In vitro development of human oocytes after parthenogenetic activation or intracytoplasmic sperm injection

In vitro development of human oocytes after parthenogenetic activation or intracytoplasmic sperm injection Alessio Paffoni, M.Sc.,a,b Tiziana A. L. Brevini, D.Pharm., Ph.D.,a,c Edgardo Somigliana, M.D.,a,b Liliana Restelli, M.Sc.,a,b Fulvio Gandolfi, D.V.M., Ph.D.,a,c and Guido Ragni, M.D.a,b a

Department of Obstetrics, Gynecology, and Neonatology, and b Infertility Unit, Department of Obstetrics, Gynecology, and Neonatology, Ospedale Maggiore Policlinico, Mangiagalli and Regina Elena; and c Biomedical Research Unit, Department of Anatomy of Domestic Animals, University of Milan, Milan, Italy

Objective: To compare directly in vitro developmental competence between parthenogenetically activated and intracytoplasmic sperm injection (ICSI)-fertilized oocytes. Design: For each patient, three metaphase II oocytes were randomized to the ICSI procedure, while n⫺3 were allocated to parthenogenetic activation. Setting: University hospital infertility unit. Patients: Thirty-eight patients, aged 35.2 ⫾ 3.3 years (mean ⫾ SD) selected for ICSI. Interventions: After 1 hour from denudation, oocytes were either fertilized by ICSI (n ⫽ 114) or chemically activated (n ⫽ 104). Fertilized and activated oocytes were cultured for up to 3 and 5 days, respectively. Main Outcome Measures: Development rate, cell number, and morphological grade during culture. Results: The two groups showed no significant differences between rates of fertilization and parthenogenetic activation, development, and blastomere number on days 2 and 3 of culture. However, parthenotes showed a lower morphological grade, and a significantly lower proportion went on cleaving to day 3, when only activated rather than total numbers of oocytes were considered. On day 5 after activation, nine oocytes (8.6%) reached the blastocyst stage, representing 12.9% of parthenotes. Conclusions: Since most parameters examined in this study were similar between activated and fertilized oocytes, parthenogenetic activation may be a useful tool for the preclinical evaluation of experimental procedures. (Fertil Steril威 2007;87:77– 82. ©2007 by American Society for Reproductive Medicine.) Key Words: Parthenogenesis, ICSI, in vitro development, blastocyst

Parthenogenesis is the process of embryonic development without the intervention of a spermatozoon that, in lower species, can lead to the generation of new individuals. In mammals, parthenotes (embryos obtained by parthenogenesis) can develop to different stages after oocyte activation, depending on the species, but never to term (1). In vivo, mammalian parthenogenesis is a rare event, and it is thought to be the primary cause of ovarian teratomas, i.e., tumors made of different cell types which originate from more than one germ layer (2). In vitro, spontaneous parthenogenesis in humans is rarely observed both in fresh (3) and aging (4) oocytes, or following cryopreservation (5). Parthenogenesis can be efficiently induced in vitro with a variety of mechanical, chemical, and electrical stimuli in several species of mice, rabbits, cows, pigs, goats, and primates (6). Human oocytes are amenable to parthenogenetic activation, but often this does not lead to parthenote devel-

Received December 27, 2005; revised and accepted May 31, 2006. Supported in part by Industrie Farmaceutiche Serono, Rome, Italy. Reprint requests: Guido Ragni, M.D., Infertility Unit, Department of Obstetrics, Gynecology, and Neonatology, Ospedale Maggiore Policlinico, Mangiagalli and Regina Elena, 20124 Milan, Italy (FAX: 00 39 025 503 4302; E-mail: [email protected]).

0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2006.05.063

opment beyond the eight-cell stage (7–16). The development of human parthenotes to the blastocyst stage was reported only recently (17–19). This important achievement, together with the establishment of parthenogenetic stem cells in nonhuman primates (20), has stimulated new interest in human parthenogenesis, because it suggests that it may be possible to eliminate the requirement to produce or disaggregate normal competent human embryos for deriving pluripotent cell lines in vitro (21,22). However, establishing an efficient protocol for the parthenogenetic activation of human eggs could have other important applications. In particular, it would provide what could be considered an ethical tool for the objective assessment of oocyte developmental competence during the preclinical screening of experimental procedures in such areas as oocyte cryopreservation or in vitro maturation. Successful activation of human oocytes and in vitro development to the blastocyst stage were previously described when using small groups of oocytes, but parthenote developmental efficiency was not directly compared with that of fertilized controls (17–19). Therefore, the aim of the present work was to test an activation protocol on a relatively large group of freshly collected oocytes, to investigate whether

Fertility and Sterility姞 Vol. 87, No. 1, January 2007 Copyright ©2007 American Society for Reproductive Medicine, Published by Elsevier Inc.

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parthenote in vitro development reflects that of embryos obtained by fertilization of a comparable group of oocytes. Previous results indicated that exposure of human oocytes to a calcium ionophore alone, or such exposure followed by a protein synthesis inhibitor, leads to high activation rates but poor parthenote development (9 –15). However, when exposure to an ionophore was followed by a protein kinase inhibitor, better developmental rates were obtained (17,18). Therefore, we applied a protocol derived from a procedure that allowed us to obtain high blastocyst rates in pigs (23), based on the sequential exposure of oocytes to the calcium ionophore ionomycin, followed by the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP). MATERIALS AND METHODS This study was conducted between October 2004 –July 2005 at the Infertility Unit of the Department of Obstetrics and Gynecology, Ospedale Maggiore Policlinico Mangiagalli e Regina Elena (Milan, Italy). In Italy, no more than three embryos per cycle can be obtained (24,25). Therefore, in our unit, patients undergoing intracytoplasmic sperm injection (ICSI) from whom we retrieve more than three good-quality oocytes are routinely offered the opportunity to cryopreserve supernumerary eggs. Patients declining this offer were asked to participate in the present study. Approval for the study was obtained by the local institutional review board. All participating women gave informed consent. Fresh oocytes were obtained following controlled ovarian hyperstimulation using a long protocol with a GnRH analogue (triptoreline, Decapeptyl 0.1; Ipsen S.p.A., Milan, Italy), or a protocol using a GnRH antagonist (Cetrotide; Serono, Milan, Italy). In both cases, ovarian hyperstimulation was achieved using recombinant FSH (rFSH, Gonal-F; Serono). Follicular development was monitored via ovarian ultrasonography and serum estradiol-17␤ assay. When at least three follicles with a mean diameter ⱖ18 mm were present, patients were instructed to take 5,000 IU of hCG (Gonasi; AMSA, Rome, Italy). Transvaginal follicular aspiration for oocyte retrieval was performed 36 hours after hCG. During ovum pickup, oocytecumulus complexes were immediately separated from follicular fluid, washed in flushing medium. and transferred to 1 mL of IVF medium (G-FERT; Vitrolife Sweden AB, Gothenburg, Sweden). Following a 2–3-hour incubation at 37°C in an atmosphere of 6% CO2 in air, cumuli oophori were completely removed from all oocytes by pipetting them through 140- or 170-␮m internal diameter pipettes (Flexipet; Cook, Bloomington, IN), after a brief exposure to 40 IU/mL of hyaluronidase (Type IV; Sigma, Aldrich Srl, Milan, Italy) in gamete handling medium (G-MOPS; Vitrolife Sweden AB). Only metaphase II (MII) oocytes showing a normal morphology with clear cytoplasm, uniform texture, and homogeneous fine granularity were included in the study. Oocytes of each patient were randomly divided into two treatment groups: oocytes in the first group were destined for 78

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routine ICSI procedure and subsequent embryo transfer; oocytes in the second group were allocated to parthenogenetic activation and in vitro culture for 5 days. Randomization was performed with the use of a specific unbalanced list of randomization for each patient. Specifically, if n suitable MII oocytes were obtained, 3 were randomized to the ICSI procedure, while n⫺3 were allocated to parthenogenetic activation. One hour after denudation, oocytes to be fertilized were microinjected using an ICSI procedure as previously described (26), and transferred to culture medium (G1; Vitrolife Sweden AB, Sweden). Eighteen to twenty hours after microinjection, oocytes were checked for the presence of two pronuclei and the extrusion of the second polar body as signs of normal fertilization. Zygotes were transferred to fresh G1 medium and cultured into single 40-␮L drops until day 3, when embryos were transferred to the patients, whereas parthenotes were cultured for 2 further days in G2 medium (Vitrolife Sweden AB). Embryos were scored on day 2 (42– 44 hours after insemination) and day 3 (66 – 68 hours after insemination), according to developmental stage and morphological quality, as specified in the grading system of Veeck (27). This classification is based primarily on equal-sized blastomeres, pattern of fragmentation, and cytoplasmic appearance. Grade 1 represents perfect morphology, and grade 5 represents the worst. Blastocysts were scored on day 5 following classification, as described by Gardner and Schoolcraft (28), based on blastocyst morphology, degree of expansion, inner cell mass (ICM), and trophectoderm development. Grade 6AA is the best score, and 1CC the worst. One hour after denudation, oocytes allocated to activation were sequentially exposed to 5 ␮M ionomycin in IVF medium for 5 minutes at 37°C, 6% CO2 in the dark, washed twice, and incubated in 2 mM 6-DMAP (Sigma-Aldrich Srl, Milan, Italy) in medium G1 for 3 hours at 37°C, 6% CO2. Oocytes were then washed three times in fresh G1 medium, placed separately in 40-␮L microdrops of the same medium under mineral oil, and cultured in standard conditions (37°C, 6% CO2). After 18 –20 hours, considering the exposure to ionomycin as time 0, oocytes were evaluated for signs of activation. Oocytes showing one enlarged pronucleus and no extrusion of the second polar body were considered activated (29,30). Parthenotes were washed twice and kept in culture in fresh medium (G1) in the same manner as sibling fertilized oocytes for 3 days, followed by a further 2 days of culture in G2 medium. Parthenote development was scored with the same timing and the same parameters described for embryos, but blastocyst formation was evaluated on day 5 (118 –120 hours after activation). The chi square test, Fisher’s exact test, and the t-test were used where appropriate to compare the two groups. To compare the efficacy of the two methods, we performed univariate analysis to obtain the odds ratios of the activation protocol compared to the fertilization protocol and their corresponding 95% confidence intervals. Analysis of data

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TABLE 1 Comparison of developmental potential between activated and fertilized oocytes. Time after activation or ICSI

Stage

0h 18–20 h

Oocyte Activated or fertilized Percentage of oocytes 42–44 h Cleaved Percentage of oocytes Percentage of activated or fertilized oocytes 66–68 h Cleaveda Percentage of oocytes Percentage of activated or fertilized oocytes 114–116 h Blastocyst Percentage of oocytes Percentage of activated or fertilized oocytes

Odds ratio (95% confidence interval)

Activation

ICSI

104 70 67.3% 64 61.5% 91.4% 47 45.2% 67.1% 9 8.6% 12.8%

114 71 62.3% 70 61.4% 98.6% 65 57.0% 91.5%

n.a. 1.25 (0.71–2.18) 1.01 (0.58–1.74) 0.15 (0.02–1.30) 0.62 (0.36–1.06) 0.19 (0.07–0.50)ⴱ n.a.

Note: n.a. ⫽ not applicable. a Includes only embryos or parthenotes that underwent further cleavage. ⴱ Statistically significant, P⬍.001. Paffoni. Human parthenogenetic development in vitro. Fertil Steril 2007.

was performed with the use of the Statistical Package for Social Sciences (SPSS, Chicago, IL). Statistical significance was set at P⬍.05.

SD of blastomeres in the two groups was 3.7 ⫾ 1.0 and 3.7 ⫾ 0.8 (range, 2– 6 for both), respectively (P⫽.63, Table 2), but a lower percentage of good morphology (grades 1 and 2) was observed among parthenotes (Table 3).

RESULTS Thirty-eight patients entered the study. The mean age ⫾ SD of these women was 35.2 ⫾ 3.3 years. Overall, 218 MII oocytes were obtained: 114 were destined for standard ICSI procedure, while 104 were allocated to parthenogenetic activation.

On day 3, 65 embryos (57.0%) had undergone further cellular division in the group of ICSI-fertilized oocytes, while in the group of parthenotes, this event was documented in 47 (45.2%) cases (P⫽.11, Table 1). However, when the rate of development was referred to the activated or fertilized oocytes, rather than to their total number, the difference between groups, which on day 2 was only a trend (P⫽.06), became statistically significant (P⫽.001). Despite this difference, the mean number ⫾ SD of blastomeres in the two groups remained comparable (6.3 ⫾ 1.5 and 6.3 ⫾ 1.4,

On day 1, 71 (62.3%) of 114 oocytes randomized to the standard ICSI procedure showed normal fertilization, while 3 (2.6%) degenerated following the microinjection procedure, and 1 (0.9%) had three pronuclei. The remaining oocytes (34.2%) did not show signs of fertilization or activation. Parthenogenetic activation was performed on 104 spare oocytes (range, 2– 6 per patient): 70 (67.3%) which activated with ionomycin and 6-DMAP showed one enlarged pronucleus and one extruded polar body, and were considered parthenotes. The remaining oocytes degenerated following the procedure (n ⫽ 4, 3.8%), or showed no signs or altered signs of pronuclear formation (n ⫽ 30, 28.9%). Altogether, there was no significant difference in the rate of fertilization compared with the rate of parthenogenetic activation (P⫽.53; Table 1). On day 2, 70 embryos (61.4%) with at least two cells were observed in the group of oocytes allocated to the ICSI procedure, while the number of parthenotes with at least two cells was 64 (61.5%, P⫽.90, Table 1). The mean number ⫾ Fertility and Sterility姞

TABLE 2 Comparison of blastomere numbers between parthenotes and embryos. Time after activation or ICSI 42–44 h 66–68 h

Blastomeres Parthenotes

Embryos

P value

3.7 ⫾ 0.8 6.3 ⫾ 1.4

3.7 ⫾ 1.0 6.4 ⫾ 1.5

0.63 0.93

Note: Values are means ⫾ SD. Comparisons determined by t-test. Paffoni. Human parthenogenetic development in vitro. Fertil Steril 2007.

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TABLE 3 Comparison of quality between parthenotes and embryos. Time after activation or ICSI 42–44 h

66–68 h

ⴱ

Morphological evaluation

Activation

ICSI

Grade I–II parthenotes/embryos Percentage of total parthenotes/embryos Percentage of total oocytes Grade I–II parthenotes/embryos Percentage of total parthenotes/embryos Percentage of total oocytes

42 65.6% 40.4% 38 80.9% 36.5%

66 94.3% 57.9% 59 90.8% 51.7%

Odds ratio (95% confidence interval) 0.12 (0.04–0.36)ⴱ 0.49 (0.29–0.84)ⴱⴱ 0.43 (0.14–1.30) 0.54 (0.31–0.92)ⴱⴱ

P⬍.001. P⬍.05.

ⴱⴱ

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respectively, P⫽.93, Table 2), but cleaved parthenotes still showed a lower percentage of good morphology (Table 3). On day 5, nine oocytes exposed to the activation protocol (8.7%) developed into blastocysts, representing 12.9% of the parthenotes (Table 1). The in vitro blastocyst formation rate could not be evaluated for oocytes randomized to the ICSI procedure, because all viable embryos were transferred on day 3. Three blastocysts reached the expanded stage, with a tightly packed ICM and a trophectoderm formed of many cells forming a tightly knit epithelium (grade 4AA). Two reached the full blastocyst stage with excellent morphology (3AA). Two reached the same stage, but with a lower morphological degree (3CB). The remaining two reached only the blastocyst stage (grade 2). Blastocyst formation was observed in eight women. In these patients, pregnancies occurred at a higher rate (3/8, 37.5%) than in women who did not develop activated blastocysts (6/30, 20.0%). This difference, however, was not statistically significant (P⫽.36). DISCUSSION Sperm entry triggers a series of intracellular calcium oscillations that occur at intervals of several minutes for the first few hours after fertilization, and lead to oocyte activation (19). Inactivation of maturation-promoting factor (MPF) and of mitogen-activated protein kinase are the events occurring downstream from calcium oscillations which enable the resumption and completion of meiosis, pronuclear formation, and DNA synthesis (30). Several activating agents, such as ethanol, Ca2⫹ ionophores, and electroporation, can induce a single, prolonged calcium rise that releases oocytes from metaphase arrest. However, MPF quickly rises again, and further development is impaired. For this reason, additional agents that can inhibit protein synthesis or protein phosporylation have been added, enabling high activation and in vitro 80

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development rates in several species, including rhesus monkey (30), rabbit (31), bovine (32), sheep (29), and pig (33). As opposed to what is observed in other species, human parthenotes often show low developmental competence, not progressing beyond the eight-cell stage. An inadequate activation stimulus is one of the possible causes. In most of the unsuccessful attempts, human oocyte activation was induced by combining an ionophore with a protein synthesis inhibitor (8 –16). In the experiments described here, on the contrary, elevation of intracellular Ca2⫹ levels with ionomycin was followed by inhibition of protein phosporylation with 6-DMAP. This resulted not only in efficient oocyte activation, but also in parthenote development to the blastocyst stage, confirming and extending previous observations performed on smaller groups of oocytes (17,18). It is not known why this combination is particularly effective. One possible explanation is that the inhibition of MPF reactivation, caused by 6-DMAP, follows a kinetic similar to that occurring after fertilization, as observed in bovine (34) and pig (35) oocytes. The importance of inhibiting MPF reactivation in order to achieve efficient blastocyst formation was indirectly suggested by injecting phospholipase C␨ (PLC␨) complement RNA (cRNA) into the human ooplasm (19). PLC␨ cRNA, in fact, induced a pattern of Ca2⫹oscillations similar to those reported after IVF or ICSI, which, in turn, are known to determine a series of molecular events leading to MPF inactivation (36). Indeed, the application of this method to 24 aged oocytes led to a 16.6% blastocyst rate that favorably compares with the results obtained in the present experiments (19). We obtained a rate of blastocyst development (8.6%) lower than that reported previously with parthenogenetically activated oocytes, which ranged from 16.6% (19) to 27.3% or 28.6% (17,18). We have no clear explanation for this lower result, but the capability of human oocytes to undergo parthenogenetic activation and cleavage to the blastocyst

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stage may not depend only on a suitable activation protocol. Previous parthenogenetic blastocysts were obtained from a total of 60 oocytes, and in only one case was the age range (24 –32 years) (17) or number of patients (18) available. The present trial was performed on 104 oocytes from 38 patients, all over 30 years of age, which reflects the typical population treated at our fertility center. It may be suggested that both the higher number of patients and their possibly higher average age may have contributed to the lower efficiency obtained in this trial. Oocyte quality is likely to be an important factor, since it is well-known that it strongly influences the success rate of IVF. Indeed, experiments performed in mice (37) and pigs (38) indicated that oocyte quality also has a strong influence on parthenogenetic development. However, no reports directly comparing in vitro development of fertilized vs. activated human oocytes are available in the literature. Furthermore, evidence that oocyte quality could influence the in vitro development of parthenogenetically activated oocytes at the same rate as that of fertilized ova is seen only in experiments performed on rhesus monkeys (30). The present work is the first to provide data on whether parthenogenetic activation may give reliable information on human oocyte quality by directly comparing the outcome of parthenogenetic activation and ICSI performed on a large group of fresh oocytes. Our results indicate that the activation rate was not statistically different from the fertilization rate. No significant differences in embryonic development at 42– 48 and 66 – 68 hours after insemination or activation were observed between the two groups. Furthermore, the blastomere number was no different between parthenotes and embryos. However, if cleavage results refer to fertilized or activated oocytes rather than to their total number, a significantly higher proportion of fertilized embryos went on cleaving to day 3, which is consistent with the lower average morphology grade rate observed among parthenotes. This was expected, because a suboptimal morphology among parthenotes is a common feature described in the mouse (39,40), bovine (41), and rhesus monkey (30). We were also interested in investigating whether blastocyst formation after parthenogenetic activation correlated with the establishment of a pregnancy following the transfer of fertilized oocytes belonging to the same patient. Current results indicate that such a correlation cannot be made. However, in our opinion, a larger sample size will be necessary to reach firmer conclusions, since oocyte quality is only one of several factors that determine the establishment of a pregnancy. Animal studies demonstrated that parthenotes obtained with an ionomycin/6-DMAP activation are mainly diploid (23,29,30,32). This is likely to be the case also with human oocytes, because, in our experiments, activated oocytes showed one enlarged pronucleus and no extrusion of the second polar body. The retention of the second polar body, in fact, means that there was no loss of chromosomes, so that Fertility and Sterility姞

the two sets of sister chromatids formed a diploid pronucleus. Experiments in other species indicate that diploid parthenotes have a higher developmental competence than their haploid counterparts (32,42– 44). Therefore, the disomy of parthenotes obtained in our experiments may explain, at least in part, their ability to reach the expanding blastocyst stage. In addition, it was suggested that the beneficial effect of 6-DMAP on parthenote development may depend on its inhibitory effect on MPF, or possibly on other protein kinases involved in the regulation of parthenogenetic development (35). Irrespective of the mechanisms involved, development to the blastocyst stage is an important feature, if human parthenotes were to be used as an alternative source for pluripotent stem cells, as was recently suggested (22). In conclusion, this study showed that an activation protocol based on the combined use of ionomycin and 6-DMAP is effective in activating fresh human oocytes, and that the resulting parthenotes can develop in vitro to the blastocyst stage. Since most parameters used for evaluating in vitro development were not significantly different between activated and fertilized oocytes, parthenogenesis may provide relevant indications for the evaluation of preclinical experimental procedures, avoiding the exposure of viable human embryos to untested practices. REFERENCES 1. Hipp J, Atala A. Tissue engineering, stem cells, cloning, and parthenogenesis: new paradigms for therapy. J Exp Clin Assist Reprod 2004;1:3. 2. Ulbright TM. Gonadal teratomas: a review and speculation. Adv Anat Pathol 2004;11:10 –23. 3. Van Blerkom J, Davis PW, Merriam J. A retrospective analysis of unfertilized and presumed parthenogentically activated human oocytes demonstrates a high frequency of sperm penetration. Hum Reprod 1994;9:2381– 8. 4. Santos TA, Dias C, Henriques P, Brito R, Barbosa A, Regateiro F, et al. Cytogenetic analysis of spontaneously activated noninseminated oocytes and parthenogenetically activated failed fertilized human oocytes—implications for the use of primate parthenotes for stem cell production. J Assist Reprod Genet 2003;20:122–30. 5. Gook DA, Osborn SM, Johnston WI. Parthenogenetic activation of human oocytes following cryopreservation using 1,2-propanediol. Hum Reprod 1995;10:654 – 8. 6. Rougier N, Werb Z. Minireview: parthenogenesis in mammals. Mol Reprod Dev 2001;59:468 –74. 7. Abramczuk JW, Lopata A. Resistance of human follicular oocytes to parthenogenetic activation: DNA distribution and content in oocytes maintained in vitro. Hum Reprod 1990;5:578 – 81. 8. Johnson MH, Pickering SJ, Braude PR, Vincent C, Cant A, Currie J. Acid Tyrode’s solution can stimulate parthenogenetic activation of human and mouse oocytes. Fertil Steril 1990;53:266 –70. 9. Taylor AS, Braude PR. The early development and DNA content of activated human oocytes and parthenogenetic human embryos. Hum Reprod 1994;9:2389 –97. 10. Winston N, Johnson M, Pickering S, Braude P. Parthenogenetic activation and development of fresh and aged human oocytes. Fertil Steril 1991;56:904 –12. 11. Yamano S, Nakagawa K, Nakasaka H, Aono T. Fertilization failure and oocyte activation. J Med Invest 2000;47:1– 8.

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