Developmental potential of human pronuclear zygotes in relation to their pronuclear orientation

Suresh Kattera1 and Christopher Chen

Centre for Reproductive Medicine and Gleneagles IVF Centre, Gleneagles Hospital, Singapore 258500

1 To whom correspondence should be addressed. e-mail: suresh.cccrm{at}pacific.net.sg


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Pronuclear zygote morphology has gained much attention recently due to its possible value in predicting implantation and pregnancy. The present study was performed to assess the developmental potential of zygotes with four different pronuclear orientations. METHODS: This prospective study involved 150 IVF and 190 ICSI patients seeking fertility treatment. Pronuclear zygotes were classified for orientation of the pronuclei in relation to the second polar body placed at the 6 o’clock position. Four types of pronuclear (PN) zygote were recognized, namely PN1, PN2, PN3 and PN4. The main outcome measures were early cleavage rate, quality of embryos, and implantation and pregnancy rates. RESULTS: The most common types of pronuclei orientations were PN1 (31.5%) and PN2 (29.3%), followed by PN3 (20.5%) and PN4 (18.5%). A significantly higher proportion of zygotes with PN1 and PN4 types of pronuclear orientation underwent early cleavage and developed into grade I embryos compared with other types (P < 0.0001). There was a tendency for higher implantation and pregnancy rates among patients who received embryos developed from PN1- and PN4-type oocytes, but this was not statistically significant. CONCLUSIONS: Zygotes exhibit four types of pronuclear orientation, and this is independent of the fertilizing spermatozoon or its entry point into the oocytes, whether IVF or ICSI is employed. Early cleavage was associated with PN1- and PN4-type zygotes, but implantation and pregnancy rates were not associated with pronuclear orientation. Implantation and pregnancy rates tended to be higher for embryos developed from PN1 and PN4 pronuclear zygotes. Further studies on a combination of pronuclear orientation and equality of nucleoli may provide a better guide to the implantation potential of embryos.

Key words: early cleavage/polar body/pregnancy/pronuclear orientation/zygote polarity


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In human IVF, the selection of embryos for transfer is based on their morphological appearance on day 2 or 3. Cleavage-stage embryos are allocated a morphological grade, which takes into account the appearance of the blastomeres, the degree of fragmentation, and various other visible features. However, morphological grading of embryos on day 2 or day 3 is not a good predictor of outcome as <30% of transferred embryos achieve their full development potential. The primary aim of any IVF programme is to achieve high implantation rates, without a high incidence of multiple pregnancies. To do this, better criteria for selection of embryos are needed, particularly when more than three grade I embryos are available in a cohort of embryos. One approach is to look for predictive parameters in pronuclear zygotes. With this objective, two scoring systems based mainly on the equality of nucleoli within each pronucleus have been reported to have a strong association with implantation rate (Scott and Smith, 1998Go; Tesarik and Greko, 1999Go; Scott et al., 2000Go; Tesarik et al., 2000Go).

During the study of pronuclei it was noted that pronuclear zygotes of both IVF and ICSI patients displayed four types of pronuclear orientation in relation to the second polar body (2PB). In order to assess the significance of this orientation, the development of each type of pronuclear zygote was observed in relation to early cleavage, embryo development and quality, and implantation and pregnancy rates.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
The study involved 150 IVF and 190 ICSI patients of mean age 35.4 ± 3.6 and 35.1 ± 3.4 years respectively.

Ovarian stimulation
All patients used Suprefact S.C. (buserelin 0.5 mg/day; Hoechst, Germany) for down-regulation of the pituitary, commencing from cycle day 21 and continuing up to the day of hCG (Profasi®; Serono, Switzerland) administration. When pituitary down-regulation was achieved (serum estradiol level <25 pg/ml; ovarian follicles <0.5 cm diameter), Metrodin HP 300 IU daily for 5 days (Serono) followed by 150 IU r-hFSH per day (Gonal F®; Serono/Puregon®; Organon, Netherlands) was administered until the day before hCG injection. Follicular development was monitored by ultrasound scanning and serum estradiol and progesterone levels. Patients received 10 000 IU hCG when three or more follicles measured more than 18–20 mm in diameter. Oocytes were obtained by using transvaginal ultrasound under sedation at 38 h after hCG injection.

Sperm preparation
Semen samples were produced by masturbation and collected into sterile containers. After liquefaction, samples were analysed for sperm density, motility and morphology. Sub-samples (1 ml) were placed into 6 ml tubes, overlaid with 1 ml Medicult IVF medium (Medicult, Denmark), and incubated at 37°C under 5% CO2. After 30–60 min, the supernatant was pelleted by centrifugation (600 g for 5 min), and then washed twice by re-centrifugation under identical conditions. The sperm pellet was suspended in 0.5–1.0 ml culture medium before use in oocyte insemination.

Severe oligoasthenoteratospermic samples and samples aspirated from the epididymis were washed twice by centrifugation at 1800 r.p.m. for 5 min with 2 ml culture medium. The pellet was then resuspended in 20–50 µl culture medium and added to sausage-shaped drops of culture medium under oil. Motile spermatozoa, which moved to the edge of the drop, were immobilized in polyvinylpyrrolidone (PVP; Medicult) under oil and used for microinjection. Samples aspirated from the testis were dissected with small scissors and washed twice by centrifugation at 600 g for 5 min. The pellet was then resuspended in 20–50 µl culture medium, and drops were made as described above.

Insemination
Oocytes were washed in Medicult IVF culture medium and placed in groups of four in 0.5 ml freshly equilibrated culture medium in 4-well Nunc dishes. At 40 h post-hCG, the oocytes were exposed to 50x103 motile spermatozoa for 3 h, and then checked for evidence of fertilization after a further 18–20 h. Any corona cells that still remained attached to the oocytes were removed using finely drawn Pasteur pipettes to allow assessment of fertilization.

ICSI
For the ICSI procedure, both holding and injection pipettes were obtained commercially (Humagen Fertility Diagnostics, USA), and the ICSI procedure was performed using Narashige micromanipulators (Narashige, Japan) under Hoffman modulation optics.

Immediately prior to the ICSI procedure, the sperm suspension was placed in a 10 µl droplet of 10% PVP at the 3 o’clock position. Injection of the oocytes was performed in microdroplets of Medicult IVF medium under mineral oil (International Medical, Netherlands). In the testicular samples, those spermatozoa which swam out in sausage-shaped drops under oil were picked up and placed into the PVP droplet before microinjection.

A single motile morphologically normal spermatozoon that had migrated to the 9 o’clock position was selected, immobilized by touching its tail with the injection micropipette, and then aspirated tail first into the pipette. The oocyte to be injected was secured with the holding pipette (9 o’clock position) adjacent to the polar body (6 o’clock position). The micropipette containing the spermatozoon was then inserted through the zona pellucida and the oolemma into the ooplasm at the 3 o’clock position of the oocyte. Penetration of the oolemma was confirmed by aspiration of some cytoplasm into the micropipette, and the spermatozoon was then slowly injected. The pipette was withdrawn gently and the oocyte released from the holding pipette.

Assessment of fertilization, pronuclear morphology and pronuclear zygote classification
At about 18–20 h after insemination/ICSI, oocytes were examined for the presence of pronuclei and polar bodies. Fertilization was considered normal when two clearly distinct pronuclei were present. The morphological parameters of pronuclear zygote evaluation included the size of pronuclei and their orientation in relation to the second polar body (2PB), the size and number of nucleoli, and their distribution within the nuclei.

Assessment of the above parameters was carried out by placing each pronuclear zygote into a drop of culture medium under oil. The pronuclear zygote (PN) was rotated using a holding pipette in order that it could be clearly seen by keeping the 2PB at the 6 o’clock position, and ensuring that the pronuclei did not overlap. The orientation and morphology of pronuclei was noted, and pronuclear zygotes were classified into four types as PN1 PN2, PN3 or PN4 based on pronuclear orientation. These examinations were completely non-invasive, and did not last for more than 10 s. PN1-type pronuclear zygotes had their pronuclei oriented at the 10:20 h or 3:50 h position, the PN2-type at the 1:40 h or 8:10 h position, the PN3-type at the 6:00 h or 12:30 h position, and the PN4-type at the 2:45 h or 9:15 h position (Figure 1).



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Figure 1. Classification of oocytes based on the orientation of pronuclei in relation to second polar body positioned at 6 o’clock.

 
The pronuclear zygotes were assessed further for early cleavage at 24, 48 and 72 h after oocyte retrieval, before being transferred into the uterus. Embryos for transfer were selected on the basis of day 3 morphology, and wherever possible a particular pronuclear zygote type was chosen if sufficient numbers of them were available. Embryos were transferred into the uterus using soft cook transfer catheters. Luteal support was started after oocyte retrieval in the form of progesterone i.m. (50 mg/day).

Serum hCG levels were measured in patients at 10 days after embryo transfer, and then serially to in order to detect any rise in titre. Implantation was further observed by the appearance of the gestational sac in the uterus, by use of transvaginal ultrasonography.

Data were expressed as mean ± SEM. The size, number and distribution of nucleoli were compared among the four types of pronuclear orientation, while early cleavage, embryo quality, and rates of implantation and pregnancy were compared between the four types of pronuclear zygote. The data were analysed using the chi-square test, and P < 0.05 was considered to be statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Among a total of 2090 oocytes retrieved from 190 ICSI patients, 109 (5.2%) were immature (germinal vesicles and metaphase I). Following ICSI, 1471 (74.2%) of the metaphase II oocytes were normally fertilized, with two pronuclei. Similarly, 1725 oocytes were retrieved from 150 IVF patients and, following insemination, 1243 (74.5%) were normally fertilized. Some 56 (3.2%) oocytes were still in the germinal vesicle stage. Each fertilized oocyte from both ICSI and IVF was assessed individually for orientation and size of pronuclei, as well as the number and equality of nucleoli. While classifying the pronuclear zygotes, the second polar body was distinguished from the first polar body by its intactness, absence of fragmentation and non-granularity. Pronuclear zygotes in which the first and second polar bodies were far apart were excluded from the study.

Oocytes of ICSI patients were injected with spermatozoa in the 3 o’clock position with the first polar body in the 6 o’clock position at 42 h after hCG injection. Oocytes of IVF patients were inseminated at 40 h after hCG administration, and spermatozoa could clearly enter these oocytes at any position. Subsequent comparison of ICSI and IVF oocytes showed that there was no difference between them in pronuclear orientation.

There was also no difference in the orientation of pronuclei when both IVF and ICSI patients were classified as being aged <38 or >38 years.

In oocytes fertilized by ICSI, the source of the spermatozoon, whether ejaculate, epididymis or testis, had no effect on the orientation of pronuclei.

As there were no significant differences between ICSI and IVF patients in the frequency distribution of the type of pronuclear orientation, equality of nucleoli, early cleavage rate and percentage of grade I embryos, the data were pooled for further analysis. The distribution of pronuclear orientations is detailed in Table I. The most common types of pronuclear orientations were PN1 and PN2, followed by PN3 and PN4 in both ICSI and IVF patients. A study of the size of pronuclei revealed that in 70% of the oocytes derived from both IVF and ICSI patients, the pronucleus located towards the second polar body was slightly smaller than that located further away, in 22% of cases this pronucleus was slightly larger, and in 8% of cases both pronuclei were of equal size.


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Table I. Distribution of pronuclear orientation, equality nucleoli, early cleavage, number of embryos and grade I embryos among the four types of pronuclear zygotes (PN) from ICSI and IVF patients
 
A comparison of the number, size and distribution of nucleoli showed the number of nucleoli in each pronucleus to range from 1 to 12, with variations both between and within oocytes, and fewer nucleoli in the female pronuclei (those toward the second polar body). The size of the nucleoli also varied between and within oocytes. In general, these could be allocated to three types—large, medium, and small/pinpoint. Interestingly, the nucleoli were polarized in 67% of female pronuclei, but in only 48% of male pronuclei. The male pronucleus was identified by its location, distal from the second polar body, and its slightly larger size compared with the female pronucleus, which was usually located close to the second polar body (Rawlins et al., 1988Go; Palermo et al., 1994Go). There was equality between the nucleoli in both pronuclei in 37% of the oocytes. Another interesting observation was the significantly higher (P < 0.0001) equality of nucleoli in both pronuclei in PN1-type oocytes compared with PN2, PN3 and PN4 (Table II).


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Table II. Statistical comparisons between pronuclear orientations
 
The pronuclear zygotes were observed for early cleavage in relation to their pronuclear orientation at 26 h after insemination/ICSI. The rate of cleavage ranged from 0 to 100%, the overall rates being 58% and 99% (ICSI) and 98.6% (IVF) of the zygotes cleaved into two cells by day 3 (Table III). The progression of pronuclear zygotes/embryos from day 1 to day 3 in both ICSI and IVF patients is detailed in Table III. In 10% of the zygotes, the pronuclei had disappeared when observed at 26 h, while in the remaining the pronuclei were still evident. None of the zygotes cleaved in 11 patients (six IVF, five ICSI) at 26 h, while in 20 patients (nine IVF, 11 ICSI) all the pronuclear zygotes had cleaved into two cells at 26 h.


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Table III. Progression of ICSI and IVF embryos from day 1 to day 3
 
Early cleavage was significantly higher (P < 0.0001) in PN1 and PN4 zygotes than in PN2 and PN3 zygotes (Tables I and II).

The numbers and percentages of grade I embryos for each type of pronuclear zygote are provided in Table I. A significantly higher percentage of grade I embryos was observed for the PN1- and PN4-type pronuclear zygotes than for PN2 and PN3 (P < 0.0001).

As there was no difference in the orientation of pronuclei, the data from IVF and ICSI patients were pooled to analyse implantation and pregnancy rates. Details of numbers of embryos transferred, numbers implanted and the numbers of pregnancies for each type of pronuclear zygotes are listed in Table IV. Higher implantation and pregnancy rates were observed for PN1 and PN4 zygotes than for PN2 and PN3 zygotes, though the differences were not statistically significant. A total of 77 patients had mixed-type transfers, and their data were excluded from the analysis.


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Table IV. Mean numbers of embryos transferreda, patients who received embryos from a particular type of zygote, implantation and pregnancy rates
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been reported that the plane of first cleavage corresponds with the sperm entry point in the mouse (Piotrowska and Zernicka-Goetz, 2001Go; Plusa et al., 2002Go). Hence, in the present study an investigation was made into the possible role of spermatozoa entry position being responsible for the four types of pronuclear orientation observed.

In IVF, the sperm may enter the oocyte from various directions (Pickering et al., 1988Go; Santella et al., 1992Go; Fulka et al., 1998Go). Thus, male pronuclei may orient with the female pronuclei in any direction, resulting in the four types of pronuclear orientation reported herein. In contrast, during ICSI, spermatozoa are introduced into the oocytes at a constant position—that is, at 3 o’clock, with the polar body positioned either at 6 o’clock or 12 o’clock. As the spermatozoa are placed in the centre or slightly away from the centre, it might be expected that all oocytes fertilized by ICSI would have only one type of pronuclear orientation. However, in the observations presented herein, pronuclear zygotes produced after either ICSI or IVF showed a similar frequency distribution of the four types of pronuclear orientation. This suggests that the spermatozoon entry position may not responsible for pronuclear orientation.

The molecular mechanism for such pronuclear orientation is not known. Within the oocyte, the centriole and some cytoplasm from the spermatozoon are drawn into the cytoplasm of the oocyte. The nucleus of the spermatozoon then swells and becomes the male pronucleus, and the centriole generates an array of microtubules which then make contact with a region of cortical cytoplasm that forms an anchor point from which the spindle can be oriented (Plusa et al., 2002Go). Similarly, the female pronucleus also makes contact and anchors to a point in the cytoplasm of the oocyte, but this is limited to an area around the polar bodies. Whilst the reason for the attraction of the array of microtubules of the male pronucleus to a specific point in the oocyte cytoplasm in four different directions (as observed in the present study) remains unknown, it might be hypothesized that certain microtubule chemotactic factors exist which are concentrated at a particular region of the cortical cytoplasm of the oocytes. These chemotactic factors may attract microtubules originating from the male pronucleus, and help them to anchor to this region. Thus, it appears that the orientation of pronuclei in a particular direction is predetermined by factors accumulated in a specific region of the oocyte, and is independent of sperm pronuclei. A similar controlling mechanism may operate in relation to female pronuclei.

Because the pronuclei must orient themselves parallel to the polar bodies before cleavage (Howlett and Bolton, 1985Go; Davies and Gardner, 2002Go; Gardner, 2002Go), it might be expected that there is an effect of pronuclear orientation on early cleavage. It has been suggested that the ooplasm and/or the pronuclei may rotate to orient the axis through opposed pronuclei toward the second polar body to prepare the zygote for cleavage (Edwards and Beard, 1997Go; Fulka et al., 1998Go). In accord with this suggestion, in observations reported herein, PN4 oocytes with pronuclei already oriented parallel to the polar bodies had a higher percentage of early cleavage than did PN2- and PN3-type zygotes (P < 0.0001; Tables I and II). The observation of a small number of oocytes with different orientations at hourly intervals did not reveal any change in orientation of the pronuclei before their disappearance prior to cleavage. Prior to cleavage, either the female pronucleus migrates to the male pronucleus or the male pronucleus migrates to the female, and the resulting fusion of the membrane of these pronuclei produces the diploid zygote nucleus, as observed in sea urchin eggs (Hinchcliffe et al., 1999Go). Thus, between the disappearance of the pronuclei and the onset of cleavage, the pronuclei/spindle must rotate in preparation for cleavage.

The larger male pronucleus in PN4-type zygotes is normally located at the 9 o’clock position, and in PN1-type zygotes it is located at the 10 o’clock position. The shorter angle between the male pronuclei of PN4 and PN1 zygotes seems to suggest that the pronuclei/spindle rotate in an anticlockwise manner rather than clockwise. The higher rate of early cleavage of pronuclear zygotes from PN1 and PN4 zygotes compared to zygotes from PN2 and PN3 zygotes thus supports this suggestion.

However, since more than 50% of zygotes from the PN2 and PN3 types also underwent early cleavage, orientation alone may not be responsible for the early cleavage of zygotes. It can be hypothesized again that there may be factors other than orientation which influence the incidence of early cleavage. These factors may expedite the process of pronuclear rotation in some zygotes and result in early cleavage.

The sizes of pronuclei were measured by the use of a digital camera (Nikon, Japan) and using image analysis software (Image Proplus, USA). In 70% of the oocytes the pronucleus, which was towards the second polar body, was slightly smaller, in 22% it was slightly larger, and in 8% both pronuclei were of equal size. The significance of this size variation is not known, but it is most likely dependent on both sperm and oocyte factors. This size difference did not differ among the four types of zygote. An analysis of the number, size and distribution of nucleoli in each pronucleus revealed no difference between the four types of oocytes. The nucleoli varied in size, and generally there was a decrease in number with an increase in size. It has been observed that nucleoli have a tendency rapidly to fuse and coalesce, and that this results in two or three very large nucleoli per nucleus (Gossens, 1984Go; Tesarik and Kopecny, 1989Go, 1990Go). The number of nucleoli was generally less in female pronuclei than in male pronuclei, but this may be due to asynchrony in the cytoplasmic and nuclear maturity of spermatozoa and oocytes, or that the male pronucleus not decondensing in a timely manner (Tesarik and Kopecny, 1989Go). Small/pinpoint nucleoli have been attributed to slow nuclear and cytoplasmic maturation (Scott et al., 2000Go).

The nucleoli were polarized in 67% of the female pronuclei, while 48% of male pronuclei and 37% of both pronuclei showed equality of nucleoli. This difference could be attributed to chromosomal normality/abnormality of the gametes. A significantly higher number of PN1 zygotes exhibited nucleolar equality between the nuclei (P < 0.001). Nucleoli equality was not used as a criterion to select embryos for transfer in the present study.

The embryos for transfer were chosen based on day 3 morphology and, as far as possible, on the pronuclear orientation when there were pronuclear zygotes. Higher implantation and pregnancy rates resulted from the transfer of embryos that developed from PN1 and PN4 pronuclear zygotes than from the transfer of embryos that developed from PN2 and PN3 pronuclear zygotes, but the difference was not statistically significant (Table IV). Thus, PN1- and PN4-type pronuclear zygotes had significant advantages over PN2- and PN3-type pronuclear zygotes during embryo development, and there was a non-significant trend towards higher implantation and pregnancy rates when compared with PN2 and PN3 pronuclear zygotes. In order to identify clearly those embryos which have a higher potential for implantation, it may be necessary to take into consideration additional parameters such as size, number and equality of nucleoli, as has been reported by others (Scott and Smith, 1998Go; Tesarik and Greko, 1999Go; Scott et al., 2000Go; Tesarik et al., 2000Go).

Hence, the following conclusions can be drawn from the present study: (i) Pronuclear orientation may be independent of the fertilizing spermatozoon or its entry point into the oocyte; (ii) although the majority of pronuclear zygotes show PN1 and PN2 types of orientation of the pronuclei, PN1- and PN4-type zygotes resulted in a significantly higher rate of early cleavage and development to grade I embryos; and (iii) embryos from PN1 and PN4 oocytes showed a non-significant trend towards higher implantation and pregnancy rates than embryos from PN2 and PN3 oocytes.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 15, 2003; resubmitted on September 11, 2003; accepted on October 9, 2003.