1 Centre for Reproductive Medicine, Walsgrave Hospital, Coventry, CV2 2DX and 2 Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK
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Abstract |
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Key words: embryo morphology/ICSI/orientation/polar bodies/pronuclear orientation
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Introduction |
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In human oocytes, a differential distribution of leptin and STAT 3 to the cortical region at the side of the oocyte containing the animal pole has been described (Antczak and Van Blerkom, 1997). These and other similarly polarized molecules (e.g. VEGF, c-kit, EGF-R, TGFß2, Bcl-x and Bax) persist into the early stages of embryo cleavage, when regular mitotic cell divisions produce blastomeres having different allocations of the polarized molecules (Antczak and Van Blerkom, 1999
). To date, no surface markers have been localized specifically to the vegetal pole. In mammals, the functional importance of polarity is not yet fully understood; however, oocyte polarization might be an early manifestation of gradients providing positional information to cells throughout fetal growth. In humans, it has been proposed that fragmentation may disturb preimplantation development by selectively removing polarized molecules from specific regions of the cleaving embryo, resulting in variable effects upon their subsequent capacity for normal implantation and growth (Almeida and Bolton, 1996
; Antczak and Van Blerkom, 1999
).
The mechanisms involved in establishing oocyte polarity, such as oocyte maturation, the site of spermatozoon entry (Roegiers et al., 1995) and ooplasmic movements (Payne et al., 1997
), also relate to the control of cleavage within the early embryo. Variations in the plane of cleavage of a polarized blastomere may result in equal or unequal proportions of polarized molecules in the daughters and may provide an important basis for subsequent development. After the first cleavage division, which bisects the animal and vegetal poles in humans, mice and other mammals, the polarity of one cell of the two-cell embryo rotates with respect to the other so that by the end of the second cleavage division (4-cell stage) the blastomeres are situated in a characteristic crosswise orientation, as described in the rabbit (Gulyas, 1975
). In humans (Payne et al., 1997
) a relationship was shown between embryo morphology and the periodicity of the `cytoplasmic wave' observed prior to extrusion of the second polar body in oocytes fertilizing after intracytoplasmic sperm injection (ICSI). They noted that the second polar body was often extruded some distance away from the first, as has been remarked upon in mice (Gardner, 1997
), possibly indicating some degree of cytoplasmic rotation. It has been suggested (Edwards and Beard, 1997
; Fulka et al., 1998
) that the pronuclei may rotate within the ooplasm, directing their axes towards the second polar body, in order to achieve a standard orientation in preparation for subsequent development. This may be observed in other work (Payne et al., 1997
; Figure 2,
G and H). In theory, embryos that do not achieve an optimal pronuclear orientation may possibly exhibit cleavage anomalies, which in the course of an in-vitro fertilization (IVF) attempt may be recorded as poor morphology, uneven cleavage, or fragmentation. If it were possible to identify, at the pronuclear stage, which embryos would be prone to poor development, it might be possible to improve pregnancy rates and reduce multiple pregnancies by better embryo selection. This, together with pronuclear embryo transfer and cryopreservation, would make embryo usage more efficient (Scott and Smith, 1998
; Edwards and Beard, 1999
).
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Materials and methods |
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For IVF, ~15 000 previously washed spermatozoa were added to each droplet and incubated overnight. For ICSI, oocytes were incubated for <30 s in 80 U/ml hyaluronidase in EBSS (Scandinavian IVF Science) as above. After moving to hyaluronidase-free medium under oil, cumulus and corona cells were removed by gentle aspiration with a fine-bore glass pipette of diameter slightly larger than the oocytes. Individual oocytes were placed in 5 µl droplets of HEPES-buffered minimal essential medium (MEM, Gibco, Paisley, Scotland) containing 10% v/v albumin solution under oil and transferred to an Olympus inverted microscope equipped with a heated stage at 37°C and Hoffman modulation contrast optics. Prepared spermatozoa were incubated in a separate microdroplet under oil, or added to a 5 µl droplet of 10% polyvinylpyrrolidone (PVP) (ICSI-100, Scandinavian IVF Science). Individual motile spermatozoa, of approximately normal morphology if possible, were selected and transferred to PVP if necessary. Single spermatozoa were immobilized by mechanically damaging the tail membrane and were then injected into the centre of a mature oocyte (Palermo et al., 1992). The first polar body was maintained at 90° from the point of entry of the microinjection pipette.
About 17 h post-insemination, the oocytes were examined to assess fertilization using an inverted microscope and transferred to a fresh pre-equilibrated droplet of Earle's medium as above. Oocytes inseminated without ICSI had their cumulus cells removed by gentle pipetting with a narrow-bore pipette before the fertilization assessment. Oocytes containing two pronuclei and two polar bodies were considered normally fertilized and eligible for this study.
The orientation of the pronuclei and polar bodies was examined under Hoffman modulation optics and an instant black and white photograph was printed. Care was taken to orient the oocyte with the pronuclei in focus and the polar bodies close to the periphery. On the photograph, a line was drawn through the axis of the pronuclei and the positions of the polar bodies were marked. Manual measurements were made to the nearest 5° of the angle subtended between this line and the nearest polar body () and between the line and the furthest polar body (ß). The angle between the two polar bodies with reference to the centre of the oocyte was termed
. Where the two polar bodies were abutting,
was recorded as 0. The different appearances of these angles occasioned by the orientation of the zygote are presented diagrammatically in Figure 1
.
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This was a prospective study, with measurements of , ß and
made before the embryos cleaved. The embryo grading was performed independently of the zygote assessment by embryologists who did not have access to the measurements made on day 1. The embryos were analysed in sets, those having morphology classed as good (grades 12), medium (grades 34) or poor (grades 56), as well as a subset of zygotes whose development arrested before cleavage. Grades were combined in this way for analysis in view of the small numbers of embryos classified to the highest and lowest grades. Subsequently, the photographs of cleaved embryos were re-evaluated to gain specific assessments of cleavage regularity (classed as regular or notably irregular) and the proportion of each embryo which had fragmented (0, <20%, 2050%, >50%).
The relationship between the embryo grade and , ß, and
was assessed for all embryos, as well as ICSI embryos and IVF embryos separately, using the KruskalWallis test. These data are presented here as notched box plots of the median, 5th, 25th, 75th, and 95th centiles. Outliers are plotted individually. Notches that do not overlap indicate significance at P < 0.05. The significance of the numbers of embryos failing to cleave or having eccentrically placed pronuclei in ICSI compared with IVF was tested using
2 with Yates' correction. Significance was considered as a probability <5%.
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Results |
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The numbers of cleavage divisions achieved by day 2 in IVF and ICSI embryos are presented in Figure 2. A total of 11 zygotes failed to cleave, 10 in the ICSI group and 1 after IVF, which was a significant difference (P < 0.05), however, similar proportions of 2-cell and 34-cell embryos were present in each group. The number of cleavages could not be clearly determined in four embryos, due to the presence of large fragments, and these are recorded in the Figure as `fragmented'.
The proportions of cleaved embryos allocated to the six different morphological grades are shown in Figure 3. The median grades did not differ significantly between ICSI and IVF embryos although an apparent increase in grade 4 versus grade 3 embryos was evident after ICSI.
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Values of and ß ranged from 0 to 90°, and
from 0 to 150°. The median values of
, ß, and
were around 30°, 60°, and 20° respectively. There were no significant differences in
, ß, or
between zygotes arising from IVF or ICSI, as shown in Figure 4
.
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Discussion |
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The first hypothesis was rejected because the magnitude of the angle between the first and second polar bodies () showed no significant difference for embryos of different grades after either ICSI or IVF. There was a very large range of values for
, in agreement with Payne et al. (Payne et al., 1997
), and as noted in mouse embryos (Gardner, 1997
). The first polar body is normally extruded around the time of ovulation, or 36 h after HCG injection (Edwards 1965
; Steptoe and Edwards, 1970
) and second polar body extrusion is the first visible manifestation of fertilization, usually occurring within ~4 h from sperm injection (Payne et al., 1997
). Non-adjacent polar bodies indicate either spindle movement within the ooplasm in the few hours between releasing the first polar body and the second, or that the polar body has moved within the perivitelline space.
Unprovoked movement of the first polar body can occur over an angle of about 30° in human zygotes, but the second polar body remains stationary on the oocyte surface (personal communication, J.Van Blerkom, University of Colorado, Boulder, CO, USA). Occasional reports show that the first polar body is not always a reliable indicator of spindle location, with ICSI resulting in sperm injection into the oocyte chromosomes in 45% of oocytes failing to fertilize (Flaherty et al., 1995), although other data are contradictory (Asada et al., 1995
, in hamsters). In mice, the location of the second polar body is regulated with respect to blastocyst formation, usually lying centrally (away from the embryonic and abembryonic poles), and in the same plane of focus when the blastocyst is viewed microscopically in its favoured orientation (i.e. because the blastocyst is slightly oval, its maximum diameter is normally visible by microscopy; Gardner, 1997
). In mice, the second polar body is tethered to the oocyte by residual cytoplasm, which must be disrupted before movement is possible (Gardner, 1997
). A similar feature has been observed in human oocytes subjected to second polar body biopsy (personal communication, Yury Verlinski, Reproductive Genetics Institute, Chicago, IL, USA). Polar body movement might have been provoked in our study by cumulus removal, although this procedure was performed as gently as possible. Oocytes for ICSI had their cumulus cells removed when only the first polar body was present (MII), and so no effect upon the second would be anticipated. In contrast, IVF zygotes had extruded both polar bodies by the time of cumulus removal. Nevertheless, the same unusual profile of
was observed in both IVF and ICSI zygotes (Figure 7
) which would not have been expected had substantial polar body movement occurred.
The alternative possibility of spindle movement within the ooplasm may also be considered. Nuclear movement is an intrinsic part of maturation and fertilization: the germinal vesicle migrates peripherally (Longo and Chen, 1985), the spermatozoon progresses through the ooplasm and pronuclei migrate together (Van Blerkom et al., 1995
). Without the presence of a spermatozoon, a female pronucleus moves randomly around the ooplasm (Payne et al., 1997
) and in ageing unfertilized oocytes, centripetal drift of the spindle occurs (Eichenlaub-Ritter et al., 1988
). However, whilst spindle movement is possible, video evidence suggests that the spindle remains in a fixed position despite cytoplasmic `waves' circling the cortex at a regular frequency in advance of second polar body extrusion (Payne et al., 1997
). The reason for the marked variability in polar body positions which we have observed is therefore unclear and cannot be explained at present; however, the unusual distribution of
in both IVF and ICSI zygotes may suggest different populations of oocytes. Edwards and Beard remark that some oocytes `almost seem to be fixed at right angles to the polar plane' (Edwards and Beard, 1997
), which is consistent with the subgroup of zygotes which we describe, having angle
of ~70°.
In our study, and ß were used arbitrarily to represent the angles subtended by the polar bodies closest to and furthest from the axis of the pronuclei respectively. Their values ranged from 0 to 90°. It has been shown (Payne et al., 1997
) that in 92% of zygotes, the female pronucleus was nearest to the second polar body, supporting earlier data (Tang et al., 1994
). The sequence of events at fertilization, which they described for ICSI oocytes, shows that the male pronucleus forms in the centre of the ooplasm and the female pronucleus subsequently moves from the point of its formation near the second polar body, towards the male pronucleus in the centre of the ooplasm. In ICSI, therefore, it is likely that the polar body nearest to the axis of the pronuclei is the second polar body, i.e. assessed by angle
.
In the case of IVF, spermatozoa can enter the cytoplasm from any direction (Pickering et al., 1988; Santella et al., 1992
), and the relationship between the pronuclear axis and the polar bodies is likely to be less clear. It has been shown (Van Blerkom et al., 1995
) that, after IVF, spermatozoa penetrate rapidly through the ooplasm towards the spindle, with pronuclear formation occurring close to the spindle if sperm penetration occurred close by, or in the centre of the oocyte if penetration was remote from the spindle.
It has been suggested (Edwards and Beard, 1997; Fulka et al., 1998
) that the ooplasm and/or the pronuclei may rotate to orient the axis through apposed pronuclei towards the second polar body. This might correct anomalies in polarity, which may arise from fertilization by spermatozoa approaching from various directions (Fulka et al., 1998
) and prepare the zygote for subsequent cleavage. The sperm centrosome may control the plane of the first mitotic cleavage and therefore the orientation and polarity of the male pronucleus may be more critical than that of the female (see Edwards and Beard, 1999
).
In our observations, the magnitude of did not relate significantly to embryo grade, but ß increased significantly with decreasing quality of embryos in the ICSI group (P < 0.05) and the total group (P < 0.01). This provides support for the second hypothesis (i.e. that the orientation of pronuclei relative to the polar bodies may relate to the grade of the embryo) but the range of angles observed was very wide. It is possible that angle ß relates to the first polar body, as discussed above. Hence, in ICSI embryos particularly, ß might reflect the degree of cytoplasmic or pronuclear rotation which has occurred since oocyte maturation. The larger the deviation from pronuclear alignment with the polar body, the greater the degree of cytoplasmic turbulence, which in theory might predispose to cleavage anomalies reflected in an embryo's morphological `grade'.
This might also be a matter of timing, since embryos which cleave rapidly after ICSI have a higher developmental potential (Sakkas et al., 1998). However, our data showed no significant difference between ICSI or IVF embryos in the magnitude of
or ß, despite pronuclear development and cleavage being expected to occur about 4 h sooner in ICSI than IVF embryos (Nagy et al., 1998
). Hypothesis (iii), that the placement of a spermatozoon by ICSI in a fixed plane relative to the first polar body may result in an altered pronuclear/polar body orientation relative to IVF, was therefore rejected. Nevertheless, other differences between IVF and ICSI zygotes were observed, notably, different proportions with cleavage failure and the tendency for eccentric pronuclear location in the ooplasm after IVF.
The higher proportion of zygotes having eccentric pronuclei after IVF compared with ICSI, and the greater chances of these undergoing irregular cleavage or cleavage failure suggest an increased uniformity of fertilization introduced by ICSI injections. An influence of the site of sperm deposition in ICSI upon embryo development was previously described (Nagy et al., 1995), although other possibilities, such as an effect of cumulus cell removal, cannot be excluded.
The increased incidence of cleavage failure after ICSI which we describe has been observed by others and relates principally to problems with sperm decondensation, sperm ejection from the oocyte or oocyte activation (Flaherty et al., 1995). Cleaving embryos after ICSI and IVF had a similar chance of initiating the second cleavage division by day 2; however, it is possible that a difference in developmental rate was masked by their being observed only once on day 2 (Gonzales et al., 1995
).
In conclusion, we have shown that polarized characteristics of human 2PN embryos relate to cleaving embryo quality and may be influenced in ICSI by the orientation of the sperm injection relative to the first polar body. The orientation of the polar bodies relative to the pronuclear axis relates to embryo morphology. The distance between the polar bodies does not relate to embryo morphology, but has an unusual distribution. ICSI and IVF embryos differ in the incidence of cleavage failure and pronuclear location in a central position.
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Acknowledgments |
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Notes |
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4 To whom correspondence should be addressed at: Department of Biological Sciences, University of Warwick, Coventry, CV4 8JL, UK
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References |
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Submitted on May 6, 1999; accepted on July 6, 1999.