1 Laboratoire d'Eylau, 55 rue Saint-Didier, 75116 Paris, France, 2 MAR & Gen, Molecular Assisted Reproduction and Genetics, Gracia 36, 18002 Granada, Spain, 3 University of Granada, Campus Fuentenueva, 18004 Granada, Spain and 4 European Hospital, Via Portuense 700, Rome, Italy
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Abstract |
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Key words: embryo/paternal effects/preimplantation development/pronuclear stage/zygote
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Introduction |
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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.
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Materials and methods |
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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: 64.2 ± 8.1%). 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 <5x105/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 16, and the male partners from the couples who shared donated oocytes with these couples are referred to as patients sharing with 16 (Table I).
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Patients
The patients involved in this study were patients 16, 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 16. The oocyte-sharing couples were never the same in the two subsequent attempts, nor did a couple sharing oocytes with one of the patients 16 in the 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 16 over the two successive assisted reproduction attempts. All patients showed abnormalities of basic sperm parameters (Table I). None of the patients 16 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 2225 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 10 000 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 14, 4 mg on days 58 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 BesinsIscovesco, Paris, France) was started, beginning with a daily dose of 200 mg which 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 36 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% CO2 in air. Fertilization was assessed 1618 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 4850 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 1618 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 15 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 morphology-grading 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 14 and their donor-sharing counterparts was evaluated by 2 test using StatView II statistical package (Abacus Concepts, Berkeley, California, USA).
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Results |
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Discussion |
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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 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 (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. 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 spermoocyte 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 aster-assembling action of sperm centrosome. The size of the sperm-derived 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 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.
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Notes |
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References |
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Asch, R., Simerly, C., Ord, T. and Schatten, G. (1995) The stages at which human fertilization arrests: microtubule and chromosomal configurations in inseminated oocytes which failed to complete fertilization and development in humans. Hum. Reprod., 10, 18971906.[Abstract]
Bolton, V.N., Hawes, S.M., Taylor, C.T. and Parsons, J.H. (1989) Development of spare human preimplantation embryos in vitro: an analysis of the correlations among gross morphology, cleavage rates and development to the blastocyst. J. In vitro Fert. Embryo Transfer, 6, 3035.[ISI][Medline]
Braude, P., Bolton, V. and Moore, S. (1988) Human gene expression first occurs between the four and eight-cell stages of preimplantation development. Nature, 332, 459461.[ISI][Medline]
Comizzoli, P., Marquant-Le Guienne, B., Heyman, Y. and Renard, J.P. (2000) Onset of the first S-phase is determined by a paternal effect during the G1-phase in bovine zygotes. Biol. Reprod., 62, 16771684.
Edwards, R.G. and Beard, H.K. (1997) Oocyte polarity and cell determination in early mammalian embryos. Mol. Hum. Reprod., 3, 863905.[Abstract]
Eid, L.N., Lorton, S.P. and Parrish, J.J. (1994) Paternal influence on S-phase in the first cell cycle of the bovine embryo. Biol. Reprod., 51, 12321237.[Abstract]
Hammadeh, M.E., Al-Hassani, S., Stieber, M. et al. (1996) The effect of chromatin condensation (aniline blue staining) and morphology (strict criteria) of human sperm on fertilization, cleavage and pregnancy rates in an intracytoplasmic sperm injection programme. Hum. Reprod., 11, 24682471.[Abstract]
Harrouk, W., Khatabaksh, S., Robaire, B. and Hales, B. (2000) Paternal exposure to cyclophosphamide dysregulates the gene activation program in rat preimplantation embryos. Mol. Reprod. Dev., 57, 214223.[ISI][Medline]
Janny, L. and Menezo, Y.J.R. (1994) Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation. Mol. Reprod. Dev., 38, 3642.[ISI][Medline]
Navara, C.S., First, N.L. and Schatten, G. (1996) Phenotypic variations among paternal centrosomes expressed within the zygote as disparate microtubular lengths and sperm aster organization. Proc. Natl Acad. Sci. USA, 93, 53845388.
Oehninger, S., Chaturvedi, S., Toner, J. et al. (1998) Semen quality: is there a paternal effect on pregnancy outcome in in-vitro fertilization/intracytoplasmic sperm injection? Hum. Reprod., 13, 21612164.[Abstract]
Parinaud, J., Mieusset, R., Vieitez, G. et al. (1993) Influence of sperm parameters on embryo quality. Fertil. Steril., 60, 888892.[ISI][Medline]
Sanchez, R., Stalf, T., Khanaga, O. et al. (1996) Sperm selection methods for intracytoplasmic sperm injection (ICSI) and andrological patients. Andrology, 13, 228233.
Scott, L., Alvero, R., Leondires, M. and Miller, B. (2000) The morphology of human pronuclear embryos is positively related to blastocyst development and implantation. Hum. Reprod., 15, 23942403.
Shoukir, Y., Chardonnens, D., Campana, A. et al. (1998) Blastocyst development from supernumerary embryos after intracytoplasmic sperm injection: a paternal influence? Hum. Reprod., 13, 16321637.[Abstract]
Tesarik, J. (1998) Oscillinreopening the hunting season. Mol. Hum. Reprod., 4, 10071009.
Tesarik, J. and Kopecny, V. (1989) Nucleic acid synthesis and development of human male pronucleus. J. Reprod. Fertil., 86, 549558.[Abstract]
Tesarik, J. and Kopecny, V. (1990) Assembly of the nucleolar precursor bodies in human male pronuclei is correlated with an early RNA synthetic activity. Exp. Cell Res., 191, 153156.[ISI][Medline]
Tesarik, J. and Sousa, M. (1995) Key elements of a highly efficient intracytoplasmic sperm injection technique: Ca2+ fluxes and oocyte cytoplasmic dislocation. Fertil. Steril., 64, 770776.[ISI][Medline]
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, 13181323.
Tesarik, J., Kopecny, V., Plachot, M. and Mandelbaum, J. (1986) Activation of nucleolar and extranucleolar RNA synthesis and changes in the ribosomal content of human embryos developing in vitro. J. Reprod. Fertil., 78, 463470.[Abstract]
Tesarik, J., Sousa, M. and Testart, J. (1994) Human oocyte activation after intracytoplasmic sperm injection. Hum. Reprod., 9, 511518.[Abstract]
Tesarik, J., Junca, A.M., Hazout, A. et al. (2000) Embryos with high implantation potential after intracytoplasmic sperm injection can be recognized by a simple, non-invasive examination of pronuclear morphology. Hum. Reprod., 15, 13961399.
Vanderzwalmen, P., Bertin Segal, G., Geerts, L. et al. (1991) Sperm morphology and IVF pregnancy rate: comparison between Percoll gradient centrifugation and swim-up procedures. Hum. Reprod., 6, 581588.[Abstract]
Wittemer, C., Bettahar-Lebugle, K., Ohl, J. et al. (2000) Zygote evaluation: an efficient tool for embryo selection. Hum. Reprod., 15, 25912597.
World Health Organization (1992) WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction, 3rd edn, Cambridge University Press, Cambridge, pp. 1107.
Submitted on May 8, 2001; accepted on September 17, 2001.