Denny Sakkas, Ph.D., a Yvonne D Arcy, B.Sc., b Gail Percival, M.Sc., b Lucinda Sinclair, R.N., b Masoud Afnan, M.D., b and Khaldoun Sharif, M.D.

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1 FERTILITY AND STERILITY VOL. 82, NO. 1, JULY 2004 Copyright 2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. Use of the egg-share model to investigate the paternal influence on fertilization and embryo development after in vitro fertilization and intracytoplasmic sperm injection Denny Sakkas, Ph.D., a Yvonne D Arcy, B.Sc., b Gail Percival, M.Sc., b Lucinda Sinclair, R.N., b Masoud Afnan, M.D., b and Khaldoun Sharif, M.D. b Assisted Conception Unit, Birmingham Women s Hospital, Edgbaston, Birmingham, United Kingdom Received July 25, 2003; revised and accepted November 19, Reprint requests: Denny Sakkas, Ph.D., Department of Obstetrics and Gynecology, Yale University School of Medicine, P.O. Box , New Haven, Connecticut (FAX: ; E- mail: denny.sakkas@yale. edu). a Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut. b Assisted Conception Unit, Birmingham Women s Hospital /04/$30.00 doi: /j.fertnstert Objective: To investigate whether sperm from different males can influence fertilization and embryo development. Design: To use an egg-sharing model, in which the eggs from one woman are shared between herself and a recipient, and different spermatozoa are used to fertilize the eggs. Setting: Assisted Conception Unit, Birmingham Women s Hospital, Edgbaston, United Kingdom. Patient(s): Infertile women undergoing egg sharing. Intervention(s): In vitro fertilization (IVF). Main Outcome Measure(s): Fertilization rates and the mean day 2 or 3 embryo score (cell number X grade) were examined for egg-sharing pairs. A comparison was also made for pairs in which intracytoplasmic sperm injection (ICSI) and IVF was used as the insemination method. A paired samples t-test was used to compare the sharer and recipient results. Result(s): Pregnancy rates did not differ between sharer and recipient couples. Interestingly, when comparing fertilization, there was a significant difference (P.05) in favor of IVF over ICSI. When comparing embryo development between egg-sharing pairs, we found that approximately 30% of patients showed a difference in mean embryo score of 5 in all embryo development and 14% in the quality of embryos available for transfer. Conclusion(s): We showed that the egg-sharing model is a successful alternative for the treatment of women who required donated eggs. More important, the egg-sharing model shows that, in a certain percentage of couples, differences in early embryo development are paternally influenced. (Fertil Steril 2004;82: by American Society for Reproductive Medicine.) Key Words: In vitro fertilization, embryo quality, paternal influence, pregnancy Egg sharing is a procedure used in numerous in vitro fertilization (IVF) centers to facilitate the treatment of patients who are unable to produce their own oocytes and, in some cases, to assist patients who are unable to afford their own treatment cycle (1 3). The system involves the sharing of eggs between a donor, who is normally prescreened to increase the chances of producing a good number of eggs and a recipient, who is unable to produce her own eggs or has failed numerous attempts even though she may have eggs. Inadvertently, by sharing eggs between two couples and inseminating them with sperm from different males, we can investigate whether there is a paternal effect on reproductive outcomes after assisted reproduction. Recently, Tesarik et al. (4) used a similar system to determine whether paternal influences on human embryo quality are detectable as early as the first cell cycle after fertilization. They found that fertilization with sperm from certain individuals repeatedly resulted in the formation of a high proportion of zygotes with abnormal pronuclear morphology that subsequently tended to cleave slowly, and showed extensive fragmentation and blastomere irregularities. 74

2 The question of whether there is a paternal effect on reproductive outcomes has been studied extensively in animal models, and numerous models have demonstrated that abnormal spermatozoa can drastically effect fertilization, embryo development, implantation, and fetal development. The paternal effect is observed in cases where the spermatozoa are considered abnormal, and numerous examples include when spermatozoa have been affected with either cyclophosphamide (5) or irradiation (6, 7). In humans, direct evidence is harder to establish; however, epidemiologic studies have shown paternal influences on fetal development. One such study was reported by Parker et al. (8), in which men working in the Sellafield nuclear reprocessing plant, United Kingdom were examined for total exposure to external ionizing radiation before conception. A significant positive association existed between a father s annual summary dose of 100 msv and stillbirth risk. In the field of assisted reproduction, the use of intracytoplasmic sperm injection (ICSI) has created concerns over the possibility of a paternal influence, first in that the technique is invasive and may cause anomalies related solely to the technique (9) or second, because of the abnormal nature of the spermatozoa being used (10 15). The use of ICSI has also led to numerous publications suggesting a link to major birth defects (16) and imprinting anomalies (17 19); however; numerous studies also suggest that the techniques are safe (20 22). The egg-sharing model allows us to examine some of these concerns. First, it allows us to assess the reproductive outcomes when spermatozoa from different patients are used to treat the sibling oocytes. Second, it allows us to examine the technical effect of ICSI, as in a number of cases either the egg sharer or the recipient is treated with ICSI, while the other couple has normal IVF. In this study, we therefore investigated whether: [1] sperm from different males can influence fertilization and embryo development and [2] eggs treated by routine IVF compared with ICSI lead to different outcomes. MATERIALS AND METHODS Patients and Stimulation The study was performed on egg-sharing patients entering the Assisted Conception Unit, Birmingham Women s Hospital, Birmingham, United Kingdom, between 1999 and The hospital ethical review board approved the egg share procedure before commencement. The patients underwent either a routine IVF or an ICSI treatment with transfer. All patients underwent an embryo transfer on day 2 or 3. In total, 65 pairs of couples both underwent routine IVF, while in an additional 47 couples, 1 couple underwent IVF and the other underwent ICSI. In an additional 7 couples, both underwent the ICSI procedure. The stimulation protocol and luteal support in the egg donors/sharers adopted by our group has been previously described (23). For the egg recipients, gonadotropin-releasing hormone (GnRH) analogue (Nafarelin; Syntex, Berkshire, United Kingdom), 200 micrograms 3 times daily by nasal spray, was used for pituitary desensitization in women with ovarian function (long protocol). This was followed by endometrial stimulation with oral estradiol valerate (Climaval; Novartis, Surrey, United Kingdom), aiming for an endometrial thickness of at least 8 mm. On the day the donor took her ovulating human chorinic gonadotropin (hcg) injection, the recipient was instructed to stop taking the nafarelin, and start vaginal progesterone (Cyclogest; Shire, Hants, United Kingdom), 400 mg twice daily. Embryo transfer was performed 2 to 3 days following oocyte retrieval. Both estradiol valerate and cyclogest were continued until the pregnancy test and, if positive, until 12 weeks of pregnancy. Egg-Sharing Procedure Egg allocation occurred according to the following criteria. The eggs from one woman were shared between herself and a recipient. A sharer with greater or equal to six eggs shared her eggs equally with a recipient; however, in the case of odd numbers of eggs, the sharer would always have the extra egg. In cases where there were less than six eggs collected, the sharer would retain all the eggs. Insemination and ICSI Insemination and sperm preparation was performed in either Scandinavian IVF media (IVF-500) for routine IVF patients or Gamete-media for ICSI patients (Scandinavian IVF, Gotheburg, Sweden). Oocytes that had undergone the ICSI procedure or collected oocytes that were inseminated overnight were cultured in Scandinavian IVF media. Insemination was routinely performed between 14:00 h and 15:00 h, while ICSI was performed between 13:00 h and 18:00 h. The following morning (day 1) the oocytes were assessed for the presence of two pronuclei and placed in groups of up to 10 in 40 l culture drops under oil (Ovoil; Scandinavian IVF) in Falcon Petri dishes (Fahrenheit, Rotheram, United Kingdom). All cultures were performed in incubators containing modules in an atmosphere of 5% CO 2,5%O 2 in N 2. On the day of transfer, all embryos were assessed for the number of cells per embryo, to ascertain their cleavage rate. The embryos were also given a quality score based on the presence of fragments and the clarity of the cytoplasm of the blastomeres, similar to that previously described by Cummins et al. (24). The ratings given to embryos for cell number were 1 for 1-cells, 2 for 2-cells, 3 for 3-cells, 4 for 4-cells, etc. The ratings given to embryos for quality were 4 points for Grade 1 embryos, 3 for Grade 2, 2 for Grade 3, and 1 for Grade 4. The embryo score was then calculated by multiplying cell number by the points score depending on grade. For example, a 4-cell Grade 1 embryo would achieve a score of , while a 3-cell Grade 2 embryo would score A mean score was then calculated for the cohort of embryos. FERTILITY & STERILITY 75

3 TABLE 1 Treatment outcomes of sharers and recipients, when both pairs underwent routine IVF. No. of pairs IVF-sharer IVF-recipient Mean no. eggs Mean fertilization Mean embryo score 61 a Mean transferred embryo 61 a score Mean no. transferred 61 a embryos Clinical pregnancy rate 61 a 24 (39.3%) 25 (41.0%) Implantation rate 29/128 (22.6%) 32/134 (23.9%) Note: Values are mean SD. a In four pairs, embryo data could not be matched for the sharer and recipient because one of the pair had all or part of their cohort of embryos frozen. Sakkas. Egg sharing evidences a paternal influence. Fertil Steril FIGURE 1 The difference in mean embryo score between transferred embryos from patients undergoing egg sharing when either (A) both patient couples underwent IVF or when (B) one couple was treated by ICSI and the other IVF. Mean embryo score was calculated as stated in the Materials and Methods. The mean embryo score for all transferred embryos was then calculated for the sharer and recipient. Subtracting the sharer and recipient score represents the difference in mean embryo score. In patients with positive pregnancy tests, an ultrasound examination was conducted 4 to 6 weeks after transfer. Patients in which the fetus or fetuses displayed a heartbeat by ultrasound examination were considered to have achieved a clinical pregnancy. The statistical evaluations used were analysis of variance (ANOVA) followed by Scheffè s F-test for comparisons of mean values and 2 analysis with continuity correction for comparison of pregnancy rates. RESULTS Couples Who Both Underwent Routine IVF The results for the couples in which both pairs underwent a routine IVF procedure are shown in Table 1. Overall, 65 pairs of couples underwent cycles in which both couples had IVF. To examine the relationship between semen parameters and embryo development, a comparison was made between the semen parameters of the partner of the sharer and recipient. When motility differed by more than 20%, no significant difference existed between the mean embryo score of all and transferred embryos. In addition, when sperm concentration differed by more than 30, 40, or 50 million per ml, no significant difference existed between the same embryo parameters. In 16 pairs of patients, the mean embryo difference was 3 between the sharer and recipient (Fig. 1A); however; in these patients, no differences were evident when we examined the partners semen parameters. Couples in Which One Underwent Routine IVF and the Other ICSI The results for the couples in which one underwent routine IVF while the other underwent ICSI are presented in Sakkas. Egg sharing evidences a paternal influence. Fertil Steril Table 2. Forty-six pairs of couples had IVF and ICSI procedures. When sperm concentration in ICSI patients was less than 10, 5, or 3 million per ml, no significant difference was found when compared with IVF patients between the mean embryo score of all embryos and transferred embryos. Interestingly, when comparing fertilization between IVF and ICSI, there was a significant difference (P.05) in favor of the IVF procedure over the ICSI procedure. The overall ICSI fertilization rate per retrieved oocyte was 61.8% 24.2% ( SD), which was significantly lower than the IVF fertilization rate: 72.0% 20.7%. The lower fertilization results were attributed mostly to ICSI patients with less than 20% motility because fertilization rates were significantly (P.05) lower in this group. 76 Sakkas et al. Egg sharing evidences a paternal influence Vol. 82, No. 1, July 2004

4 TABLE 2 Treatment outcomes of sharers and recipients, when one pair underwent intracytoplasmic sperm injection (ICSI) and the other underwent routine IVF. No. of pairs ICSI IVF Mean no. eggs Mean fertilization 45 a b Mean embryo score 43 a Mean transferred embryo 43 a score Mean no. transferred 43 a embryos Clinical pregnancy rate 43 a 20 (46.5%) 17 (39.5%) Implantation rate 22/90 (24.4%) 21/82 (25.6%) Note: Values are mean SD. a In one pair, fertilization did not occur, and in another two pairs, embryo data could not be matched for the sharer and recipient because all the embryos were frozen. b Significantly different P.05. Sakkas. Egg sharing evidences a paternal influence. Fertil Steril In 10 pairs of patients, the difference in the mean embryo score was 5 (Fig. 1B). No significant difference in the quality of the ICSI semen samples could be found when examining ICSI patients who had differences in the mean embryo score of 5 compared with those who had a difference of 5. Embryo Differences in All Pairs of Patients A further investigation of embryo differences was performed, regardless of whether the patients were the sharers or recipients. When comparing the pairs of patients who only underwent IVF, the overall total mean embryo score differed by 3 or more in 16 out of 61 pairs of patients (26.2%), while it differed by more than 5 in 9 out of 61 (14.8%) pairs. When examining for differences in the transferred embryos, which would represent the best embryos available for the patients, the mean embryo score differed by 3 or more in 21 out of 61 pairs of patients (34.4%), while it differed by 5 or more in 12 out of 61 (19.7%) pairs (Fig. 1). We could not find any significant difference when comparing sperm concentration and motility in the patients who showed a mean embryo score difference of 3 or 5. When differences were examined, regardless of whether the patients underwent IVF or ICSI, a similar number of patients exhibited differences in their mean embryo scores. The overall total mean embryo score differed by 3 or more in 12 out of 43 pairs of patients (27.9%), while it differed by more than 5 in 7 out of 43 (16.3%) pairs. The difference in transferred embryos is given in Figure 1. Finally, when all patients were taken into account, the total mean embryo score differed by 3 or more in 28 out of 104 pairs of patients (26.9%), while it differed by more than 5 in 16 out of 104 (15.4%) pairs. When examining for differences in the transferred embryos, the mean embryo score differed by 3 or more in 31 out of 104 pairs of patients (29.8%), while it differed by 5 or more in 15 out of 104 (14.4%) pairs. DISCUSSION This study has demonstrated that implantation rates, pregnancy rates, and overall mean embryo development rates did not differ when comparing the overall results of sharer and recipient couples using conventional IVF and when one couple was treated with IVF and the other ICSI. This substantiates previous studies indicating that the sharer recipient model is a beneficial treatment of choice (3). Interestingly, when comparing couples where one couple had IVF and the other had ICSI, the overall fertilization rates were not improved with the use of ICSI and, indeed, were significantly higher in IVF compared with ICSI. This would indicate that the argument that ICSI is beneficial for treating all patients (25) may not be applicable. Based on this series, where the maternal factor does not play a role, fertilization rates were higher in IVF patients, but there was no difference in pregnancy rates. In addition, distinct from the clinical benefits of performing egg sharing, a second interest lies in the ability to distinguish if differences occur in the embryos generated from sibling oocytes fertilized with sperm from different males. When ignoring the differences between sharer and recipient and concentrating solely on the patient pairs, we found a substantial number of patients who did show differences in their overall embryo development and in the quality of embryos available for transfer. The majority of embryos in this study were only cultured until day 2, thus a greater impact of any paternal effect may be more pronounced if the culture periods of the embryos were extended postembryonic genome activation (26, 27). It is evident that a number of couples do show distinct differences in embryo development. In comparing both IVF-IVF and IVF-ICSI patients, around 30% of them had a mean difference in embryo score 3, even when examining only the transferred embryos. When we examined the semen parameters of the patients who showed a large difference in embryo development, no apparent pattern was observed. This, however, does not preclude that a paternal effect may have been responsible for differences in embryo development. In the clinic, the sharing of eggs was performed randomly between the two patients, and this difference could not be explained solely by egg quality. A standard semen analysis is limited to examining numbers, motility, and morphology, but a more in-depth analysis may be more informative for these patients. The failure to find a relation to the standard parameters indicates that more subtle analyses may provide more information. FERTILITY & STERILITY 77

5 Examination of the sperm can be performed at various levels: nuclear, cytoskeletal, and organelle. Anomalies of spermatozoa at the nuclear level have been linked to distinct effects on embryo development and overall reproductive outcomes (15, 28, 29). The inheritance of defective centrosomes has also been postulated to lead to abnormal cleavage and contribute to infertility (30 33). The possible role that oxidative stress may also have on spermatozoa, particularly in asthenozoospermic patients, has been extensively reported (34 36). This may provide a plausible reason for the lower fertilization rates in ICSI patients with less than 20% motility. Furthermore, subtle effects related to the action of the sperm have been shown in numerous studies where there are no effects on fertilization, however, later development is abnormal. For example, in the bovine, even though fertilization rates were similar using sperm from two different bulls, the onset of the first S-phase was found to differ, and subsequent development to the blastocyst stage differed considerably (37, 38). Upon closer analysis, it was concluded that the onset of the first S-phase is determined by an epigenetic effect related to the sperm during the G1-phase in bovine zygotes. Other studies in the bull have also shown a paternal effect on the first cleavage stage postinsemination (39). In conclusion, we have demonstrated that clinically, the egg-sharing model is a successful alternative for the treatment of women requiring donated eggs. More important, the egg-sharing model has indicated that differences in early embryo development may be paternally influenced. What the contributing paternal mechanism effecting embryo development is remains unidentified, but alerts us that the quality of spermatozoa fertilizing the egg may play a more important role than once thought. Finally, a more in-depth molecular dissection of the spermatozoa is needed in relation to the influence that its various components (i.e., nucleus, cytoskeleton, and organelles) have on embryo development and viability. References 1. Kolibianakis EM, Tournaye H, Osmanagaoglu K, Camus M, Van Waesberghe L, Van Steirteghem A, et al. Outcome for donors and recipients in two egg-sharing policies. Fertil Steril 2003;79: Blyth E. Subsidized IVF: the development of egg sharing in the UK. Hum Reprod 2002;17: Sharif KM, Afnan M, Hammadieh N, Sinclair L., D Arcy Y, Percival G. Does donating half your eggs reduce your chances of success in in vitro fertilization: a comparative study using the egg sharing model. ASRM Fort Lauderdale USA 2001: Tesarik J, Mendoza C, Greco E. 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6 33. Sathananthan AH. Paternal centrosomal dynamics in early human development and infertility. J Assist Reprod Genet 1998;15: Agarwal A, Saleh RA, Bedaiwy MA. Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril 2003;79: Aitken RJ, Sawyer D. The human spermatozoon not waving but drowning. Adv Exp Med Biol 2003;518: Ford WC. Male infertility: tales of progress and frustration. Hum Fertil (Camb) 2002;5:S Comizzoli P, Marquant-Le Guienne B, Heyman Y, Renard JP. Onset of the first S-phase is determined by a paternal effect during the G1-phase in bovine zygotes. Biol Reprod 2000;62: Comizzoli P, Urner F, Sakkas D, Renard JP. The formation of the male pronucleus controls/determines the up regulation of glucose metabolism and the onset of the S phase in bovine zygotes. Biol Reprod 2003;68: Ward F, Rizos D, Corridan D, Quinn K, Boland M, Lonergan P. Paternal influence on the time of first embryonic cleavage post insemination and the implications for subsequent bovine embryo development in vitro and fertility in vivo. Mol Reprod Dev 2001;60: FERTILITY & STERILITY 79