/408 (31.4%) in sibling round embryos (P = 0.013). A possible relationship between blastocyst formation and RI did not reach statistical significance (P = 0.09), but once more, cleavage pattern on Day 2 was predictive of further development (P = 0.0008). In more detail, regularly cleaved ovoid four-cell embryos had a higher blastocyst formation rate (15/52) on Day 5 when compared with irregular ones of abnormal shape (0/32). The latter group showed a 12.5% blastulation rate on Day 6 (4/32).
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Discussion |
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In primordial follicles, oocytes are surrounded by a monolayer of granulosa cells. As the follicles develop, the granulosa cells multiply and establish extensive processes toward the oocytes. In the cleft between the two ZP, an acellular glycoprotein layer forms. Despite considerable speculation about the origin of this shell (15–20 µm), there is evidence that all zona proteins are synthesized by the oocyte in a coordinate manner (Epifano et al., 1995
). Experiments in a mouse model have led to the conclusion that ZP in mammalian oocytes consists of three zona proteins. In detail, filaments are constructed of repeating zona protein 2 and 3 units which are cross-linked by zona protein 1 (Wassarman, 1988), thus contributing to the structural integrity of the zona matrix. Recently, characterization of the genes involved has demonstrated that there are in fact four zona protein genes (Hughes and Barratt, 1999) and, consequently, four ZP glycoproteins are expressed in the human (Lefiévre et al., 2004).
Around fertilization, ZP has several functions including species-specific sperm binding (Tsubamoto et al., 1999) and prevention of polyspermy (Hoodbhoy and Dean, 2004). After fertilization, ZP assists the oviductal transport and plays a key role in protecting the integrity of the developing embryo (Herrler and Baier, 2000).
Last but not least, a certain shaping function is attributed to the outer shell of the concepti. A spherical shape of the ZP ensures maximal contact between the blastomeres of the embryo. In this event, the resulting embryo will cleave as expected, giving a crosswise arrangement of four cells with three blastomeres lying side by side (Edwards et al., 1970). An intense contact between the loosely attached blastomeres of a four-cell embryo appears beneficial in this critical period of precompaction stage, e.g. for the E-cadherin-induced cytoskeleton redistribution (Neganova et al., 2000). In addition, an increase in the number of contact points will facilitate compaction because of a larger number of tight junctions available (Ding et al., 1999).
Cleaving embryos deriving from ovoid oocytes, on the other hand, would face a reduced chance to express an optimal cell association. Moreover, theoretically, a negative correlation could exist between the degree of distortion and the number of blastomere contacts. Indeed, present data show that ovoid zonae (but not ovoid ooplasms) favor generation of atypical cleavage patterns, resulting in delayed compaction and blastocyst formation.
This scenario somewhat resembles the situation found in zona-free embryos (Suzuki et al., 1995, Ding et al., 1999; Ebner et al., 2004). In these case reports, elongated appearance of the embryo and slightly delayed development was due to a failure in the shaping function of the ZP.
In this respect, Graham and Deussen (1978) were able to show that at the second cleavage in zona-free embryos, daughter cells usually move during cleavage, so that after completion of the four-cell stage, a variety of patterns may exist and can be distinguished by the actual number of cell-to-cell contacts. Suzuki et al. (1995) found that 87% of zona-intact mouse embryos cleaved regularly (six contact points at four-cell stage) with another 13% showing five blastomere contacts. In zona-free embryos, a significant shift toward 3 (14%) and 4 (29%) cell contacts was observed. Interestingly, only 15% showed an optimal cleavage pattern without the shaping function of the ZP (Suzuki et al., 1995). These data are in line with the present findings (Table II). However, it has to be emphasized that embryos deriving from spherical oocytes may also show delayed development presumably because the cleavage pattern is suboptimal, e.g. due to specific arrangements of fragmentation that may hinder blastomere contact.
Two possible mechanisms may account for the occurrence of ovoid oocytes. First, mechanical stress during oocyte puncture and/or the denudation process could deform the oocyte. Unpublished observations from this study indicate that this unwanted occurrence causes ovoid gametes with both ooplasm and ZP being affected; however, a strong tendency toward recovery within a day was observed in these artificially damaged gametes. Thus, for the vast majority of ovoid ova, it can be assumed that the deformation is a pre-existing anomaly caused by problems having taken place in the follicle, e.g. during patterning and secretion of the zona proteins (Shen et al., 2005).
Thus, although not being essential for the development of the embryo in vitro, ZP shape and in parallel perivitelline space size may influence the maintenance of normal preimplantation development. This would definitely hold for the phenomenon of splitting of the ZP that was found to be associated with failed IVF cycles (Shen et al., 2005). Although the close-fitting inner layer of the ZP performs its shaping function in this type of ovoid oocytes (Fig. 1b), it is unclear whether such alterations in zona structure may affected hatching and/or implantation. It could be that assisted hatching might rescue some of the ovoid embryos/blastocysts in terms of implantation. In the present study, however, at least two pregnancies could be achieved with ovoid embryos deriving from oocytes showing zona splitting (with no assisted hatching applied).
It can be summarized that embryo/blastocysts from ovoid gametes can show different cleavage patterns. This is probably related to the dimension of the perivitteline space offering blastomeres enough room to move within ovoid ZP. Thus, suboptimal arrangement of the blastomeres at precompaction stages may occur and delay further development up to blastocyst. Once this stage is reached and the resulting blastocyst is of adequate quality, transfer of such concepti can be considered to be quite safe. This is supported by data from animal (Suzuki et al., 1995) and human studies (Esfandiari et al., 2005) as well as by the data presented.
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Submitted on March 28, 2007; resubmitted on June 14, 2007; accepted on August 13, 2007.
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