Monozygotic twinning is not increased after single blastocyst transfer compared with single cleavage-stage embryo transfer Evangelosa G. Papanikolaou, M.D., Ph.D., a,b Human Fatemi, M.D., Ph.D., a Christos Venetis, M.D., b Pato Donoso, M.D., a Efstratios Kolibianakis, M.D., Ph.D., b Herman Tournaye, M.D., Ph.D., a Basil Tarlatzis, M.D., Ph.D., b and Paul Devroey, M.D., Ph.D. a a Centre for Reproductive Medicine, University Hospital, Vrije Univestiteit Brussel, Brussels, Belgium; and b Unit for Human Reproduction, 1st Department of Obstetrics and Gynecology, Papageorgiou General Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece Objective: To compare the incidence of monozygotic twinning between cleavage-stage and blastocyst-stage embryo transfer in a large cohort of patients undergoing single embryo transfer. Design: Retrospective study. Setting: Dutch-speaking Free University of Brussels. Patient(s): This study covered the period between July 2003 and December 2005. 1,951fresh IVF/ICSI cycles in which single embryo transfer was performed were retrospectively reviewed. Only the first cycle of each patient was included. Intervention(s): Five hundred eighty seven (n ¼ 587) cycles that resulted in clinical pregnancies were identified; 308 after single day-3 embryo transfer and 271 after single blastocyst transfer. Main Outcome Measure(s): The incidence of monozygotic twinning. Result(s): Overall, 13 cases (2.2%) of monozygotic twinning were observed, 2.6% in the cleavage-stage group (n ¼ 8/308) and 1.8% in the blastocyst group (n ¼ 5/271). No statistically significant differences were observed in the probability of monozygotic twinning between the Cleavage-stage and the Blastocyst group (difference: þ0.8%; 95% CI, 1.97 to þ3.41). All of these pregnancies resulted in the delivery of 24 healthy babies. The crude odds ratio for the incidence of monozygotic twinning after day-5 embryo transfer was calculated to be 0.71 (95% CI, 0.23 2.18). Conclusion(s): To investigate the potential association between the day of embryo transfer (day 3 or 5) and the incidence of monozygotic twinning, the clinical pregnancies analyzed should have been established after single embryo transfer. The current study represents the first methodologically appropriate study attempting to investigate the above research question. Our findings support that opting for blastocyst transfer does not increase the probability for monozygotic twins. (Fertil Steril Ò 2010;93:592 7. Ó2010 by American Society for Reproductive Medicine.) Key Words: Monozygotic twinning, cleavage-stage, blastocyst stage, single embryo transfer, IVF, risks In recent years, extending embryo culture to day 5 of embryo development has become the most frequent strategy for improved embryo selection, resulting in a higher implantation and pregnancy rate (1). Nevertheless, a high multiple-pregnancy rate might be expected when more than one blastocyst was replaced; thus, single embryo transfer (SET) has been suggested. It has been recently shown that under the terms of an SET policy in women less than 36 years old, the transfer Received August 4, 2008; revised December 14, 2008; accepted December 16, 2008; published online February 24, 2009. E.G.P. has nothing to disclose. H.F. has nothing to disclose. C.V. has nothing to disclose. P.D. has nothing to disclose. E.K. has nothing to disclose. H.T. has nothing to disclose. B.T. has nothing to disclose. P.D. has nothing to disclose. Reprint requests: Papanikolaou Evangelos, M.D., Ph.D., Centre for Reproductive Medicine, Vrije Univestiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium (TEL: þ32 2 4261042, þ32 2 477 6607) (E-mail: drvagpapanikolaou@yahoo.gr, Evangelos.Papanikolaou@vub.ac.be). of a day-5 embryo achieves 10% more deliveries than a single day 3 embryo without increasing the risk for monozygotic twins (2). Concerns have been raised, earlier however, regarding an increased incidence of monozygotic twinning (MZT) after blastocyst transfer compared with cleavage-stage embryos (3, 4). This has been attributed to a hardening in the zona pellucida or to a disorder in the cell adhesion process secondary to the culture media. It has also been proposed that intracytoplasmic sperm injection (ICSI) itself might cause splitting of the inner cell mass through the artificial gap (5), although this has not been confirmed by other authors (4). The prevalence of monozygotic twins in the general population is 0.42%, and MZT is associated with threefold to fivefold higher perinatal morbidity and mortality than dizygotic twins; hence, an increase in this type of multiple pregnancies would constitute a major drawback for blastocyst transfer. 592 Fertility and Sterility â Vol. 93, No. 2, January 15, 2010 0015-0282/10/$36.00 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2008.12.088
Nevertheless, data reporting on augmented frequency of monozygotic twinning after blastocyst transfer has considered only monochorionic/monoamniotic monozygotic twins, because more than one embryo had been transferred and therefore only this type of twinning could be certainly assigned as originating from the same single embryo (3, 4, 6). This methodology, however, results in an underestimation of the overall frequency of monozygotic twins, because monozygotic/dichorionic twins arise within the first three days of embryo-development and are disregarded; this leads to an overestimation of the proportion of MZT developed by day-5 embryos, because monochorionic monozygotic twins arise between day-5 to day-14). Therefore, the best approach to study this possible complication is to compare only cycles in which SET has been performed (7). The aim of the present study is to compare the incidence of monozygotic twinning between cleavage-stage and blastocyst-stage embryo transfer in a large cohort of patients undergoing single embryo transfer. MATERIALS AND METHODS Population This study covered the period of July 2003 to December 2005. All nondonor, fresh IVF/ICSI cycles in which SET was performed (n ¼ 1,951) were retrospectively reviewed. Only the first cycle of these women was included in the analysis. Five hundred seventy nine (n ¼ 579) cycles that resulted in clinical pregnancies were identified. Consequently, 579 clinical pregnancies resulting from IVF/ICSI-SET cycles were analyzed. Female age ranged from 19 36 years and most of the women (n ¼ 516) had at most two previous IVF/ICSI attempts. The most frequent indications for treatment were combination of male and female factor (n ¼ 264) and male factor alone (n ¼ 198). Cases of assisted hatching or preimplantation genetic diagnosis were excluded. Treatment Characteristics Ovarian stimulation was performed using recombinant follicle stimulating hormone (rfsh; n ¼ 374) or human menopausal gonadotrophin (hmg; n ¼ 205), where LH surge was inhibited with the use of either GnRH antagonists (n ¼ 394) or GnRH agonists (n ¼ 185). Gonadotrophin starting dose ranged from 75 450 IU, although in the majority of the cases (n ¼ 465) it was 150 225 IU. Ovarian stimulation was performed with a fixed gonadotrophin dose in 392 cases (67.7%). Final oocyte maturation was induced in all cases by the administration of 10,000 IU of human chorionic gonadotrophin as soon as three follicles of R17 mm in mean diameter were present at transvaginal ultrasound. Three hundred ninety-seven (n ¼ 397) and 182 (n ¼ 182) clinical pregnancies were observed after the transfer of an embryo inseminated with ICSI or conventional IVF, respectively. Embryo culture was performed with the use of two types of sequential media; Media A (Medicult, Jyllinge, Denmark; n ¼ 330) and Media B (Vitrolife, Goteborg, Sweden; n¼249). Luteal support was performed with the vaginal administration of 600 mg of micronized progesterone per day. Assessment of Clinical Pregnancy and Twinning According to the standard policy of our center, all early pregnancies are closely and regularly monitored, and data are collected regarding their evolution and final outcome. Clinical pregnancy was defined as the presence of fetal cardiac activity, as assessed by transvaginal ultrasound performed at 7 weeks gestation. Because SET was performed in all cases, twinning was defined as the presence of more than one fetal hearts. Data regarding chorionicity/amnionicity was retrieved by a study nurse after contacting the patient and/or the treating obstetrician. Data Collection Required information was retrieved from the electronic database of our centre. All reviewed data were prospectively collected for the purposes of various ongoing randomized controlled trials of that period. An institutional review board approval was not obtained because the data was retrospectively analyzed. Statistical Analysis In order to investigate the potential association between the day of embryo transfer (day 3 or 5) and the incidence of monozygotic twinning, the clinical pregnancies analyzed were divided in two groups: [1] cleavage-stage group, those clinical pregnancies resulting after the transfer of a single day 3 embryo (n ¼ 308) and [2] blastocyst group, those resulting after the transfer of a single day 5 embryo (n ¼ 271). Statistical comparisons between groups were performed with the use of the independent samples Student s t test or Mann-Whitney U test for normally and nonnormally distributed variables, respectively, for continuous variables, and with the use of the chi-square or Fisher s exact test, where appropriate, for categorical variables. The crude odds ratio (OR) for the incidence of monozygotic twinning after day-5 embryo transfer with the corresponding 95% confidence intervals (CIs) was calculated through logistic regression analysis. The presence of more than one fetal heart, as assessed by a transvaginal ultrasound at 7 weeks gestation, represented the dichotomous dependent variable, and the day of embryo transfer (day 3 or 5) was the independent variable. In order to control for potential confounding effects, a multivariable logistic regression analysis was also performed and an adjusted odds ratio (AOR) was calculated. The following variables were analyzed as independent covariates: female age, indication for treatment, number of previous assisted reproduction attempts, GnRH analogue protocol used for LH inhibition, type (rfsh or hmg) and total dose of Fertility and Sterility â 593
gonadotrophins administered, duration of ovarian stimulation, number of cumulus-oocytes complexes (COCs) retrieved, type of insemination (conventional IVF or ICSI), and type of media used for embryo culture. Moreover, the robustness of the results was assessed with an additional multivariable logistic regression in which variables were included as potential confounders only if they presented statistically significant differences between the two groups compared. All statistical tests were two-sided and statistical significance was set at P % 0.05. All statistical analyses were performed with the use of SPSS 15.0 for Windows (SPSS Inc., Chicago, IL). RESULTS Details and formal statistical comparisons between the two groups regarding population and treatment characteristics, as well as outcome data, are presented in Tables 1, 2, and 3, respectively. In brief, the patients in the blastocyst group were younger compared with the patients of the cleavage-stage group (difference, þ0.9 years; 95% CI, 0.35 1.37). In addition, higher total dose of gonadotrophins was required for ovarian stimulation (cleavage-stage group, 1,800 [1,450 2,250] vs. blastocyst group, 1,650 (1,350 2,100); P¼0.041] and fewer COCs were retrieved (difference, 1.87 COCs; 95% CI, 2.86 to 0.89) in the cleavage-stage group compared with the blastocyst group. No significant differences were present among the two groups regarding number of previous IVF/ICSI attempts, indication for treatment, type of gonadotrophin used, total duration of ovarian stimulation, GnRH analogue protocol used for LH inhibition, type of insemination method, and media used for embryo culture. Overall, 13 cases (2.2%) of monozygotic twinning were observed, of which eight (n ¼ 8) were in the cleavage-stage group and five (n ¼ 5) were in the blastocyst group (Table 2). In one case of monozygotic twinning in the blastocyst group, three fetal hearts were observed. This couple decided to undergo an elective termination of the pregnancy. In all the remaining twelve cases of monozygotic twinning, two fetal hearts were identified. Only one case in the cleavage-stage group was monochorionic monoamniotic, one case was monochorionic diamniotic, and the remaining six were dichorionic diamniotic. In the blastocyst group, two cases were monochorionic diamniotic, and the next two were monochorionic monoamniotic. No statistically significant differences were observed in the probability of monozygotic twinning between the cleavagestage and the blastocyst group (difference, þ0.8%; 95% CI, 1.97 to þ3.41). Group sample sizes of 308 in group one and 271 in group two achieve 10% power to detect a difference between the group proportions of 0.0080, when the proportion in group one (the treatment group) is assumed to be 0.0260 under the null hypothesis and 0.0180 under the alternative hypothesis. In order to achieve 80% power to detect such a low difference (0.8%) as significant, more than 5,000 patients should be included in each group. The crude OR for the incidence of monozygotic twinning after day-5 embryo transfer was calculated to be 0.71 (95% CI, 0.23 2.18). When all variables presented in Tables 1 and 2 were entered in the analysis as additional covariates, the adjusted OR was 0.81 (95% CI, 0.23 2.83). When only those variables that were significantly different were entered in the logistic regression analysis as additional covariates (i.e., female age, total dose of gonadotrophins administered, number of COCs retrieved) the results were not materially altered (AOR ¼ 0.89; 95% CI, 0.27 2.92). In both logistic regression models tested, none of the covariates were found to be a significant predictor of monozygotic twinning. TABLE 1 Demographics and infertility etiology. Cleavage-stage group (n [ 308) Blastocyst group (n [ 271) P level Female age (years) 30.8 3.03 29.9 3.21 0.001 a Previous IVF/ICSI attempts 0.92 1.5 0.87 1.4 0.651 a Indication for treatment 0.859 b Male factor 108 (35.1) 90 (33.2) Tubal factor 15 (4.9) 14 (5.2) Endometriosis 10 (3.2) 14 (5.2) PCOS and/or anovulation 12 (3.9) 7 (2.6) Idiopathic 24 (7.8) 21 (7.7) Combined 139 (45.1) 125 (46.1) All values are presented as mean SD or cases (percentage), unless stated otherwise. a P level obtained with independent samples Student s t test. b P level obtained with Fisher s exact test. Papanikolaou. Monozygotic twinning in SET. Fertil Steril 2010. 594 Papanikolaou et al. Monozygotic twinning in SET Vol. 93, No. 2, January 15, 2010
TABLE 2 Treatment characteristics. Cleavage-stage group (n [ 308) Blastocyst group (n [ 271) P level Type of gonadotrophin used 0.338 a for ovarian stimulation hmg 115 (37.3) 90 (33.2) rfsh 193 (62.7) 181 (66.8) Gonadotrophin consumption (IU) 1800 (1450-2250) b 1650 (1350-2100) b 0.041 c Duration of stimulation (days) 10.50 2.83 10.55 2.80 0.844 d GnRH analogue protocol used 0.119 a for LH inhibition Antagonist day 6 200 (64.9) 194 (71.6) Agonist long day 1 11 (3.6) 13 (4.8) Agonist long day 21 84 (27.3) 59 (21.8) Agonist short protocol 13 (4.2) 5 (1.8) Number of COCs retrieved 10.4 5.9 12.3 6.2 <0.001 d Insemination method 0.929 a IVF 96 (31.2) 86 (31.7) ICSI 212 (68.8) 185 (68.3) Embryo culture media 0.276 a Media A 169 (54.9) 161 (59.4) Media B 139 (45.1) 110 (40.6) All values are presented as mean SD or cases (percentage), unless stated otherwise. a P level obtained with Fisher s exact test. b Median (interquartile range). c P level obtained with Mann-Whitney U test. d P level obtained with independent samples Student s t test. Papanikolaou. Monozygotic twinning in SET. Fertil Steril 2010. All of these pregnancies resulted in the delivery of 24 healthy babies. Gestational age at delivery was not significantly different between the cleavage-stage and the blastocyst groups (difference, þ0.29 weeks; 95% CI, 2.87 to þ3.44). DISCUSSION The incidence of MZT is higher in pregnancies conceived after assisted reproduction than after natural conception (8), and the current study confirms that by reporting an overall TABLE 3 Pregnancy outcome data. Cleavage-stage group (n[308) Blastocyst group (n[271) P level Monozygotic twinning 8 (2.6%) 5 (1.8%) 0.587 a (>1 fetal hearts) 2 fetal hearts 8/8 4/5 3 fetal hearts 0/8 1/5 Babies delivered 16 8 Gestational age at delivery (weeks) 35.2 2.25 34.9 2.46 0.843 b All values are presented as mean SD or cases (percentage), unless stated otherwise. a P level obtained with Fisher s exact test. b P level obtained with independent samples Student s t test. Papanikolaou. Monozygotic twinning in SET. Fertil Steril 2010. Fertility and Sterility â 595
2.2% of monozygotic twinning after single embryo transfer. The true incidence of monozygotic twinning after IVF, however, is still unknown. Counting cases, in which the number of fetuses exceeds the number of transferred embryos, is less accurate than zygosity, as this method disregards likesexed monozygotic-dichorionic twins and leads to an underestimation of the true frequency of zygosity. In this view, the current study represents the first attempt in the literature to appropriately describe the incidence of MZT in the setting of SET policy, abolishing the inherent bias of previous studies in which more than one embryo were transferred. The rate of MZT after blastocyst transfer in SET seems not to be increased compared with patients undergoing a cleavage stage SET (1.8% vs. 2.6%, respectively). It is important to note that assisted hatching and blastocyst coculture have not been performed in our series. Therefore, the benefit of higher delivery rates after blastocyst-stage transfer (9) should not be offset by some reported increase of MZT in the literature (3, 4, 6, 10 13), because of the previously mentioned limitations of the design of the studies (14, 15). However, the fact that six of eight MZT cases observed with day 3 SET were dichorionic should be mentioned, because the obstetrical risks with dichorionic monozygotic twins are significantly lower compared with monochorionic twins. The largest retrospective study ever published, the 1999 and 2000 Society of Assisted Reproductive Technology (SART) data compared 7,921 pregnancies from day 5 embryo transfer with 29,144 from day 3 embryo transfer (12). Among the resulting 226 monzygotic pregnancies (only 31 after single embryo transfer) there was a fourfold increased likelihood of monzygotic twinning with day 5 embryo transfer compared with day 3 embryo transfer, after controlling for multiple treatment and patient factors. Nevertheless, the authors acknowledge that such pregnancies might not entirely be monzygotic multiple gestations as their definition (in which the number of fetal hearts on ultrasound exceeds the number of embryos transferred) allowed for the inclusion of pregnancies with a mix of monozygotic and dizygotic fetuses (12). According to the first and most extended theory, monozygosity is caused by a breach in the integrity of the zona pellucida, herniation of the blastomeres, and splitting of the embryo (16, 17). The exact etiology of monozygosity after IVF is not known; however, there are speculations on the manipulation of the zona pellucida (i.e., spermatic microinjection and assisted hatching) (5, 18, 19). However, recent studies failed to confirm an association between ICSI and/ or assisted hatching and monozygotisity (12, 15). An additional factor associated with monozygosity that has been proposed is prolonged embryo culture (3, 4). On the contrary, other researchers support that it is not prolonged culture time but in vitro culture conditions, such as high levels of glucose in the medium (20), causing the production of more free radicals that induce apoptosis leading to disruption of the inner cell mass and presumably splitting into identical twins (21). Steinman (22) proposed that the long exposure time of a blastocyst to lower calcium levels in culture predisposes inner cell mass (ICM) division as the intracellular bonds are destabilized. Moreover, familial clustering also has been described, suggesting a role of genetic factors as well (18). The gold standard for determination of monozygosity is DNA analysis. However, none of the published data concerning the monozygosity used a DNA analysis as a diagnostic tool of monozygosity (7). Future studies should focus on the monozygotism in IVF/ICSI after preimplantation genetic diagnosis (PGD), which involves making a small artificial opening in the zona pellucida of a fertilized embryo through chemical, laser, or mechanical manipulation. Furthermore, accounting for the low incidence of MZT, future studies on SETs should have higher sample sizes to achieve a higher power to detect such small differences. With a growing tendency toward the transfer of a reduced number of embryos (23) and with the improvements in culture systems (24), the prevalence of a monozygotic twin pregnancy might be decreasing. Interestingly, Moayeri et al. (25) have described a decrease in the incidence of MZT with blastocysts over an 8-year period, attributed to improvement of blastocyst culture media. The current study s results suggest that although the incidence of monozygotic twining is increased following IVF compared to natural conception, the extension of the embryo culture to the blastocyst stage has no effect on this increase. There is a need for larger trials to assess the causes of monozygotic twining after assisted reproductive technology (26) to be able to accurately counsel eventual patients about their risks. Acknowledgments: We appreciate the effort of the nursing and embryology staff of our unit, who made it possible to carry out the IVF treatment of the patients included. REFERENCES 1. Papanikolaou EG, D haeseleer E, Verheyen G, Van de Velde H, Camus M, Van Steirteghem A, et al. Live birth rate is significantly higher after blastocyst transfer than after cleavage-stage embryo transfer when at least four embryos are available on day-3 of embryo culture. A randomized prospective study. Hum Reprod 2005;20:3198 203. 2. Papanikolaou EG, Camus M, Kolibianakis EM, Van Lunduyt L, Van Steirteghem A, Devroey P. Transfer of a single blastocyst-stage embryo as compared with a single cleavage-stage embryo for in vitro fertilization. N Engl J Med 2006;354:1139 46. 3. da Costa Al AL, Abdelmassih S, de Oliveira FG, Abdelmassih V, Abdelmassih R, Nagy ZP, et al. Monozygotic twins and transfer at the blastocyst stage after ICSI. Hum Reprod 2001;16:333 6. 4. Milki AA, Jun SH, Hinckley MD, Behr B, Giudice LC, Westphal LM. Incidence of monozygotic twinning with blastocyst transfer compared to cleavage-stage transfer. Fertil Steril 2003;79:503 6. 5. Tarlatzis BC, Qublan HS, Sanopoulou T, Zepiridis L, Grimbizis G, Bontis J. Increase in the monozygotic twinning rate after intracytoplasmic sperm injection and blastocyst stage embryo transfer. Fertil Steril 2002;77:196 8. 6. Behr B, Fisch JD, Racowsky C, Miller K, Pool TB, Milki AA. Blastocyst- ET and monozygotic twinning. J Assist Reprod Genet 2000;17:349 51. 7. 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8. Toledo MG. Is there increased monozygotic twinning after assisted reproductive technology? Aust N Z J Obstet Gynaecol 2005 Oct;45(5):360 4. 9. Papanikolaou EG, Kolibianakis EM, Tournaye H, Venetis CA, Fatemi H, Tarlatzis B, et al. Live birth rates after transfer of equal number of blastocysts or cleavage-stage embryos in IVF. A systematic review and metaanalysis. Hum Reprod 2008;23:91 9. 10. Peramo B, Ricciarelli E, Cuadros-Fernandez JM, Huguet E, Hernandez ER. Blastocyst transfer and monozygotic twinning. Fertil. Steril 1999;72:1116 7. 11. Sheiner E, Har-Vardi I, Potashnik G. The potential association between blastocyst transfer and monozygotic twinning. Fertil Steril 2001;75:217 8. 12. Wright V, Schieve LA, Vahratian A, Reynolds MA. Monozygotic twinning associated with day 5 embryo transfer in pregnancies conceived after IVF. Hum Reprod 2004;19:1831 6. 13. Skiadas CC, Missmer SA, Benson CB, Gee RE, Racowsky C. Risk factors associated with pregnancies containing a monochorionic pair following assisted reproductive technologies. Hum Reprod 2008;23:1366 71. 14. Elizur SE, Levron J, Shrim A, Sivan E, Dor J, Shulman A. Monozygotic twinning is not associated with zona pellucida micromanipulation procedures but increases with high-order multiple pregnancies. Fertil Steril 2004;82:500 1. 15. Schachter M, Raziel A, Friedler S, Strassburger D, Bern O, Ron-El R. Monozygotic twinning after assisted reproductive techniques: a phenomenon independent of micromanipulation. Hum Reprod 2001;16:1264 9. 16. Alikani M, Noyes N, Cohen J, Rosenwaks Z. Monozygotic twinning in the human is associated with the zona pellucida architecture. Hum Reprod 1994;9:1318 21. 17. Carrillo-Vadillo R, Garcıa-Lozano JC, Lozano Arana MD, Molinı Rivera JL, Sanchez Martın P, Anti~nolo G. Two sets of monozygotic twins after intracytoplasmic sperm injection and transfer of two embryos on day 2. Fertil Steril 2007;88(1676):e3 5. 18. Abusheikha N, Salha O, Sharma V, Brinsden P. Monozygotic twinning and IVF/ICSI treatment: a report of 11 cases and review of literature. Hum Reprod Update 2000;6:396 403. 19. Saito H, Tsutsumi O, Noda Y, Ibuki Y, Hiroi M. Do assisted reproductive technologies have effects on the demography of monozygotic twinning? Fertil Steril 2000;74:178 9. 20. Cassuto G, Chavrier M, Menezo Y. Culture conditions and not prolonged culture time are responsible for monozygotic twinning in human in vitro fertilization. Fertil Steril 2003;80:462 3. 21. Menezzo YZ, Sakkas D. Monozygotic twinning: is it related to apoptosis in the embryo? Hum Reprod 2002;17:247 8. 22. Steinman G. Mechanisms of twinning. II. Laterality and intercellular bonding in monozygotic twinning. J Reprod Med 2001;46:473 9. 23. Fauser BC, Devroey P. Reproductive biology and IVF: ovarian stimulation and luteal phase consequences. Trends Endocrinol Metab 2003;14: 236 42. 24. Lane M, Gardner DK. Embryo culture medium: which is the best? Best Pract Res Clin Obstet Gynaecol 2007;21:83 100. 25. Moayeri SE, Behr B, Lathi RB, Westphal LM, Milki AA. Risk of monozygotic twinning with blastocyst transfer decreases over time: an 8-year experience. Fertil Steril 2007;87:1028 32. 26. Hall J. Twinning. Lancet 2004;362:735 43. Fertility and Sterility â 597