Paternal effects acting during the first cell cycle of human preimplantation development after ICSI

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1 Human Reproduction Vol.17, No.1 pp , 2002 Paternal effects acting during the first cell cycle of human preimplantation development after ICSI Jan Tesarik 1,2,5, Carmen Mendoza 2,3 and Ermanno Greco 4 1 Laboratoire d Eylau, 55 rue Saint-Didier, Paris, France, 2 MAR & Gen, Molecular Assisted Reproduction and Genetics, Gracia 36, Granada, Spain, 3 University of Granada, Campus Fuentenueva, Granada, Spain and 4 European Hospital, Via Portuense 700, Rome, Italy 5 To whom correspondence should be addressed at: MAR & Gen, Gracia 36, Granada, Spain. cmendoza@ugr.es BACKGROUND: The ability of human embryos to undergo normal development has been shown previously to be subject to strong paternal (sperm-derived) effects. This study was undertaken to determine whether paternal influences on human embryo quality are detectable as early as the first cell cycle after fertilization. METHODS: The quality of zygotes and cleaving embryos resulting from sibling donor oocytes fertilized by sperm from different patients were compared in a donor oocyte-sharing programme. RESULTS: Fertilizations with sperm from certain individuals repeatedly resulted in the formation of high proportions of zygotes with abnormal pronuclear morphology that subsequently tended to cleave slowly and to show extensive fragmentation and blastomere irregularities. This phenomenon was observed with oocytes from two different donors for each of these individuals and contrasted with normal developmental performance of embryos resulting from sibling oocytes fertilized by sperm from other men with similar basic sperm characteristics. Fertilization rates were not related to these differences. CONCLUSIONS: These data point to a very early onset of paternal effects that condition human embryo development. These effects may be both of genetic (related to the minor gene activity of the male pronucleus) or epigenetic (related to the sperm-derived oocyte-activating factor or sperm centrosome) origin. Key words: embryo/paternal effects/preimplantation development/pronuclear stage/zygote Introduction It is well-known that sperm quality influences not only fertilization results, but also the subsequent embryo development. In humans, these paternal effects have been shown to affect embryo cleavage speed and morphology, the rate of invitro blastocyst formation and implantation rates after embryo transfer, both after conventional IVF and after ICSI (Vanderzwalmen et al., 1991; Parinaud et al., 1993; Janny and Menezo, 1994; Hammadeh et al., 1996; Sanchez et al., 1996; Shoukir et al., 1998). The mechanism underlying these phenomena is not known. In particular, it remains to be determined whether the paternal effects become manifest only after the major activation of embryonic gene expression, which occurs between the 4-cell and the 8-cell stage in humans (Tesarik et al., 1986; Braude et al., 1988), or whether they already appear before this important developmental milestone. In this study we made use of particular conditions created by sharing sibling oocytes coming from the same oocyte donors between different infertile couples in whom oocyte donation was indicated because of advanced maternal age or precocious menopause. In certain of these cases, the quality of embryos developing after fertilization with sperm from one of the oocyte-sharing patients was markedly inferior as compared with the sibling oocytes fertilized by sperm from another patient. Moreover, a new treatment attempt with shared donor oocytes was made in some of these cases, which made it possible to analyse embryo development after fertilization with sperm from these low-fertility patients with oocytes from two different donors, and to compare the results with the performance of sibling oocytes from the same donors fertilized with sperm from two other men. To determine the onset of the detectable paternal effect, we combined recently described non-invasive criteria for evaluation of pronuclear stage zygotes (Tesarik and Greco, 1999) with standard criteria of cleaving embryo quality, based on evaluation of cleavage speed and morphology on days 2 and 3 after fertilization. Materials and methods Study group and design In our assisted reproduction programme, we routinely determine zygote quality at the pronuclear stage by a single, non-invasive examination using criteria based on the number and distribution of nucleolar precursor bodies within the two pronuclei (Tesarik and Greco, 1999). These criteria were simplified as described previously (Tesarik et al., 2000), by grouping together individual abnormal patterns of pronuclear morphology, referred to as patterns 1 5 in the original criteria description (Tesarik and Greco, 1999), resulting in a 184 European Society of Human Reproduction and Embryology

2 Paternal effect on zygote development Table I. Basic sperm characteristics of participants of this study 1 Patient Sperm count Motility Normal 10 6 (%) forms (%) Sharing with 1 in 1st attempt Sharing with 1 in 2nd attempt Sharing with 2 in 1st attempt Sharing with 1 in 2nd attempt Sharing with 3 in 1st attempt Sharing with 3 in 2nd attempt Sharing with 4 in 1st attempt Sharing with 4 in 2nd attempt Sharing with 5 in 1st attempt Sharing with 5 in 2nd attempt Sharing with 6 in 1st attempt Sharing with 6 in 2nd attempt Patients 1 6 and male partners of other infertile couples sharing oocytes from the same donors in two successive attempts. single category referred to as poor-morphology zygotes. On the other hand, zygotes showing the normal pattern of pronuclear morphology (pattern 0 in the original criteria description) are referred to as goodmorphology zygotes in this study. The examination of pronuclei was performed h after ICSI. In 93 ICSI attempts using donor oocytes, carried out over 16 months, the overall percentage of good-morphology zygotes, calculated from all oocytes subjected to ICSI including those that failed to fertilize, was high (mean SD: %). However, there was a small sub-population of couples for which this percentage was repeatedly low. Such couples were included in this study when they fulfilled simultaneously the following criteria. Firstly, they shared oocytes from the same donor with another couple undergoing the same assisted reproduction treatment, each of the two couples being attributed at least six donor oocytes. Secondly, ICSI was used for fertilization in both of the donor oocyte-sharing couples. The choice of ICSI rather than conventional IVF was study-independent and reflected sperm quality. ICSI was used when the total number of sperm showing progressive motility after 4 h of incubation in capacitating medium was /ml or when the percentage of normal sperm forms in the original sample, according to World Health Organization criteria (World Health Organization, 1992), was 40%. Only cases allocated to the ICSI group according to these criteria are included in this study. Thirdly, the number of good-morphology zygotes in the first assisted reproduction attempt with shared donor oocytes did not exceed 25% of oocytes microinseminated. Fourthly, a second assisted reproduction attempt with shared donor oocytes, fulfilling the three criteria above, was performed within 12 months following the first attempt. After application of these inclusion criteria to the 93 treatment attempts using donor oocytes, six couples (6.5%) were retained for this study. The respective male partners are referred to as patients 1 6, and the male partners from the couples who shared donated oocytes with these couples are referred to as patients sharing with 1 6 (Table I). In the first part of this study, the yield of good- and poormorphology zygotes resulting from fertilizations of sibling oocytes with sperm from patients 1 6 and their respective oocyte-sharing counterparts are compared. In the second part, the relationship between the morphological quality of the pronuclear zygotes resulting from the shared donor oocytes and their subsequent developmental characteristics (cleavage speed and cleavage-stage embryo morphology) is analysed. Patients The patients involved in this study were patients 1 6, selected according to criteria described above, and male partners from other infertile couples who shared donor oocytes in the first and in the second treatment attempt performed for patients 1 6. The oocytesharing couples were never the same in the two subsequent attempts, nor did a couple sharing oocytes with one of the patients 1 6 inthe first attempt share oocytes with any of these six patients in the second attempt. Accordingly, a total of 12 different couples shared oocytes with patients 1 6 over the two successive assisted reproduction attempts. All patients showed abnormalities of basic sperm parameters (Table I). None of the patients 1 6 had fathered a child, whereas one of the oocyte-sharing patients (sharing with patient 3 in the second attempt) had had a child with his previous wife. Oocyte donors Oocyte donors participating in this study were healthy women aged years, without any apparent reproductive or other pathology. Ovarian stimulation Oocyte donors were stimulated either with high-purity urinary FSH (Neofertinorm; Serono, Rome, Italy) or with recombinant human FSH (Puregon; Organon, Oss, The Netherlands) after pituitary suppression with triptorelin (Decapeptyl; Ipsen Pharma, Barcelona, Spain) started in the midluteal phase. The initial dose of urinary and recombinant FSH was, respectively, 225 IU/day and 200 IU/day during the first 3 days and 150 IU/day on day 4. Beginning with day 5, the daily dose of FSH was adjusted every second day taking into account the evolution of serum estradiol concentration and the number and size of ovarian follicles determined by vaginal ultrasonography. When at least three follicles had reached a diameter of 18 mm, ovulation was induced with IU HCG (Profasi; Serono). Ultrasound-guided transvaginal follicular aspiration was performed 36 h later. Oocyte recipient synchronization The ovarian activity of oocyte recipients was suppressed by pituitary desensitization with triptorelin (Decapeptyl; Ipsen Pharma) started in the midluteal phase. When serum estradiol concentration was 45 pg/ml and there was no cystic structure of 10 mm detectable by ultrasonography in the patient s ovaries, the desensitization was considered complete, and the patient was ready for the beginning of endometrial growth stimulation. This condition was maintained until the respective oocyte donor was ready for ovarian stimulation. Accordingly, the period between the start of triptorelin treatment and embryo transfer varied between 32 and 45 days. Triptorelin was first applied in a sole i.m. injection of a long-acting preparation (Decapeptyl LP, 3.75 mg) followed, when necessary, by daily s.c. injections of a short-acting preparation of triptorelin (Decapeptyl, 0.1 mg). Stimulation of endometrial growth was started one day before the beginning of ovarian stimulation of the respective donor. It was performed by oral estradiol valerate (Progynova; Schering, Madrid, Spain) at daily doses of 2 mg on day 1 4, 4 mg on days 5 8 and 6 mg from day 9 until one day after the injection of HCG in the respective oocyte donor, when the daily dose of estradiol valerate was reduced to 2 mg. On the same day, intravaginal application of natural micronized progesterone (Utrogestan; Laboratoires Besins Iscovesco, Paris, France) was started, beginning with a daily dose of 200 mg which 185

3 J.Tesarik, C.Mendoza and E.Greco was augmented on the two following days to 400 and 600 mg respectively. The daily dose of 600 mg micronized progesterone was maintained until the pregnancy test, and in case of pregnancy this medication was prolonged through the 2 months following embryo transfer. The treatment with 2 mg estradiol valerate was maintained until the negative pregnancy test or, in case of pregnancy, until serum estradiol concentration had reached 1000 pg/ml. ICSI, embryo culture and transfer ICSI was performed 3 6 h after oocyte recovery by using previously described methods and instruments (Tesarik and Sousa, 1995). After ICSI, injected oocytes were placed individually on plastic cell culture dishes in 25 µl drops of IVF medium (Scandinavian IVF Science, Gothenborg, Sweden) covered with embryo-culture tested light mineral oil (Ovoil; Scandinavian IVF Science) and cultured at 37 C and 100% humidity under a gas phase of 5% CO 2 in air. Fertilization was assessed h after ICSI. Normal fertilization was characterized by the presence of two pronuclei and two polar bodies at this time point. Fertilized oocytes were transferred individually to freshly prepared drops of IVF medium and cultured under the same conditions for an additional h. Two to three embryos were transferred to the uterus of oocyte recipients on day 3 following ICSI. Evaluation of zygote and cleaving embryo quality Zygote quality was assessed h after ICSI using a simplified version (Tesarik et al., 2000) of previously described scoring criteria (Tesarik and Greco, 1999). Briefly, good-morphology zygotes were characterized by the following criteria: (i) the difference in the number of NPBs between both pronuclei does not exceed three; (ii) the distribution of NPBs (random versus polarized) is the same in both pronuclei; and (iii) there are at least three NPBs in each pronucleus. Zygotes that did not fulfil these criteria, corresponding to patterns 1 5 of the original classification (Tesarik and Greco, 1999), were grouped together as poor-morphology zygotes. The quality of cleaving embryos was scored using criteria based on the cleavage speed and morphology (blastomere regularity and the volume occupied by anucleate cell fragments). The latter was quantified with the used of previously described embryo morphologygrading criteria (Bolton et al., 1989). Statistics Significance of differences in the proportion of good-morphology and poor-morphology zygotes originating from sibling oocytes microinseminated with sperm from patients 1 4 and their donor-sharing counterparts was evaluated by χ 2 test using StatView II statistical package (Abacus Concepts, Berkeley, California, USA). Results Paternal-derived differences in embryo quality detectable during the first embryonic cell cycle Out of the six cases in which 25% donor oocytes developed into good-morphology zygotes (see inclusion criteria in the Study design section of Materials and methods), the percentage of good-morphology zygotes resulting from the repeated treatment attempt using oocytes from a different donor was never 29% (Table II). Over the two attempts it ranged from 0 29% with a mean value of 16%. In contrast, the percentage of good-morphology zygotes resulting from oocytes from the same donors but microinseminated with sperm from the donorsharing patients ranged from 50 88% with a mean value of %. The overall percentages of good-morphology and poormorphology zygotes resulting from the two successive treatment attempts were significantly different (P 0.001) for patients 1 6 and for the 12 patients who shared oocytes from the same donors with them (Table II). Interestingly, this difference was not caused by a reduced fertilizing ability of sperm from patients 1 4, because both these patients and their donor-sharing counterparts achieved similar and high fertilization rates of 81 and 83% respectively. Relationship between zygote and embryo quality The poor embryo quality, detected as early as the first cell cycle after fertilization, continued to be manifest during subsequent embryo development over the following 2 days. In fact, embryos developing from the good-morphology zygotes cleaved faster (Figure 1) and showed better morphology (Figure 2) as compared with those developing from the poormorphology zygotes, irrespective of the source of sperm used for fertilization Paternal effects on pregnancy outcomes The first treatment attempt failed to establish a clinical pregnancy in any of the patients 1 6. The second attempt resulted in a singleton clinical pregnancy in two of these patients (patients 2 and 6). The overall implantation rate calculated for the two treatment attempts in patients 1 6 was 5% (two implantations out of 40 embryos transferred). In contrast, three of the donor-sharing patients achieved a clinical pregnancy (two singleton and one twin) in the first attempt and four (three singleton and one twin) in the second attempt, with an overall implantation rate of 29% (nine implantations out of 31 embryos transferred). This difference was statistically significant (P 0.05). Discussion Various studies have reported observations suggesting the existence of paternal (sperm-derived) effects on human embryo quality (Vanderzwalmen et al., 1991; Parinaud et al., 1993; Janny and Menezo, 1994; Hammadeh et al., 1996; Sanchez et al., 1996; Shoukir et al., 1998). However, previous reports only dealt with the capacity of human embryos to develop to the blastocyst stage or to implant after transfer to the mother s uterus. To determine the stage of embryo development at which the paternal effects begin to be manifest, we analysed individual embryos as early as the first cell cycle after fertilization by applying recently established criteria based on the evaluation of pronuclear morphology (Tesarik and Greco, 1999; Tesarik et al., 2000). With the use of these criteria, this study shows unequivocally that paternally-derived problems of embryo cleavage during preimplantation development can be traced back to a very early post-fertilization period, before the occurrence of the first cleavage division. The sequential evaluation of embryos during the first and subsequent cell cycles after fertilization showed a strong linkage between abnormalities detected during the first cell cycle and the impairment of further development. In this regard, our data are in agreement with previous reports

4 Paternal effect on zygote development Table II. Zygote development from donor oocytes microinseminated with sperm from patients 1 6 and from sibling oocytes from the same donors microinseminated with sperm from donor-sharing patients No. (%) of good- and poor-morphology zygotes from microinseminated oocytes First attempt Second attempt Total Patient Oocytes Good zygotes Poor zygotes Oocytes Good zygotes Poor zygotes Oocytes Good zygotes Poor zygotes (14) 5 (71) 8 2 (25) 5 (63) 15 3 (20) 10 (67) Sharing with (67) 1 (17) 8 6 (75) 1 (13) (71) 2 (14) (20) 6 (60) 7 1 (14) 4 (57) 17 3 (18) 10 (59) Sharing with (60) 1 (10) 8 4 (50) 2 (25) (56) 3 (17) (25) 4 (50) 7 1 (14) 5 (71) 15 3 (20) 9 (60) Sharing with (88) 1 (13) 7 4 (57) 2 (29) (73) 3 (20) (22) 5 (56) 7 2 (29) 5 (71) 16 4 (25) 10 (63) Sharing with (50) 3 (38) 7 6 (86) 0 (0) (67) 3 (20) (13) 4 (50) 8 0 (0) 6 (75) 16 1 (6) a 10 (63) Sharing with (67) 2 (33) 7 4 (57) 1 (14) 13 8 (62) a 3 (23) (0) 5 (83) 7 1 (14) 5 (71) 13 1 (8) a 10 (77) Sharing with (50) 3 (38) 7 6 (86) 0 (0) (67) a 3 (20) Total (17) b 29 (60) a 44 7 (16) b 30 (68) c (16) c 59 (64) c Total sharing (64) b 9 (20) a (67) b 6 (14) c (66) c 15 (17) c a,b,c Differences between patients 1 6 and their respective donor-sharing patients are significant ( a P 0.05; b P 0.01; c P 0.001). Figure 1. Distribution of embryos with different numbers of blastomeres on days 2 and 3 after ICSI, developing from zygotes showing good and poor morphology at the pronuclear stage. (A) Patients 1 6. (B) Donor-sharing patients. Statistically significant differences between percentages of embryos with a given blastomere number developing from the good-morphology and poor-morphology zygotes are indicated above the corresponding column pairs (*P 0.05; **P 0.01). showing that embryos developing from zygotes with pronuclear abnormalities are at a higher risk of developmental arrest, blastomere multinucleation and fragmentation (Tesarik and Greco, 1999; Scott et al., 2000; Wittemer et al., 2000) and have a lower chance of achieving the blastocyst stage in vitro Figure 2. Distribution of embryos with different morphology grade on days 2 and 3 after ICSI, developing from zygotes showing good and poor morphology at the pronuclear stage. (A) Patients 1 6. (B) Donor-sharing patients. Statistically significant differences between percentages of embryos with a given morphology grade developing from the good-morphology and poor-morphology zygotes are indicated above the corresponding column pairs (*P 0.05; **P 0.01). (Scott et al., 2000) and of implanting in the uterus after transfer (Scott et al., 2000; Tesarik et al., 2000; Wittemer et al., 2000) as compared with zygotes with normal-appearing pronuclei. However, this is the first study demonstrating that this impaired developmental potential can be transmitted exclusively by the fertilizing spermatozoon without any maternal contribution. 187

5 J.Tesarik, C.Mendoza and E.Greco These observations do not exclude, of course, that in other conditions, similar developmental abnormalities can be related to poor oocyte quality. The distinction between morphological manifestations of paternally and maternally derived anomalies of human preimplantation development is a challenge for further research and may enable, in the future, a substantial improvement of treatment choices and counselling for infertile couples with repeated assisted reproduction failures. The early manifestation of paternal influences on human preimplantation development raises the question of the underlying molecular mechanisms. The use of ICSI for the production of all embryos involved in this study excludes the implication of factors intervening with sperm oocyte interactions, other than those occurring after sperm penetration into the oocyte cytoplasm. Interestingly, fertilization rates achieved with sperm that subsequently transmitted a paternal developmental disadvantage to the embryo were not lower as compared with those giving rise to embryos with normal developmental potential. In rat preimplantation embryos, paternal drug exposure temporally and spatially dysregulates embryonic gene activation, altering the developmental clock (Harrouk et al., 2000). However, the major activation of the human embryonic genome occurs only between the 4-cell and 8-cell stages of preimplantation development (Tesarik et al., 1986; Braude et al., 1988), which is much later than the first manifestation of the paternal effects on early human embryo development. Defective expression of paternally derived genes whose transcription is activated during this period can thus be excluded as the primary cause of negative paternal effects on the early human embryo development. However, a weak transcriptional activity has been detected in human male pronuclei (Tesarik and Kopecny, 1989; Ao et al., 1994), and this early transcriptional activity is crucial for nucleolar development (Tesarik and Kopecny, 1990). It remains to be determined whether an early transcriptional failure of the male pronucleus, leading to a lag of male pronuclear development behind that of the female pronucleus, is at the origin of the observed paternal-derived developmental impairment. In addition to the possible implication of early-transcripted paternal genes, the early paternal effects on human embryo development may also be caused by epigenetic factors. One such sperm-derived factor, whose molecular nature is still under debate (Tesarik, 1998), is responsible for oocyte activation and acts by promoting a particular, oscillatory pattern of calcium signalling. Abnormal calcium signalling patterns can lead to abortive oocyte activation and a failure of pronuclear development (Tesarik et al., 1994). Another developmentally important epigenetic mechanism is responsible for the asterassembling action of sperm centrosome. The size of the spermderived aster in the cytoplasm of freshly fertilized bovine oocytes has been shown to be correlated with male fertility (Navara et al., 1996). The sperm-derived aster is responsible for the proper apposition of both pronuclei (Asch et al., 1995) which, in its turn, is necessary for further spatial rearrangements and polarization of the pronuclear structure (Edwards and Beard, 1997). It remains to be determined whether one or several of the above factors are implicated in the paternal 188 effects that are responsible for impaired preimplantation development of human embryos. Although the early manifestation of paternally derived effects on preimplantation development is described here for the first time for the human species, these observations are in agreement with recent findings obtained with animal embryos. For instance, the onset and the duration of the first S-phase is determined by a paternal effect in bovine zygotes (Eid et al., 1994; Comizzoli et al., 2000). On the other hand, the consistently poor embryo quality after fertilization with sperm from certain individuals does not appear to be related to any of the basic sperm parameters, in agreement with a previous study in which no differences in pregnancy, implantation or miscarriage rates were found between patients with oligoasthenoteratozoospermia treated by ICSI and normozoospermic patients treated by conventional IVF in an oocyte donation programme (Oehninger et al., 1998). In conclusion, the present data show that fertilization with sperm from certain individuals consistently leads to the formation of embryos with developmental abnormalities which are detectable as early as the first cell cycle following fertilization. This sperm deficiency does not appear to be related to any of the conventional parameters of sperm quality and does not lead to a decrease of fertilization rates after ICSI. Further research is needed to determine the mechanism of this impaired developmental performance and to design pertinent diagnostic procedures and treatment strategies. References Ao, A., Erickson, R.P., Winston, R.M.L. and Handyside, A.H. (1994) Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage. Zygote, 2, Asch, R., Simerly, C., Ord, T. and Schatten, G. 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