Pronuclear orientation, polar body placement, and embryo quality after intracytoplasmic sperm injection and in-vitro fertilization: further evidence for polarity in human oocytes?

C. Garello1,2,3, H. Baker1, J. Rai1, S. Montgomery1, P. Wilson1, C.R. Kennedy1 and G.M. Hartshorne1,2,4

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Three hypotheses were tested: (i) the distance between first and second polar bodies (PB) may relate to embryo morphology, (ii) that the orientation of pronuclei (PN) relative to PB may relate to embryo morphology, (iii) that the placement of a spermatozoon in a fixed plane relative to the first PB [intracytoplasmic sperm injection (ICSI)] may alter PN/PB orientation relative to in-vitro fertilization (IVF). A total of 251 two pronuclear (2PN) embryos (124 ICSI, 127 IVF) from 64 patients was studied. Angles were measured between the PN axis and the nearest PB ({alpha}), the furthest PB (ß), and between the two PB ({gamma}). On day 2, the morphological grades of embryos were recorded. {gamma} ranged from 0 to 150° and was not significantly different for ICSI or IVF embryos of different grades; however, an unusual distribution of {gamma} suggested different populations of oocytes. The first hypothesis was rejected. {alpha} and ß ranged from 0 to 90°: {alpha} did not relate significantly to embryo grade, but ß increased significantly with decreasing quality of ICSI embryos (P < 0.05) and the total group (P < 0.01), supporting hypothesis (ii). The difference in ß between ICSI and IVF embryos was not significant, so hypothesis (iii) was unproven. Significant differences between ICSI and IVF embryos in PN positions, irregular cleavage, and cleavage failure were noted.

Key words: embryo morphology/ICSI/orientation/polar bodies/pronuclear orientation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oocyte polarity is well known in many non-mammalian species, where the animal pole (containing nuclear material) and the vegetal pole (containing yolk) can be clearly distinguished (see Edwards and Beard, 1997Go). There is also evidence of oocyte polarity in mammals, where particular spatial arrangements of surface structures, organelles (notably mitochondria) and RNA, may vary at different maturational stages (Calarco 1995Go; Ji et al., 1996Go). The animal pole of the oocyte may be estimated by the location of the first polar body, whereas after fertilization, the second polar body marks the so-called embryonic pole (Gardner, 1997Go).

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, 1997Go). 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, 1999Go). 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, 1996Go; Antczak and Van Blerkom, 1999Go).

The mechanisms involved in establishing oocyte polarity, such as oocyte maturation, the site of spermatozoon entry (Roegiers et al., 1995Go) and ooplasmic movements (Payne et al., 1997Go), 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, 1975Go). In humans (Payne et al., 1997Go) 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, 1997Go), possibly indicating some degree of cytoplasmic rotation. It has been suggested (Edwards and Beard, 1997Go; Fulka et al., 1998Go) 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., 1997Go; Figure 2, GoG 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, 1998Go; Edwards and Beard, 1999Go).



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Figure 2. Comparison of numbers of cleavage divisions in IVF (n = 127) and ICSI (n = 120) embryos on day 2, presented as percentages.

 
During physiological fertilization or conventional IVF, the spermatozoa can enter the oocyte at almost any position; however, in ICSI, the position of the spermatozoa relative to the first polar body is approximately constant. In this study, this difference has been used as a means of assessing whether the injection of a spermatozoon in a particular manner (ICSI) may influence pronuclear orientation, polar body placement, and embryo quality in comparison with IVF. We have made observations of two-pronuclear zygotes on day 1 after insemination, and cleaved embryos on day 2, to test the hypotheses that: (i) the placement of the second polar body relative to the first relates to the morphology of the embryo, (ii) the orientation of pronuclei relative to the polar bodies relates to the morphology of the embryo, (iii) the placement of a spermatozoon in a fixed plane relative to the first polar body (ICSI) results in an altered pronuclear/polar body orientation relative to IVF.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study group comprised patients attending the Centre for Reproductive Medicine at the Walsgrave Hospitals NHS Trust, and undergoing an IVF or ICSI cycle between July and October 1997. ICSI was offered to patients in preference to IVF if their sperm count was <1x106/ml and/or the proportion of morphologically normal spermatozoa was <5% (Kruger et al., 1986Go; WHO, 1992) and/or they had undergone a previous IVF cycle with <50% fertilization. All patients were eligible for inclusion provided that they had at least one two-pronucleate (2PN) embryo on day 1 after insemination. 2PN embryos were included only if they were cultured singly, to enable individual follow-up of embryo development. Information on the patient groups is presented in Table IGo.


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Table I. Clinical details of the patients whose two-pronucleate (2PN) embryos were studied
 
Patient preparation, oocyte collection, and embryological procedures were performed using standard protocols as described below. Tissue culture reagents were pretested using a mouse `2 cell to blastocyst' biotoxicity assay. Patients received a superactive analogue of gonadotrophin-releasing hormone (GnRH; Synarel; Searle, High Wycombe, UK; 200 µg/nostril twice daily) from day 21 of a menstrual cycle, to down-regulate endogenous pituitary gonadotrophins. This was followed from day 2 of withdrawal bleeding by stimulation with follicle stimulating hormone (FSH, urinary or recombinant preparations), in an individualized dosage according to the patient's age and previous history. A single dose of human chorionic gonadotrophin (HCG; 10 000 U, Pregnyl, Organon, Cambridge, UK) was administered when at least three follicles had reached 17 mm diameter, determined by transvaginal ultrasound scanning. Oocytes were collected transvaginally under ultrasound guidance 36 h after HCG injection. The oocytes were collected into Ham's F10 medium (Gibco, Paisley, Scotland) supplemented with heparin (3000 U/l, Unihep, Leo Labs, Princes Risborough, UK) and then allocated to microdrops of ~150 µl Earle's balanced salt solution (EBSS, prepared in bulk weekly from 10x concentrate, Gibco), supplemented with sodium pyruvate (0.22 mmol/l, Sigma, Poole, Dorset, UK), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 285 mOsm, and containing 10% v/v of a 5% w/v injectable preparation of human serum albumin (Immuno, Thetford, Norfolk, UK). The drops had been pre-incubated overnight in embryo-tested Petri dishes (Falcon, Fahrenheit, Milton Keynes, UK) under paraffin oil (Scandinavian IVF Science, Gothenburg, Sweden), and pre-equilibrated at 37°C with 5% CO2 in air.

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., 1992Go). 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 ({alpha}) 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 {gamma}. Where the two polar bodies were abutting, {gamma} was recorded as 0. The different appearances of these angles occasioned by the orientation of the zygote are presented diagrammatically in Figure 1Go.



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Figure 1. Diagram showing the measurement of {alpha}, ß, and {gamma}. {alpha} is the smallest angle between a line drawn through the axis of the pronuclei and the closest polar body. ß is the smallest angle between the same line and the furthest polar body. {gamma} is the angle between the two polar bodies using the centre of the oocyte for reference. The placement of these angles may appear differently depending upon the angle of observation and the orientation of the polar bodies.

 
On the morning of day 2, the embryo was photographed again. Embryos were graded according to the standard practice of the Centre, by visual assessment of morphology, from grade 1 (excellent) to grade 6 (very poor). The criteria for embryo grading were features visible by inverted Hoffman microscopy, including degree of fragmentation, evenness of division, cell shape and orientation, membrane appearance and zona shape. Two or three embryos with the best available morphology were selected for transfer to the patient's uterus, and any remaining embryos of grades 1–3 were cryopreserved (Lasalle et al., 1985Go).

This was a prospective study, with measurements of {alpha}, ß and {gamma} 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 1–2), medium (grades 3–4) or poor (grades 5–6), 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%, 20–50%, >50%).

The relationship between the embryo grade and {alpha}, ß, and {gamma} was assessed for all embryos, as well as ICSI embryos and IVF embryos separately, using the Kruskal–Wallis 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 {chi}2 with Yates' correction. Significance was considered as a probability <5%.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study group comprised 251 embryos from 64 patients was studied, originating from 36 IVF cycles, 27 ICSI cycles, and one cycle where oocytes were shared between IVF and ICSI. There were 124 ICSI zygotes and 127 IVF zygotes, but four ICSI zygotes (from two patients) were excluded from the analysis because they were cryopreserved before cleavage. A comparison of the embryos in the IVF and ICSI groups is given in Table IIGo.


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Table II. Comparison of ICSI and IVF embryos
 
At the time of the fertilization check, all the zygotes had pronuclei which were apposed. These were eccentrically located in the ooplasm of 4/120 ICSI zygotes compared with 15/127 IVF zygotes (Table IIIGo; P < 0.05). Of the four in the ICSI group, three occurred in one patient who had five 2PN embryos in total. These three zygotes failed to cleave, although the patient had a biochemical pregnancy after transfer of the other two with central pronuclei which had cleaved to produce `good' 4-cell embryos. The fourth ICSI zygote with pronuclei located towards the periphery of the ooplasm cleaved to a `fair' regular 2-cell embryo.


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Table III. Relationship between pronuclear placement and cleavage regularity in IVF and ICSI embryos
 
In the IVF group, 7/15 (46.7%) zygotes with eccentric pronuclei cleaved to form embryos with clearly irregular sized blastomeres, compared to 22/111 (19.8%) arising from IVF zygotes with centrally located pronuclei (P < 0.05). Similar proportions of IVF and ICSI embryos with central pronuclei cleaved to irregular embryos (22/111 (19.8%) versus 13/109 (11.9%) respectively, not significantly different). These results are summarized in Table IIIGo.

The numbers of cleavage divisions achieved by day 2 in IVF and ICSI embryos are presented in Figure 2Go. 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 3–4-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 3Go. 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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Figure 3. Comparison of morphological grades allocated to cleaving embryos after IVF (n = 126) and ICSI (n = 110) on day 2, presented as percentages.

 
Data from six IVF zygotes and three ICSI zygotes were unusable for calculations of {alpha}, ß and {gamma} because the polar bodies were not both in focus in the same plane as the pronuclei. These embryos did not show any other particular distinguishing characteristics, having various relative positions of the polar bodies and a range of grades after cleavage. Thus the data set used for subsequent calculations included 121 IVF embryos and 117 ICSI embryos.

Values of {alpha} and ß ranged from 0 to 90°, and {gamma} from 0 to 150°. The median values of {alpha}, ß, and {gamma} were around 30°, 60°, and 20° respectively. There were no significant differences in {alpha}, ß, or {gamma} between zygotes arising from IVF or ICSI, as shown in Figure 4Go.



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Figure 4. Notched box plot comparing angles {alpha}, ß, and {gamma} in zygotes arising from ICSI (n = 117) or IVF (n = 121). {alpha} represents the angle between the pronuclear axis and the nearest polar body, ß represents the angle between the pronuclear axis and the furthest polar body, and {gamma} represents the angle between the first and second polar bodies (see Figure 1Go).

 
The distribution of {alpha} according to embryo morphology in the IVF and ICSI groups is shown in Figure 5Go. The magnitude of {alpha} did not relate significantly to the grade of the embryo. Nevertheless, a tendency for angle {alpha} to increase with decreasing embryo quality was more noticeable in the ICSI group. Uncleaved embryos did not differ from cleaved embryos in the magnitude of {alpha}.




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Figure 5. Notched box plots of the distribution of angle {alpha} in (a) IVF (n = 121) or (b) ICSI (n = 117) zygotes that cleaved to embryos with good, medium, and poor morphology, or which failed to cleave. {alpha} represents the angle between the pronuclear axis and the nearest polar body.

 
The distribution of ß according to embryo morphology is shown in Figure 6Go. Angle ß increased significantly (P < 0.05) with decreasing morphological quality of cleaved embryos in the ICSI group. A similar but insignificant trend was observed for the IVF embryos. This relationship was more significant when the IVF and ICSI groups were combined (P < 0.01). ß for uncleaved embryos was not significantly different from that for cleaved embryos.




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Figure 6. Notched box plots of the distribution of angle ß in (a) IVF (n = 121) or (b) ICSI (n = 117) zygotes that cleaved to embryos with good, medium, and poor morphology, or which failed to cleave. ß represents the angle between the pronuclear axis and the furthest polar body. Angle ß increased significantly (P < 0.05) with decreasing quality of cleaved embryos in the ICSI group. When all zygotes (IVF + ICSI) were considered together, a similar relationship was found (P < 0.01).

 
Values of {gamma} ranged from 0° to 150° and there was an unusual distribution, as shown in Figure 7Go. Most zygotes had {gamma} ~20–30°; however, a distinct subgroup occurred at {gamma} ~70°. This distribution was present in both the IVF and the ICSI groups. There was no significant difference in {gamma} for ICSI or IVF embryos of different grades, as shown in Figure 8Go.



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Figure 7. Frequency distribution of angle {gamma} in ICSI (––––––-; n = 117) and IVF (––––; n = 121) embryos. {gamma} represents the angle between the first and second polar bodies.

 



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Figure 8. Notched box plots of the distribution of angle {gamma} in (a) IVF (n = 121) or (b) ICSI (n = 117) zygotes that cleaved to embryos with good, medium, and poor morphology, or which failed to cleave. {gamma} represents the angle between the first and second polar bodies.

 
IVF and ICSI embryos had similar proportions of fragmentation, but the data suggested increasing {alpha} and ß with increasing fragmentation in ICSI but not IVF embryos (data not shown), with no such tendency observed for {gamma}. Since fragmentation was not independent of the morphological grading, a repeat statistical analysis was not done.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The data presented have bearing upon the possible existence of oocyte polarity in humans. We have attempted to quantify features of zygotes and to relate these to subsequent embryo development. In human IVF, cleaving embryos are usually allocated a morphological `grade' which takes into account the regularity of appearance of the embryo, its degree of fragmentation and various other visible features. On the contrary, pronuclear orientation, which may reflect cytoplasmic or pronuclear rotation relative to the polar bodies, is rarely assessed. We have measured the angle subtended by the pronuclei and each polar body as a measure of orientation. We have observed zygotes and embryos derived by both ICSI and IVF to test three hypotheses: (i) the placement of the second polar body relative to the first relates to the morphology of the embryo, (ii) that the orientation of pronuclei relative to the polar body relates to the morphology of the embryo, and (iii) that the placement of a spermatozoon in a fixed plane relative to the first polar body, using ICSI, results in an altered pronuclear/polar body orientation relative to IVF.

The first hypothesis was rejected because the magnitude of the angle between the first and second polar bodies ({gamma}) showed no significant difference for embryos of different grades after either ICSI or IVF. There was a very large range of values for {gamma}, in agreement with Payne et al. (Payne et al., 1997Go), and as noted in mouse embryos (Gardner, 1997Go). The first polar body is normally extruded around the time of ovulation, or 36 h after HCG injection (Edwards 1965Go; Steptoe and Edwards, 1970Go) and second polar body extrusion is the first visible manifestation of fertilization, usually occurring within ~4 h from sperm injection (Payne et al., 1997Go). 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 4–5% of oocytes failing to fertilize (Flaherty et al., 1995Go), although other data are contradictory (Asada et al., 1995Go, 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, 1997Go). In mice, the second polar body is tethered to the oocyte by residual cytoplasm, which must be disrupted before movement is possible (Gardner, 1997Go). 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 {gamma} was observed in both IVF and ICSI zygotes (Figure 7Go) 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, 1985Go), the spermatozoon progresses through the ooplasm and pronuclei migrate together (Van Blerkom et al., 1995Go). Without the presence of a spermatozoon, a female pronucleus moves randomly around the ooplasm (Payne et al., 1997Go) and in ageing unfertilized oocytes, centripetal drift of the spindle occurs (Eichenlaub-Ritter et al., 1988Go). 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., 1997Go). 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 {gamma} 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, 1997Go), which is consistent with the subgroup of zygotes which we describe, having angle {gamma} of ~70°.

In our study, {alpha} 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., 1997Go) that in 92% of zygotes, the female pronucleus was nearest to the second polar body, supporting earlier data (Tang et al., 1994Go). 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 {alpha}.

In the case of IVF, spermatozoa can enter the cytoplasm from any direction (Pickering et al., 1988Go; Santella et al., 1992Go), 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., 1995Go) 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, 1997Go; Fulka et al., 1998Go) 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., 1998Go) 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, 1999Go).

In our observations, the magnitude of {alpha} 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., 1998Go). However, our data showed no significant difference between ICSI or IVF embryos in the magnitude of {alpha} or ß, despite pronuclear development and cleavage being expected to occur about 4 h sooner in ICSI than IVF embryos (Nagy et al., 1998Go). 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., 1995Go), 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., 1995Go). 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., 1995Go).

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.


    Acknowledgments
 
Presented in part at the Alpha International Congress on Development of the Human Embryo in vitro, Serono Symposium, Sorrento, Italy, 31 October to 3 November 1997, and the British Fertility Society annual meeting, Newcastle, UK, 14–16 April 1999. The authors are grateful to all members of the Centre for Reproductive Medicine, Walsgrave Hospitals NHS Trust, Coventry, who participated in the patients' treatment, and to the Mason Medical Research Fund, and the Gift of a Life Appeal, which supported the purchase of the equipment used.


    Notes
 
3 Present address: LIVET, C.so Vinzaglio 4, 10131 Torino, Italy Back

4 To whom correspondence should be addressed at: Department of Biological Sciences, University of Warwick, Coventry, CV4 8JL, UK Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Almeida, P.A. and Bolton, V.N. (1996) The relationship between chromosomal abnormality in the human preimplantation embryo and development in vitro. Reprod., Fertil., Dev., 8, 235–241.[ISI][Medline]

Antczak, M. and Van Blerkom, J. (1997) Oocyte influences on early development: the regulatory proteins leptin and STAT 3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embryo. Mol. Hum. Reprod., 3, 1067–1086.[Abstract]

Antczak, M. and Van Blerkom, J. (1999) Temporal and spatial aspects of fragmentation in early human embryos: possible effects on developmental competence and association with the differential elimination of regulatory proteins from polarized domains. Hum. Reprod., 14, 429–447.[Abstract/Free Full Text]

Asada, Y., Baka, S.G., Hodgen, G.D. and Lanzendorf, S.E. (1995) Evaluation of the meiotic spindle apparatus in oocytes undergoing intracytoplasmic sperm injection. Fertil. Steril., 64, 376–381.[ISI][Medline]

Calarco, P.G. (1995) Polarisation of mitochondria in the unfertilised mouse oocyte. Dev. Genet., 16, 36–43.[ISI][Medline]

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Submitted on May 6, 1999; accepted on July 6, 1999.