Abstract. Introduction. RBMOnline - Vol 10. No Reproductive BioMedicine Online; on web 18 January 2005

Transcription

1 RBMOnline - Vol 10. No Reproductive BioMedicine Online; on web 18 January 2005 Article Paternal effects on cell division in the human preimplantation embryo Jan Tesarik obtained his MD degree in 1979 and PhD in He realized the first successful gamete intra-fallopian transfer (GIFT) (1982) and the first childbirths after oocyte fertilization with round spermatids (1995) and with in-vitro cultured spermatids from a man with meiotic maturation arrest (1998). He developed an original technique for nuclear transfer in mature human oocytes (2000). He is author or co-author of >280 scientific publications. At present he is director of MAR&Gen (Molecular Assisted Reproduction and Genetics) in Granada (Spain) and scientific consultant for the Laboratoire d Eylau (Paris, France) and the European Hospital (Rome, Italy). Dr Jan Tesarik Jan Tesarik MAR&Gen, Molecular Assisted Reproduction and Genetics, Gracia 36, Granada, Spain, Laboratoire d Eylau, 55 rue Saint Didier, Paris, France Correspondence: Fax: ; cmendoza@ugr.es Abstract Cell divisions in the human preimplantation embryo can be compromised by deficiencies in sperm nuclear genome or sperm-derived developmentally relevant cytoplasmic factors, oocyte activating substance and centriole. Sperm nuclear deficiencies are usually not detected before the 8-cell stage of embryo development, when a major expression of spermderived genes has begun. Sperm cytoplasmic deficiencies can be detected as early as the 1-cell zygote and then throughout the preimplantation development. The terms late paternal effect and early paternal effect have been suggested to denote these two pathological conditions. The late paternal effect is associated with an increased incidence of sperm DNA fragmentation. No association with sperm DNA damage has been found for the early paternal effect. The diagnosis of the late paternal effect is thus based on the examination of sperm DNA integrity, which should be performed in cases of repeated assisted reproduction failure even if morphologically normal embryos result from fertilization with the patient s spermatozoa. The only element leading to the diagnosis of the early paternal effect is poor zygote and embryo morphology and low cleavage speed. The absence of increased sperm DNA damage does not exclude the presence of this pathology. ICSI with testicular spermatozoa has recently been shown to be an efficient treatment for the late paternal effect. The use of oral antioxidant treatment in this indication has also given promising results. Keywords: cell division, paternal effect, preimplantation embryo, programmed cell death, sperm DNA fragmentation 370 Introduction With the current state of the art of assisted reproduction techniques fertilization and preimplantation development of several embryos can be achieved in most cases. However, the implantation potential of the embryos after transfer to the uterine cavity is still relatively low. Implantation and early post-implantation development can be negatively influenced by the technique of embryo transfer and a complex of locally acting factors that are responsible for uterine receptivity. Moreover, they are conditioned by the viability of each embryo transferred which, in its turn, depends on the biological quality of the spermatozoon and the oocyte at the given embryo s origin and on technical aspects of the laboratory procedures used to assist fertilization. Consequences of the actions of sperm-derived factors on preimplantation embryo development, referred to as paternal effects, have been shown to be responsible for repeated failures of assisted reproduction attempts (Vanderzwalmen et al., 1991; Parinaud et al., 1993; Janny and Menezo, 1994; Hammadeh et al., 1996; Sanchez et al., 1996; Shoukir et al., 1998). Oocyte donation programmes offer a unique opportunity to analyse this condition by making it possible to form matched patient groups sharing sibling donor oocytes and thus avoiding the bias related to superimposition of paternal and oocyte-derived effects on embryo quality. This paper reviews the data obtained with this model as well as those derived from other human and animal models. Possible mechanisms of different types of paternal effect on embryo development are also discussed. Finally, an approach to clinical management of infertility caused by paternal effects is suggested.

2 Control of cell division in the human preimplantation embryo After syngamy, leading to fusion between the paternal and the maternal genome, mitotic division of the resulting 1-cell zygote is started and embryo cleavage begins. Mitotic divisions, in general, are subjected to stringent controls at what is called cell cycle checkpoints. These are specific phases of the cell cycle at which the completeness of events that took place at previous phases of the cell cycle is checked. If an anomaly is detected, the progression of the cell cycle is withheld and reparatory mechanisms are activated; if the latter fail to restore normality, the cell enters an autodestruction pathway of programmed cell death (PCD) (Weinert, 1998; Amon, 1999; Taylor, 1999). How these checkpoint controls apply to the human preimplantation embryo has been the subject of a recent review (Fulka et al., 2000). It appears that at least certain controls are established very early in preimplantation development, but these controls are relatively leaky as evidenced by the relatively high frequency of chromosomal abnormalities in preimplantation human embryos (Fulka et al., 2000). If a major anomaly is detected in a blastomere at a cell cycle checkpoint, the cell in question does not divide, which leads to the observation of a lower number of cells in the embryo than expected for a given time point. If the existing problem cannot be resolved, the cell in question is ultimately removed by fragmentation, resulting in an impairment of embryo morphology grade according to current cleaving embryo scoring systems. In view of the recent hypothesis suggesting that trophectoderm arises from a single founder blastomere of the 4-cell embryo (Hansis and Edwards, 2003; Hansis et al., 2004), such partial blastomere losses may be compatible with embryo implantation in some cases and incompatible in others, depending on whether the trophectoderm founder cell is or is not concerned. Limited production of fragments detaching from blastomeres may also occur as part of the remodelling mechanisms involved in cellular reparatory processes (Martinez et al., 2002), which means that fragments may appear in human preimplantation embryos even if none of its blastomeres is ultimately lost. Both the slow cleavage and the poor morphology grade of cleaving embryos are thus likely to be consequences of active autodefence mechanisms employed by the embryo in its fight against aneuploidy and DNA damage in general. Respective roles of the spermatozoon and the oocyte in preimplantation development The early post-fertilization phases of mammalian development are characterized by the absence of gene expression, and the information required for the control and coordination of early developmental events is derived from a stock of maternal mrna accumulated in the oocyte during its growth and maturation (Telford et al., 1990). Expression of the embryonic genome, which is a blend of the sperm and the oocyte contribution, starts between the 4- cell and 8-cell stage of human preimplantation development (Tesarik et al., 1986, 1988; Braude et al., 1988). Eventual disruption of sperm-derived genes is thus unlikely to become manifest between fertilization and the 4-cell stage. Though transcriptionally inactive, the spermatozoon delivers two essential epigenetic contributions to the early postfertilization development, oocyte activating factor and centriol. Oocyte activating factor is responsible for specific modifications of cell components controlling oocyte intracellular calcium homeostasis (calcium pumps, calcium channels and calcium-binding proteins), resetting them in a way that generates and sustains oscillations of free intracellular calcium concentration. This particular pattern of calcium signalling is produced in mammalian oocytes shortly after fusion with the fertilizing spermatozoon, and is responsible for oocyte exit from metaphase II arrest and initiation of the mitotic cell cycle and the early developmental processes (Cuthbertson and Cobbold, 1985; Jones, 1998). At the molecular level, several sperm proteins have been suggested as candidates for oocyte activating factor, but none of them has been confirmed with certainty (Tesarik, 1998). The sperm-derived centriol is entirely responsible for the nucleation of microtubules and the function of the mitotic spindle in early human embryos, while centrosome of oocyte origin is inactivated after fertilization (Sutovsky and Schatten, 2000). Experimental work with human embryos has indeed confirmed that nuclear syngamy and the mitotic potential of the preimplantation embryo are paternally inherited (Sathananthan et al., 1996; Palermo et al., 1997). Even though it is widely held that oocyte-derived transcripts are responsible for the control of preimplantation development until the major activation of embryonic gene expression, it has been shown that the sperm-derived genome is not completely silent in the period between fertilization and the first cleavage division. In fact, RNA synthesis has been detected in human male pronuclei (Tesarik and Kopecny, 1989), and this early transcription of paternal genes has been shown to be required for the proper assembly of nucleolar precursor bodies (NPB) (Tesarik and Kopecny, 1990). Studies on mouse pronuclear zygotes have shown that ongoing rate of gene transcription is 4 5 times greater in the paternal pronucleus as compared with the maternal one (Aoki et al. 1997). This difference is supposed to be due to chromatin-mediated repression of promoter activity, which is imposed on maternal pronuclei, but not on paternal pronuclei (Van Blerkom, 1981; Nothias et al., 1995). The information about the nature of the genes that are transcribed in paternal pronuclei is limited. Chromosome Y-linked transcripts are expressed in paternal pronuclei in human zygotes (Ao et al., 1994), and the heat shock protein hsp70.1 is among the genes transcribed in 1-cell mouse zygotes (Christians et al., 1995). After the major gene activation, which begins as early as the S and G2 phases of the 2-cell stage in mouse embryos, a large array of genes, including genes involved in cell line differentiation into the inner cell mass and trophectoderm, genes controlling growth factors, cytokines, cell cell communication, general housekeeping and cytoskeletal and surface secretory functions, are expressed in this species (reviewed in Edwards and Beard, 1997). The nature of genes expressed after the major gene activation in human embryos, 371

3 372 which occurs between the 4-cell and the 8-cell stage (Tesarik et al., 1986, 1988; Braude et al., 1988) remains to be determined. Presumptive mechanisms of paternal effects on human preimplantation development A number of studies have demonstrated an elevated percentage of spermatozoa with damaged DNA in the ejaculate of infertile men as compared with healthy subjects (Filatov et al., 1999; Host et al., 2000; Larson et al., 2000; Morris et al., 2002; Tomsu et al., 2002; Benchaib et al., 2003). The pattern of DNA damage (fragmentation) observed in these cases resembles that resulting from the classical programmed cell death (PCD) pathway in somatic cells (Gandini et al., 2000). On the other hand, spermatozoa with damaged DNA lack many other markers of an active PCD process. For instance, no strict relationship has been found between sperm DNA damage and Fas, p53 or Bcl-x expression (Sakkas et al., 2002), supporting the suggestion that sperm DNA damage results from an atypical or abortive PCD pathway (Sakkas et al., 1999). Other studies have suggested the activity of the classical PCD pathway in the human seminiferous tubules (Francavilla et al., 2002; Tesarik et al., 2002b; Cayli et al., 2004). However, these activities appear to prevent abnormal germ cells from reaching the ejaculate rather than promote DNA damage in ejaculated spermatozoa, and most of the germ cells concerned are dismantled by Sertoli cells (Tesarik et al., 2004a). Thus, ejaculated sperm DNA damage has been suggested to be a sequela of oxidative damage occurring to spermatozoa after their release from Sertoli cell support (Tesarik et al., 2004a). A relationship between the proportion of spermatozoa with damaged DNA in the ejaculate and fertility has been evaluated by several recent studies (Filatov et al., 1999; Host et al., 2000; Larson et al., 2000; Morris et al., 2002; Tomsu et al., 2002; Benchaib et al., 2003). The threshold values above which fertility is seriously compromised will obviously depend on several aspects of the technique used for DNA damage detection. For instance, by using terminal deoxyribonucleotidyl transferase-mediated nick-end labelling (TUNEL), Benchaib et al. (2003) have shown that pregnancy rate after intracytoplasmic sperm injection (ICSI) is significantly reduced when the percentage of spermatozoa with damaged DNA in the ejaculate is >10%, and no pregnancy was obtained in men with >20% of spermatozoa with damaged DNA. These observations raise the question whether sperm DNA damage is the only responsible for the adverse paternal affect on the early embryonic development and, if so, what is the mechanism of this effect. To address this point, we need to know more about the period of the early post-fertilization development at which the adverse paternal effect begins to be manifest and about the cytological picture of these early manifestations. Early and late paternal effects and sperm DNA damage It has been shown previously that irregularities of cell division during human preimplantation development can sometimes be predicted as early as the the pronuclear zygote stage by examining the number and spatial distribution of NPB in the pronuclei (Tesarik and Greco, 1999; Scott et al., 2000; Wittemer et al., 2000). Moreover, the same criteria can be used to predict the chance of implantation and pregnancy (Tesarik et al., 2000; Scott, 2003) and the risk of chromosomal abnormalities in the embryo (Coskun et al., 2003; Gamiz et al., 2003; Balaban et al., 2004). Even though all of these anomalies can also be of oocyte origin, a recent study has shown clearly, by comparing developmental potential of sibling oocytes from young donors shared between couples with previous implantation failures suspected to be of paternal origin and other couples undergoing their first treatment attempt, that the paternal effect can become manifest as early as the 1-cell zygote (Tesarik et al., 2002a). This early paternal effect subsequently causes delayed cleavage divisions and increases the degree of cleaving embryo fragmentation (Tesarik et al., 2002a). It is important to note that no relationship between this early paternal effect and sperm DNA damage could be demonstrated (Tesarik et al., 2004b). On the other hand, there are also infertile couples in whom a paternal effect on embryonic development was clearly demonstrated, by comparing outcomes of treatment attempts with sibling donor oocytes, in the absence of any detectable impairment of pronuclear development, cleavage speed or cleaving embryo morphology (Tesarik et al., 2004b). Unlike the early paternal effect, this late paternal effect was positively correlated with the incidence of sperm DNA fragmentation (Tesarik et al., 2004b). These observations suggest that the early and the late paternal effect on embryo development may represent two distinct pathologies with different timing of developmental disturbances, aetiology and pathogenesis. Abnormalities related to the early paternal effect appear for the first time in the 1-cell human zygote. Thus, their first appearance coincides with the minor gene transcriptional activity previously detected at this stage (Tesarik and Kopecny, 1989, 1990; Table 1). It may be interesting to determine whether this activity is disturbed or delayed in zygotes derived from spermatozoa from men with the early paternal effect and, if so, whether eventual gene expression disturbances are the cause or a consequence of the developmental impairment. Alternatively, the early paternal effect may be caused by abnormalities of sperm centriol or of the sperm-derived oocyte activating factor. This would explain the absence of a relationship between the early paternal effect and sperm DNA damage (Table 2). The late paternal effect may be primarily caused by sperm DNA damage because an association between both phenomena has been demonstrated (Tesarik et al., 2004b). The absence of detectable zygote and early cleaving embryo abnormalities in these cases (Table 2) also supports this hypothesis because a major expression of sperm-derived genes does not occur at these early developmental stages (Table 1).

4 Table 1. Typical timing of sperm-derived genetic and epigenetic activities during preimplantation development. Time after gamete Genetic activity Epigenetic activity fusion (h) 0 12 Unknown Oocyte activation 1,2 MTOC assembly Limited gene transcription 6,7 Syngamy 8 NPB formation 7 Cell cycle activation Unknown Unknown Activation of gene transcription Unknown and translation Further genes transcribed 12 Unknown and translated References: 1 Cuthbertson and Cobbold, 1985; 2 Jones, 1998; 3 Palermo et al., 1994; 4 Sathananthan et al., 1996; 5 Sutovsky and Schatten, 2000; 6 Tesarik and Kopecny, 1989; 7 Tesarik and Kopecny, 1990; 8 Yanagimachi, 1994; 9 Tesarik et al., 1986; 10 Braude et al., 1988; 11 Tesarik et al., 1988; 12 Edwards and Beard, Table 2. Early and late adverse paternal effects on embryo development and their relationship with sperm DNA fragmentation 1. Paternal Pronuclear Cleaving Cleavage Increased effect morphology embryo speed sperm DNA morphology fragmentation Early Impaired Impaired Decreased Not detected Late Unaffected Unaffected Normal Detected 1 Compiled from Tesarik et al., 2002a and Tesarik et al., 2004b. Clinical management of infertility caused by paternal effects on preimplantation development Diagnosis Our data (Tesarik et al., 2004b) have suggested that there are at least two different mechanisms leading to what is commonly referred to as adverse paternal effect on early embryo development. For the early paternal effect, no association with any available diagnostic test result for sperm quality has been found. Consequently, the history of repeated previous assisted reproduction failures with spermatozoa from the man in question, particularly when they were associated with unexplained poor zygote and cleaving embryo morphology or a slow cleavage, should direct our attention towards this diagnosis. This suspicion is reinforced if the same kind of zygote and embryo deficiencies are detected in assisted reproduction attempts using donor oocytes. The late paternal effect, on the other hand, may easily escape attention because of the usually good morphology and cleavage speed of preimplantation embryos. However, this type of paternal effect is often associated with a high incidence of sperm DNA fragmentation (Tesarik et al., 2004b). A test of sperm DNA integrity should thus be performed in all cases of repeated assisted reproduction failure, and especially in those in which embryos fail to implant in the absence of an impairment of zygote and cleaving embryo morphology. Treatment In cases of repeated assisted reproduction failure in which the paternal effect is likely to be the cause, assisted reproduction with donor spermatozoa can be envisaged. However, this solution may not be easily acceptable by many couples. Moreover, it is not easy to decide to what extent the paternal effect lowers the couple s chance of conceiving with husband s own spermatozoa in each individual case. A relative measure of the importance of the existing reproductive disadvantage is possible in cases of the late paternal effect when the degree of the underlying sperm DNA damage can be objectively assessed. So, a recent study has suggested that ongoing pregnancies do not occur when ICSI is performed with spermatozoa from men in whom the percentage of DNA-fragmented spermatozoa in ejaculated sperm samples is >20% (Benchaib et al., 2003). However, another recent study calculated a cut-off value of 24.3% TUNEL-positive spermatozoa in the ejaculate (Henkel et al., 2003). Thus, the question of clinically relevant threshold for sperm DNA fragmentation still remains open. Interestingly, sperm DNA fragmentation appeared to have less impact on ART outcomes when conventional in-vitro 373

5 374 fertilization was used as compared with ICSI (Benchaib et al., 2003). These observations suggest that some kind of selection against spermatozoa with damaged DNA may act during sperm passage through the cumulus oophorus or the zona pellucida. If this is confirmed, the study of mechanisms responsible for this natural selection might be at the origin of the development of in-vitro sperm selection techniques with which sperm populations used for ICSI can be enriched with healthy sperm cells. The recourse to testicular spermatozoa is a new treatment option to be envisaged in cases of late paternal effect. A recent study has suggested that sperm DNA damage in patients with primary testiculopathies is mainly produced after completion of spermatogenesis, when late elongated spermatids release from Sertoli cells (Tesarik et al., 2004a). Moreover, the recovery of healthy spermatozoa from testicular biopsy samples has been shown to be facilitated after in-vitro incubation of partially dissected seminiferous tubule segments (Tesarik et al., 2001). A recent prospective controlled clinical trial has indeed demonstrated that outcomes of ICSI with testicular spermatozoa are significantly better than those of ICSI with ejaculated spermatozoa in men with 15% of DNAfragmented spermatozoa in the ejaculate (Greco et al., 2005a). It remains to be determined whether further improvement can be obtained by in-vitro incubation of testicular spermatozoa before their use in ICSI. A more conservative approach, using oral treatment with two antioxidants, vitamins C and E, has been shown recently to decrease significantly the proportion of DNA-fragmented spermatozoa in the ejaculate of men with pathologically increased sperm DNA damage (Greco et al., 2005b). The potential clinical usefulness of this treatment must be determined by further trials. As to the early paternal effect, which is not associated with the impairment of any known sperm parameter, the evaluation of clinical relevance is even more problematic. The nature of the sperm components whose damage underlies this effect and the mechanism leading to this damage are completely unknown. Further research is needed to suggest a specific treatment for this recently recognized pathology. References Amon A 1999 The spindle checkpoint. Current Opinions in Genetics and Development 9, Ao A, Erickson RP, Winston RM, Handyside AH 1994 Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage. Zygote 2, Aoki F, Worrad DM, Schultz RM 1997 Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo. Developmental Biology 181, Balaban B, Yakin K, Urman B et al Pronuclear morphology predicts embryo development and chromosome constitution. Reproductive BioMedicine Online 8, Benchaib M, Braun V, Lornage J et al Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Human Reproduction 18, Braude P, Bolton V, Moore S 1988 Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 332, Cayli S, Sakkas D, Vigue L, Demir R, Huszar G 2004 Cellular maturity and apoptosis in human sperm: creatine kinase, caspase- 3 and Bcl-XL levels in mature and diminished maturity sperm. Molecular Human Reproduction 10, Christians E, Campion E, Thompson EM, Renard JP 1995 Expression of the HSP 70.1 gene, a landmark of early zygotic activity in the mouse embryo, is restricted to the first burst of transcription. Development 121, Coskun S, Hellani A, Jaroudi K et al Nucleolar precursor body distribution in pronuclei is correlated to chromosomal abnormalities in embryos. Reproductive BioMedicine Online 7, Cuthbertson KS, Cobbold PH 1985 Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca 2+. Nature 316, Edwards RG, Beard HK 1997 Oocyte polarity and cell determination in early mammalian embryos. Molecular Human Reproduction 3, Filatov MV, Semenova EV, Vorob eva OA et al 1999 Relationship between abnormal sperm chromatin packing and IVF results. Molecular Human Reproduction 5, Francavilla S, D Abrizio P, Cordeschi G et al Fas expression correlates with human germ cell degeneration in meiotic and post-meiotic arrest of spermatogenesis. Molecular Human Reproduction 8, Fulka Jr J, Tesarik J, Loi P, Moor RM 2000 Manipulating the human embryo: cell cycle checkpoint controls. Cloning 2, 1 7. Gamiz P, Rubio C, de los Santos MJ et al 2003 The effect of pronuclear morphology on early development and chromosomal abnormalities in cleavage-stage embryos. Human Reproduction 18, Gandini L, Lombardo F, Paoli D et al 2000 Study of apoptotic DNA fragmentation in human spermatozoa. Human Reproduction 15, Greco E, Scarselli F, Iacobelli M et al. 2005a Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa. Human Reproduction 20, Greco E, Iacobelli M, Rienzi L et al. 2005b Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment. Journal of Andrology, in press. Hammadeh ME, al-hasani S, Stieber M et al The effect of chromatin condensation (aniline blue staining) and morphology (strict criteria) of human spermatozoa on fertilization, cleavage and pregnancy rates in an intracytoplasmic sperm injection programme. Human Reproduction 11, Hansis C, Edwards RG 2003 Cell differentiation in the preimplantation human embryo. Reproductive BioMedicine Online 6, Hansis C, Grifo J, Krey L 2004 Candidate lineage marker genes in human preimplantation embryos. Reproductive BioMedicine Online 8, Henkel R, Kierspel E, Hajimohammad M et al DNA fragmentation of spermatozoa and assisted reproduction technology. Reproductive BioMedicine Online 7, Host E, Lindenberg S, Smidt-Jensen S 2000 The role of DNA stand breaks in human spermatozoa used for IVF and ICSI. Acta Obstetrica et Gynecologica Scandinavica 79, Janny L, Menezo YJR 1994 Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation. Molecular Reproduction and Development 38, Jones KT 1998 Ca 2+ oscillations in the activation of the egg and development of the embryo in mammals. International Journal of Developmental Biology 42, Larson KL, DeJonge CJ, Barnes AM et al Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproduction techniques. Human Reproduction 15, Martinez F, Rienzi L, Iacobelli M et al Caspase activity in preimplantation human embryos is not associated with apoptosis. Human Reproduction 17, Morris ID, Ilott S, Dixon L, Brison DR 2002 The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development. Human Reproduction 17,

6 Nothias JY, Majumder S, Kaneko KJ, De Pamphilis ML 1995 Regulation of gene expression at the beginning of mammalian development. Journal of Biological Chemistry 270, Palermo G, Munne S, Cohen J 1994 The human zygote inherits its mitotic potential from the male gamete. Human Reproduction 9, Parinaud J, Mieusset R, Vieitez G et al 1993 Influence of sperm parameters on embryo quality. Fertility and Sterility 60, Sakkas D, Mariethoz E, St John JC 1999 Abnormal sperm parameters in humans are indicative of an abortive apoptotic mechanism linked to the Fas-mediated pathway. Experimental Cell Research 251, Sakkas D, Moffatt O, Manicardi GC et al Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biology of Reproduction 66, Sanchez R, Stalf T, Khanaga O et al Sperm selection methods for intracytoplasmic sperm injection (ICSI) and andrological patients. Journal of Assisted Reproduction and Genetics 13, Sathananthan AH, Ratnam SS, Ng SC et al The sperm centriole: its inheritance, replication and perpetuation in early human embryos. Human Reproduction 11, Scott L 2003 Pronuclear scoring as a predictor of embryo development. Reproductive BioMedicine Online 6, Scott L, Alvero R, Leondires M et al The morphology of human pronuclear embryos is positively related to blastocyst development and implantation. Human Reproduction 15, Shoukir Y, Chardonnens D, Campana A, Sakkas D 1998 Blastocyst development from supernumerary embryos after intracytoplasmic sperm injection: a paternal influence? Human Reproduction 13, Sutovsky P, Schatten G 2000 Paternal contribution to the mammalian zygote: fertilization after sperm egg fusion. International Review of Cytology 195, Taylor SS 1999 Chromosome segregation: dual control ensured fidelity. Current Biology 9, R562 R564. Telford NA, Watson AJ, Schultz GA 1990 Transition from maternal to embryonic control in early mammalian development: a comparison of several species. Molecular Reproduction and Development 26, Tesarik J 1998 Oscillin reopening the hunting season. Molecular Human Reproduction 4, Tesarik J, Greco E 1999 The probability of abnormal preimplantation development can be predicted by a single static observation on pronuclear stage morphology. Human Reproduction 14, Tesarik J, Kopecny V 1989 Nucleic acid synthesis and development of human male pronucleus. Journal of Reproduction and Fertility 86, Tesarik J, Kopecny V 1990 Assembly of the nucleolar precursor bodies in human male pronuclei is correlated with an early RNA synthetic activity. Experimental Cell Research 191, Tesarik J, Kopecny V, Plachot M, Mandelbaum J 1986 Activation of nucleolar and extranucleolar RNA synthesis and changes in the ribosomal content of human embryos developing in vitro. Journal of Reproduction and Fertility 78, Tesarik J, Kopecny V, Plachot M, Mandelbaum J 1988 Early morphological signs of embryonic genome expression in human preimplantation development as revealed by quantitative electron microscopy. Developmental Biology 128, Tesarik J, Junca AM, Hazout A et al Embryos with high implantation potential after intracytoplasmic sperm injection can be recognized by a simple, non-invasive examination of pronuclear morphology. Human Reproduction 15, Tesarik J, Greco E, Mendoza C 2001 Assisted reproduction with invitro-cultured testicular spermatozoa in cases of severe germ cell apoptosis: a pilot study. Human Reproduction 16, Tesarik J, Mendoza C, Greco E 2002a Paternal effects acting during the first cell cycle of human preimplantation development after ICSI. Human Reproduction 17, Tesarik J, Martinez F, Rienzi L et al 2002b In-vitro effects of FSH and testosterone withdrawal on caspase activation and DNA fragmentation in different cell types of human seminiferous epithelium. Human Reproduction 17, Tesarik J, Ubaldi F, Rienzi L et al. 2004a Caspase-dependent and - independent DNA fragmentation in Sertoli and germ cells from men with primary testicular failure: relationship with histological diagnosis. Human Reproduction 19, Tesarik J, Greco E, Mendoza C 2004b Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation. Human Reproduction 19, Tomsu M, Sharma V, Miller D 2002 Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters. Human Reproduction 17, Van Blerkom J 1981 Structural relationship and posttranslational modification of stage-specific proteins synthesized during early preimplantation development in the mouse. Proceedings of the National Academy of Sciences USA 78, Vanderzwalmen P, Bertin-Segal G, Geerts L et al Sperm morphology and IVF pregnancy rate: comparison between Percoll gradient centrifugation and swim-up procedures. Human Reproduction 6, Weinert T 1998 DNA damage checkpoints update: getting molecular. Current Opinion in Genetics and Development 8, Wittemer C, Bettahar-Lebugle K, Ohl J et al Zygote evaluation: an efficient tool for embryo selection. Human Reproduction 15, Yanagimachi R 1994 Mammalian fertilization. In: Knobil E, Neill JD (eds) The Physiology of Reproduction, 2nd edn, Raven Press, New York, USA, pp Paper based on contribution presented at the International Serono Symposium From the Oocyte to the Embryo: a Pathway to Life in Stresa, Milan, Italy, September 24 25, Received 2 September 2004; refereed 21 September 2004; accepted 21 December