The impact of the source of spermatozoa used for ICSI on pronuclear morphology

L.C. Demirel1,3, O. Evirgen2, K. Aydos1 and C Ünlü1

1 Center for Research in Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Ankara University and 2 Department of Histology and Embryology, Ankara University, 06590, Ankara, Turkey


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The aim of this prospective study was to find out whether the source of spermatozoa used for intracytoplasmic sperm injection (ICSI) has an impact on the morphological features of pronucleate zygotes, which make up the basis of a pronuclear scoring system for the selection of the most viable embryos for transfer. METHODS AND RESULTS: The study group consisted of 194 two pronucleate (2PN) ICSI zygotes, of which 144 originated from ejaculated (ES) and 50 from testicular spermatozoa (TS). At 18 h postinjection, 2PN zygotes were assessed for pronuclear alignment, polarity in nucleoli and cytoplasmic appearance; all of which were found to exhibit similar patterns of distribution between the ES and TS groups (P = not significant). At 25 h, the presence of first cleavage was similar for both groups; 11% of zygotes in the ES and 10% of those in the TS group underwent early cleavage (P = not significant). At 48 h, a quality score was obtained for cleaving embryos by multiplying the number of blastomeres with the grade of the embryo. Pronuclear scoring in both groups of spermatozoa correlated with embryo quality score at 48 h postinjection. There was a trend for a higher incidence of early cleavage and a lower incidence of pronuclear arrest with better pronuclear scoring embryos for both types of spermatozoa. CONCLUSION: The morphological features of pronucleate zygotes at 18 h after microinjection with ES and TS are similar to each other.

Key words: ICSI/pronuclear morphology/spermatozoa


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In recent years, there has been growing interest in the assessment of pronuclear morphology to select the most viable and competent embryos. It has been shown that pronuclear morphology assessment correlates well with implantation and pregnancy rates (Scott and Smith, 1998Go; Tesarik and Greco, 1999Go).

In contrast to the common policy of transferring embryos obtained after IVF to the uterus on day 2 after oocyte retrieval, the option of transfer at the pronuclear stage has been adopted by some centres and has proved to yield comparable pregnancy rates (Ahuja et al., 1985Go; Smith et al., 1993Go; Scott and Smith, 1998Go). Data have also accumulated to show a correlation between pronuclear embryo morphology and implantation and delivery rates (Scott and Smith, 1998Go; Tesarik and Greco, 1999Go).

In this regard, IVF-generated human pronucleate zygotes were scored on the basis of pronuclear alignment, polarity of nucleoli, cytoplasmic heterogeneity, and cortical and peri-pronuclear halos, along with the presence of early first cleavage (Scott and Smith, 1998Go). This scoring system correlated positively with the implantation and delivery rates in pronuclear stage transfers. Therefore it seems likely that the selection of competent and viable embryos at a stage as early as pronuclear development, when paternal and maternal genomes are still physically separated, is possible. This raises the question of whether blastocyst culture and pronucleate zygote scoring are equally effective in selecting the most viable embryos. If so, this would obviate the need for extended culturing periods as in blastocyst stage transfers.

In this prospective study, we tried to find out whether the source of spermatozoa used for intracytoplasmic sperm injection (ICSI) has an impact on the morphological features of pronucleate zygotes, which make up the basis of a pronuclear scoring system for the selection of the most viable embryos for transfer.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study group consisted of 194 two pronucleate (2PN) zygotes of which 144 originated from ICSI with ejaculated (ES) and 50 from ICSI with testicular spermatozoa (TS) in consecutive IVF cycles for male factor infertility. The indications for microinjection were ES with very poor sperm parameters, previous poor or failed fertilization with conventional IVF and non-obstructive azoospermia (NOA). Very poor sperm parameters were defined as a sperm count of <1x106/ml or a recovery rate of <0.5x106 motile spermatozoa, and the proportion of morphologically normal spermatozoa according to Kruger's strict criteria being <5%. Poor fertilization with conventional IVF cycles denotes a rate of <25% fertilization. None of the couples had more than one cycle of treatment in this study.

Ovarian stimulation
For ovarian stimulation, 150–300 IU human menopausal gonadotrophin (HMG, Menogon, Ferring, Kiel, Germany) injections were started daily after verification of pituitary suppression with the gonadotrophin-releasing hormone agonist triptorelin (Decapeptyl 0.5–0.1 mg/day, Ferring, Kiel, Germany) which was started daily on day 21 of the menstrual cycle preceding the treatment cycle. The criteria for pituitary down-regulation were <50 pg/ml serum oestradiol level and <6 mm endometrial thickness on transvaginal sonography. The dosage of HMG injections were individualized after day 5 according to ultrasonographic and hormonal follow-ups of the follicular growth. Once at least two follicles >18 mm in diameter were achieved, 10 000 IU of human chorionic gonadotrophin (Choragon, Ferring, Kiel, Germany) were administered and transvaginal ultrasound guided oocyte recovery was scheduled for 36 h later.

Preparation of spermatozoa
Ejaculated spermatozoa were prepared early on the morning of oocyte retrieval with the mini swim-up method (Rabe et al., 1997Go). After liquefaction, the ejaculate was diluted with Earle's balanced salt solution (EBSS) supplemented with 10% human serum albumin (HSA) (Sigma Chemical Co., St Louis, MO, USA) in 1:1 (v/v) and centrifuged at 550 g for 5 min. The pellet was resuspended with 1ml EBSS in an Eppendorf tube and centrifuged. The pellet was overlaid with 50 µl of medium and spermatozoa were allowed to swim up for 60–90 min. The swim-up fraction was then recovered for the ICSI procedure.

In cases with NOA, microdissection testicular sperm extraction was performed (Schlegel, 1999Go). Briefly, a 0.5–1.5 cm measuring incision was made on an avascular region of tunica albuginea, selected with the aid of optical magnification of an operating microscope. Direct examination of the testicular parenchyma was carried out at high power magnification to identify enlarged seminiferous tubules. Small volumes of testicular samples, including these tubules, were extracted by traction and excised sharply.

Testicular tissue samples were transferred into a Falcon tube containing 1 ml spermrinse-20 medium supplemented with HSA and 0.05 mg/ml gentamicin (Scandinavian IVF Science, Sweden). The testicular tissue was then minced with the aid of two hypodermic needles in a Petri dish containing 4 ml of Spermrinse-20 medium. After the tissue had been finely shredded, the presence of spermatozoa was searched for under an inverted microscope at x320 magnification. The suspensions were then transferred to a conical centrifuge tube, vortexed for 1 min and centrifuged at 450 g through a HEPES buffered human tubal fluid containing discontinuous gradient solution (Purewash, Nidacon International AB, Sweden) in 0.3 ml 47% upper and 1.5 ml 90% lower layers for 20 min. The lower fraction under the band with debris was aspirated slowly and washed for 10 min at 450 g. The pellet was resuspended in IVF-50 medium (Scandinavian IVF Science) and stored at room temperature until the moment of injection.

ICSI procedure
At the conclusion of retrieval, oocytes were exposed to 80 IU/ml hyaluronidase (type VIII, Sigma Chemical Co., St Louis, MO, USA) in EBSS medium supplemented with 10% HSA for <30 s and mechanically denuded of their cumulus and corona radiata cells by being pipetted in and out through a flame polished Pasteur pipette in hyaluronidase free medium on the heated stage of a laminar air flowhood. Denuded oocytes were cultured for up to 3 h prior to microinjection. Only metaphase II (MII) oocytes were used for ICSI. ICSI was performed as previously described (Al-Hasani et al., 1999Go). Briefly, each MII oocyte was maintained singly in a pre-equilibrated 20 µl IVF-50 droplet under mineral oil (M-8410, Sigma) in a Petri dish (Falcon, Fahrenheit, Milton Keynes, UK) and injected with a motile spermatozoon, the tail of which had been crushed with the injection pipette in a central 10% polyvinylpyrolidine droplet (ICSI-100, Scandinavian IVF Science). The oolemma was penetrated at 3 o'clock position while positioning the polar body at 6 o'clock with the opening of the injection pipette facing downwards. Following aspiration of the ooplasm into the injection pipette, the spermatozoon was deposited into the cytoplasm. The whole procedure was performed on the heated stage of an inverted microscope (Diaphot, Nikon Corporation, Tokyo, Japan) at x200 magnification using a phase contrast optic system equipped with micromanipulators (Narishige, Tokyo, Japan). The oocytes were then checked for signs of degeneration and cultured singly in 20 µl microdrops of IVF-50 under mineral oil at 37°C in a humidified atmosphere with 5% CO2.

Evaluation of pronuclear morphology
At 18 h postinjection, normally fertilized, two pronucleus-harbouring zygotes were assessed by one of the authors (L.C.D.) on the heated stage of the inverted microscope at x400 magnification with Hoffman modulation contrast optics. L.C.D. was blinded to the source of spermatozoa used and the clinical characteristics of the female patient. In accordance with the scoring system suggested previously (Scott and Smith, 1998Go), 1 to 5 points were given for the following morphological features: (i) pronuclear alignment: if both pronuclei were aligned closely or met each other so that they appear flattened at the interphase, a score of 5 points was given. If they were far from each other or varied greatly in size, a score of 1 point was given. (ii) polarity in nucleoli: if nucleoli within their respective pronuclei were grouped longitudinally in a row at the pronuclear junction, the distribution was considered as polarized and a score of 5 points was given. If they were in the process of alignment but not yet completed, a score of 4 points was awarded. If they were randomly distributed within the pronuclei, the score was 3 points. (iii) cytoplasmic appearance: a score of 5 points was given for an heterogeneous cytoplasm with a clear cortical and/or a peri-pronuclear halo. A clear, homogeneous, pitted and/or darkened cytoplasm received 3 points. A total score was calculated for each embryo by adding up all the points received.

To enable a complete assessment of both pronuclei and the cytoplasm, occasional focus adjustments were required during the evaluation. In the next step, evaluated 2PN zygotes were placed singly in 20 µl G1 medium culture drops under mineral oil to enable individual follow-up of embryo development. At 25 h postinjection, the presence of cleavage to 2-cell stage was checked for. Embryos that achieved cleavage by this time point were designated as early cleaving embryos.

At 48 h postinjection, a quality score was obtained for cleaving embryos by multiplying the number of blastomeres with the grade of the embryo. Embryos with equal sized blastomeres and no fragmentation were graded as 4; those with asymmetrical blastomeres and/or <10% cytoplasmic fragmentation as 3; those with 10–50% fragmentation as 2 and those with >50% fragmentation as 1. The embryo grading was performed by the author O.E., who had no prior knowledge about the results of pronuclear morphology assessment. Zygotes that failed to pass beyond the pronuclear stage at subsequent observations were regarded as developmentally arrested.

Statistical analysis
Data are expressed as mean ± SD. For comparison of means, Student's t-test or Mann–Whitney U-test were used. One way analysis of varience with Duncan's test was used when >2 groups were compared. For comparison of differences between percentages, {chi}2 test was used. To correlate pronuclear scoring with embryo quality score at 48 h, Spearman's correlation analysis was employed. Statistical significance was set at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mean age of the women, length of stimulation cycle, number of HMG ampoules used and fertilization rates were similar in ICSI cycles with ES and TS (Table IGo).


View this table:
[in this window]
[in a new window]
 
Table I. The characteristics of intracytoplasmic sperm injection cycles with ejaculated and testicular spermatozoa
 
Pronuclei were in close apposition in 70.6% of all zygotes examined at 18 h after microinjection, while in 29.4% male and female pronuclei were either well separated or exhibited size discrepancy. At this time point, polarization of nucleoli was evident in 38.1% of zygotes examined. In the rest, nucleoli were either beginning to align at the pronuclear junction (37.6%) or were randomly distributed within the pronuclei (24.2%). As for the cytoplasmic appearance, heterogeneous cytoplasm with a cortical and/or peri-pronuclear halo was observed in 88.7% of 2PN zygotes. At 25 h postinjection, 10.8% of all 2PN zygotes had completed their first cleavage. At the pronuclear stage, 11.3% of zygotes were developmentally arrested.

When the morphological features of pronucleate zygotes at 18 h were compared with regard to the source of fertilizing spermatozoa, i.e. ES versus TS, the pronuclear alignment, polarity of nucleoli and cytoplasmic appearance were found to exhibit similar patterns of distribution (Table IIGo).


View this table:
[in this window]
[in a new window]
 
Table II. The analysis of the morphological features of two pronucleate oocytes at 18 h postinjection with respect to the source of spermatozoa used for intracytoplasmic sperm injection
 
The early cleavage rate of zygotes at 25 h postinjection did not differ between ES and TS groups (11.1 and 10% respectively, P = not significant). Similarly, the percentage of zygotes developmentally arrested at pronuclear stage was no different in ES and TS groups (10.4 and 14.0% respectively, P = not significant).

Despite the fact that a consistent statistical significance could not be outlined, a greater proportion of high PN morphology-scoring zygotes underwent early cleavage as compared with low PN scoring zygotes for both ES and TS groups. The only exception to this trend was for cytoplasmic appearance in the TS group, where heterogeneous cytoplasm with a clear cortical halo ended up with a lower incidence of early cleavage (Table IIIGo). Similarly, developmental arrest at pronuclear stage was more frequently encountered if the pronuclear morphological features had received lower scores in both groups of spermatozoa (data not shown).


View this table:
[in this window]
[in a new window]
 
Table III. The percentage of early cleavage at 25 h postinjection with regard to pronuclear morphological features in different types of spermatozoa used for intracytoplasmic sperm injection
 
The mean ± SD embryo quality score at 48 h was similar for the ES and TS groups (9.5 ± 6.1 versus 7.4 ± 5.1, P = not significant). The mean ± SD pronuclear score was also similar between ES and TS groups (12.7 ± 2.4 versus 12.5 ± 2.7 respectively). Pronuclear scoring in both groups correlated positively with embryo quality score at 48 h postinjection.

When the effects of individual pronuclear morphological parameters and early cleavage on embryo quality at 48 h were evaluated, close apposition of pronuclei and early cleavage proved to predict better quality embryos for the ES group, while polarity of nucleoli and cytoplasmic appearance failed to yield a correlation with embryo quality. Although statistically insignificant, there was a higher embryo quality score when pronuclei were closely aligned in the TS group (Table IVGo).


View this table:
[in this window]
[in a new window]
 
Table IV. The embryo quality at 48 h postinjection with regard to individual pronuclear morphological features and early cleavage in different types of spermatozoa used for intracytoplasmic sperm injection
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In 1998, the correlation between pronuclear embryo morphology and implantation rates was retrospectively evaluated for 114 conventional IVF cycles utilizing a day 1 embryo transfer policy (Scott and Smith, 1998Go). In this study, favourable pronuclear morphological features for implantation, at 16–18 h post-insemination, were closely apposed pronuclei, nucleoli aligned at the pronuclear interface and the perinuclear condensation of the cytoplasm. These pronuclear features had previously been shown to correlate with improved pregnancy rates (Van Blerkom, 1990Go; Wright et al., 1990Go). A scoring system based on the evaluation of these pronuclear morphological features was found to correlate with the chance of achieving pregnancy (Scott and Smith, 1998Go). Similarly, the correlation between the morphological transformations of zygotes at pronuclear stage and their implantation potential after ICSI was well documented (Tesarik and Greco, 1999Go).

In an effort to clarify potential factors that may have an impact on pronuclear scoring, it was found that younger female partners had yielded higher pronuclear scores compared with older ones (Scott and Smith, 1998Go). We tried to find out whether the source of fertilizing spermatozoa had an impact on the morphological features of pronucleate zygotes with an ICSI model. ICSI serves as an excellent tool to study if an influence exists or not, since with this procedure, the site of spermatozoa entrance relative to the first polar body is kept constant and the polarized planes of oocytes are traversed similarly. This is particularly important because the site of microinjection has been shown to influence pronuclear orientation and embryo quality (Nagy et al., 1995Go; Garello et al., 1999Go). Therefore, with an ICSI model the only difference that could be observed in the sequence and timing of early events at fertilization might be attributed to the characteristics of spermatozoa. Furthermore the timing of spermatozoa–oocyte interaction with ICSI is well defined in contrast to conventional IVF, where the time required for sperm penetration through the zona is unpredictable.

After fertilization, the pronuclei display a dynamic mode of action within the cytoplasm. In an elegant study of the sequence of events during fertilization after ICSI (Payne et al., 1997Go), it was shown that after the formation of a male pronucleus in the centre of the ooplasm, the female pronucleus gradually moves from the point of its formation near the second polar body towards the male pronucleus until the two meet.

In the earlier phases of pronuclear development, the nucleoli are small, abundant and randomly distributed within the pronuclei. With time their number decreases, they become larger and align alongside the interphase of closely apposed pronuclei in a so-called polarized fashion (Tesarik and Kopecny, 1989Go; Tesarik and Greco, 1999Go). During the pronuclear growth the organelles contract from the cortex towards the centre of the oocyte leaving a clear cortical zone.

The stabilization of sperm chromatin is provided by protamine dephosphorylation at the end of spermiogenesis and by the formation of intra- and intermolecular disulphide bridges between protamines during the epididymal transit (Martin-Ponthieu et al., 1994Go). The difference between ES and TS in nuclear DNA packing may influence the timing of the fertilization process. Furthermore, the hypothetical difference in the concentrations of oocyte-activating sperm factors (Stice and Robl, 1990Go) in ES and TS may result in differences in the timing of oocyte activation.

Actually, the timing of pronuclear formation is found not to differ between the microinjection of TS and ES (Nagy et al., 1998Go). The very similar nature of second polar body extrusion and appearance of pronuclei after ICSI with either ES or TS indicates that there is actually no important influence of the spermatozoon's origin on the timing of the fertilization process (Dadoune, 1995Go; Nagy et al., 1998Go).

Pronuclear movement is regulated by the sperm astral microtubules (Longo, 1976Go; Schatten, 1994Go). Pronuclear migration and apposition is under the control of the newly formed zygotic centrosome, the oocyte's microtubule organizing region (Schatten, 1994Go). The lack of pronuclear apposition may therefore be due to a defective sperm centrosome, either the quality or quantity of which has a direct effect on the fertilization process (Schatten, 1994Go). The failure of pronuclear growth is also a common phenomenon during fertilization with immature sperm cells (Ogura et al., 1994Go; Tesarik and Mendoza, 1996Go). The failure in pronuclear migration and growth prevents normal development (Tesarik and Greco, 1999Go). A prominent size discrepancy between the two pronuclei may suggest an asynchrony between the developmental stages of the two.

In this study, mean pronuclear scores of the zygotes were similar for both types of spermatozoa used for ICSI. The individual pronuclear features displayed a similar distribution for either group of spermatozoa. Therefore, there seems to be no difference between ES and TS in terms of pronuclear morphological features at 18 h after ICSI. The correlation of pronuclear score with the embryo quality score at 48 h favours the role of pronuclear morphology assessment as an early selection tool.

Conventional IVF-generated embryos that display poor pronuclear morphology do not cleave as fast as those having the favourable characteristics described above (Scott and Smith, 1998Go). Also in this study, embryos generated by ICSI with both ES and TS had a clear trend for early cleavage at 25 h if high scoring pronuclear features were displayed. This is particularly important because embryos that cleave rapidly after ICSI have a higher developmental potential (Sakkas et al., 1998Go) and those with nuclear membrane breakdown and cleavage by 24–26 h have higher pregnancy rates (Shoukir et al., 1997Go). It was previously reported that the delayed or failed alignment of pronuclei and nucleoli was associated with delayed or failed pronuclear membrane breakdown and first cleavage (Van Blerkom, 1990Go). In a parallel fashion, low scoring pronucleate zygotes in our study were more prone to developmental arrest at this stage regardless of the source of spermatozoa used for ICSI. Pronucleate zygotes from both TS and ES origin experienced a similar proportion of developmental arrest in this study.

In a study (Nagy et al., 1994Go) which examined the kinetics of fertilization after ICSI, 11% of the fertilized oocytes had cleaved to 2-cell by 20 h postinjection which is a comparable figure with ours, whereas in another study, an early cleavage rate of 30.5% at 27 h after ICSI has been reported (Sakkas et al., 1998Go).

The nucleoli within their respective pronuclei were longitudinally grouped along the edge of the pronucleus only when the two pronuclei were closely apposed. Occasionally polarization of nucleoli could be observed in discrepant-sized pronuclei, but never in well separated pronuclei for the study as a whole. This may be due to the fact that the female pronucleus in some studies was found to be smaller than the male (Schatten, 1994Go; Dieguez et al., 1995Go; Payne et al., 1997Go), which points to a normal variation in assessment rather than a developmental asynchrony between the two.

The apposition of pronuclei at 18 h and early cleavage at 25 h were the features that predicted the emergence of good quality embryos in the case of ES. This points to the fact that, in the normal sequence of development, the nucleoli would eventually align at the pronuclear junction and become polarized once both pronuclei had adhered to each other. That is why in this study, 70.6% of all zygotes had pronuclei closely apposed at 18 h postinjection, while polarity of nucleoli was evident in only 38.1% for the study group as a whole. Close pronuclei at 18 h denote an advanced stage of development as compared with separate pronuclei which may eventually become apposed. It has been reported (Wright et al., 1990Go) that the incidence of equatorially aligned nucleoli was ~60% in firmly adhered pronuclei. In our study, this figure was 43% for ES and 58% for TS group.

It has been shown that a single, time-independent examination of the number and distribution of nucleoli in each pronucleus proves to be a good indicator of further developmental progression (Tesarik and Greco, 1999Go). By a single static observation 12–20 h after IVF or ICSI, it was possible to define a group of zygotes that have a high implantation potential. These were the zygotes with more or less the same number of nucleoli within each pronucleus, with polarization of nucleoli when their numbers are <7 or non-polarization when >7 and with nucleoli that were either polarized or non-polarized in both pronuclei. In this study, zygotes having pronuclei with prominent size discrepancy or separation either did not cleave or developed into embryos that were later consistently cleavage-arrested. In our study such zygotes displayed both developmental arrest or cleavage, though the quality of embryos were mostly poor for both types of spermatozoa.

The evaluation of cytoplasmic appearance at pronuclear stage did not correlate with the cleaving embryo quality for both types of spermatozoa used in our study. It was stated that the halo effect or perinuclear condensation of the cytoplasm was associated with high implantation rates (Scott and Smith, 1998Go). This may be due to the preferential accumulation of mitochondria around the pronuclei which act as the most metabolically active site of the developing zygote (Van Blerkom and Runner, 1984Go; Muggleton-Harris and Brown, 1988Go; Tokura et al., 1994Go). In conclusion, it would be more appropriate to evaluate such relocations by mitochondrial staining for instance, therefore this point of pronuclear morphology assessment should be considered inadequate and subjective.

Our study has certain limitations, such as the inadequacy of evaluation of a dynamic process between two groups of spermatozoa used to induce fertilization by a static time point assessment and the inadequacy of the number of parameters used to assess pronuclear morphology. For instance, pronuclear orientation relative to polar bodies might be an additional feature to study, since this orientation is believed to be a manifestation of cytoplasmic rotation which has an influence on the embryo development (Garello et al., 1999Go). It would also be interesting to compare the pronuclear morphological characteristics after fertilization with fully mature ejaculated spermatozoa and immature spermatids that have neither completed their nuclear protein transition nor acquired complete centriole function.

In conclusion, the morphological features of pronucleate zygotes at 18 and 25 h after microinjection with ejaculated and testicular spermatozoa were similar to each other.


    Notes
 
3 To whom correspondence should be addressed at: Defne Sitesi 9. Blok No: 52, 06530 Ümitköy, Ankara, Turkey. E-mail: cemdemirel{at}hotmail.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ahuja, K.K., Smith, W., Tucker, M. et al. (1985) Successful pregnancies from the transfer of pronuclear embryos in an outpatient in vitro fertilization program. Fertil. Steril., 44, 181–184.[ISI][Medline]

Al-Hasani, S., Demirel, L.C., Schöpper, B. et al. (1999) Pregnancies achieved after frozen–thawed pronuclear oocytes obtained by ICSI with spermatozoa extracted from frozen–thawed testicular tissues from non-obstructive azoospermic men. Hum. Reprod., 14, 2031–2035.[Abstract/Free Full Text]

Dadoune, J.P. (1995) The nuclear status of human sperm cells. Micron, 26, 323–345.[ISI][Medline]

Dieguez, L., Soler, C., Perez-Sanchez, F. et al. (1995) Morphometric characterization of normal and abnormal human zygotes. Hum. Reprod., 10, 2339–2342.[Abstract]

Garello, C., Baker, H., Rai, J. et al. (1999) Pronuclear orientation, polar body placement, and embryo quality after intracytoplasmic sperm injection and in-vitro fertilization: a further evidence for polarity in human oocytes? Hum. Reprod., 14, 2588–2595.[Abstract/Free Full Text]

Longo, F.J. (1976) Sperm aster in rabbit zygotes; its structure and function. J. Cell Biol., 69, 539–547.[Abstract]

Martin-Ponthieu, A., Wouters-Tyrou, D., Pudlo, B. et al. (1994) Isolation and characterization of a small putative zinc finger protein from cuttlefish epididymal sperm cells. Eur. J. Biochem., 220, 463–468.[Abstract]

Muggleton-Harris, A.L. and Brown, J.J.G. (1988) Cytoplasmic factors influence mitochondrial reorganization and resumption of cleavage during culture of early mouse embryos. Hum. Reprod., 3, 1020–1028.[Abstract]

Nagy, Z.P., Liu, J., Joris, H. et al. (1994) Time-course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection. Hum. Reprod., 9, 1743–1748.[Abstract]

Nagy, Z.P., Liu, J., Joris, H. et al. (1995) The influence of the site of sperm deposition and mode of oolemma breakage at intracytoplasmic sperm injection on fertilization and embryo development rates. Hum. Reprod., 10, 3171–3177.[Abstract]

Nagy, Z.P., Janssenswillen, C., Janssens, R. et al. (1998) Timing of oocyte activation, pronucleus formation and cleavage in humans after ICSI with testicular spermatozoa and after ICSI or in-vitro fertilization on sibling oocytes with ejaculated spermatozoa. Hum. Reprod., 13, 1606–1612.[Abstract]

Ogura, A., Matsuda, J. and Yanagimachi, R. (1994) Birth of normal young after electrofusion of mouse oocytes with round spermatids. Proc. Natl Acad. Sci. USA, 91, 7460–7462.[Abstract]

Payne, D., Flaherty, S.P., Barry, M.F. et al. (1997) Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography. Hum. Reprod., 12, 532–541.[ISI][Medline]

Rabe, T., Diedrich, K., Eberhardt, I. and Küpker, W. (1997) In vitro Fertilization. In Rabe, T., Diedrich, K. and Runnebaum, B. (eds), Manual on Assisted Reproduction. Springer-Verlag, Berlin, pp. 297.

Sakkas, D., Shoukir, Y., Chardonnens, D. et al. (1998) Early cleavage of human embryos to the two-cell stage after intracytoplasmic sperm injection as an indicator of embryo viability. Hum. Reprod., 13, 182–187.[Abstract]

Schatten, G. (1994) The centrosome and its mode of inheritance: the reduction of the centrosome during gametogenesis and its restoration during fertilization. Dev. Biol., 165, 299–335.[ISI][Medline]

Schlegel, P.N. (1999) Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum. Reprod., 14, 131–135.[Abstract/Free Full Text]

Scott, L.A. and Smith, S. (1998) The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum. Reprod., 13, 1003–1013.[Abstract]

Shoukir, Y., Campana, A., Farley, T. et al. (1997) Early cleavage of in-vitro fertilized human embryos to the 2-cell stage: a novel indicator of embryo quality and viability. Hum. Reprod., 12, 1531–1536.[Abstract]

Smith, S., Scott, L.A. and Hosid, S. (1993) Combined intrauterine triplet and ectopic pregnancy following pronuclear transfer in a patient with elevated serum progesterone during ovulation induction. J. Assist. Reprod. Genet., 10, 478–480.[ISI][Medline]

Stice, S.L. and Robl, J.M. (1990) Activation of mammalian oocytes by a factor obtained from rabbit sperm. Mol. Reprod. Dev., 25, 272–280.[ISI][Medline]

Tesarik, J. and Kopecny, V. (1989) Development of human male pronucleus: ultrastructure and timing. Gamete Res., 24, 135–149.[ISI][Medline]

Tesarik, J. and Mendoza, C. (1996) Spermatid injection into human oocytes. I. Laboratory techniques and special features of zygote development. Hum. Reprod., 11, 772–779.[Abstract]

Tesarik, J. and Greco, E. (1999) The probability of abnormal preimplantation development can be predicted by a single static observation on pronuclear stage morphology. Hum. Reprod., 14, 1318–1323.[Abstract/Free Full Text]

Tokura, T., Noda, Y., Soto, Y. et al. (1994) Sequential observation of mitochondrial distribution in mouse oocytes and embryos. J. Assist. Reprod. Genet., 10, 417–426.

Van Blerkom, J. (1990) Occurrence and developmental consequences of aberrant cellular organization in meiotically mature human oocytes after exogenous ovarian hyperstimulation. J. Electron Microsc. Tech., 16, 324–346.[ISI][Medline]

Van Blerkom, J. and Runner, M.N. (1984) Mitochondrial reorganisation during resumption of arrested meiosis in the mouse oocyte. Am. J. Anat., 171, 335–355.[ISI][Medline]

Wright, G., Wiker, S., Elsner, C. et al. (1990) Observations on the morphology of pronuclei and nucleoli in human zygotes and implications for cryopreservation. Hum. Reprod., 5, 109–115.[Abstract]

Submitted on January 30, 2001; accepted on August 10, 2001.