Laser-assisted removal of necrotic blastomeres from cryopreserved embryos that were partially damaged

FERTILITY AND STERILITY威 VOL. 77, NO. 6, JUNE 2002 Copyright ©2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.

Laser-assisted removal of necrotic blastomeres from cryopreserved embryos that were partially damaged Laura Rienzi, B.Sc.,a Zsolt Peter Nagy, M.D., Ph.D.,b Filippo Ubaldi, M.D.,a Marcello Iacobelli, B.Sc.,a Reno Anniballo, M.D.,c Jan Tesarik, M.D.,d and Ermanno Greco, M.D.a Centre for Reproductive Medicine, European Hospital, Rome, Italy; Clinica de Reproduca˜o Humana, Sa˜o Paulo, Brazil; Andrology Centre John MacLeod, Naples, Italy; and MAR&Gen, Molecular Assisted Reproduction and Genetics, Granada, Spain

Received July 24, 2001; revised and accepted February 21, 2002. Presented in part at the 55th Annual Meeting of the American Society for Reproductive Medicine, Toronto, Ontario, Canada, September 25–30, 1999. Reprint requests: Laura Rienzi, B.Sc., Centre for Reproductive Medicine, European Hospital, Via Portuense 700, 00149 Rome, Italy (FAX: 39-0665975727; E-mail: rienzi. [email protected]). a Centre for Reproductive Medicine, European Hospital. b Clinica de Reproduca˜o Humana. c Andrology Centre John MacLeod. d MAR&Gen, Molecular Assisted Reproduction and Genetics. 0015-0282/02/$22.00 PII S0015-0282(02)03109-6

1196

Objective: To examine whether the developmental potential of embryos that were partially damaged after freezing and thawing can be improved by removal of necrotic blastomeres before embryo transfer. Design: Prospective pilot study and observational clinical series. Setting: Private hospital. Patient(s): Two hundred thirty-five infertile couples undergoing frozen embryo transfer. Intervention(s): Removal of necrotic blastomeres from frozen-thawed human embryos. Main Outcome Measure(s): Pregnancy and implantation rates. Result(s): Removal of necrotic blastomeres from partially damaged frozen-thawed embryos before transfer increased rates of pregnancy (45.7% vs. 17.1%), ongoing pregnancy (40.0% vs. 11.4%) and ongoing implantation (16.2% vs. 4.3%) compared with the control group, in which necrotic blastomeres were not removed. A similarly high implantation rate (16.7%) was seen a subsequent clinical series in which necrotic blastomeres were removed from all partially damaged embryos. Conclusion(s): The viability of partially damaged frozen-thawed embryos can be improved by removal of necrotic blastomeres before embryo transfer. (Fertil Steril威 2002;77:1196 –201. ©2002 by American Society for Reproductive Medicine.) Key Words: Cryopreservation, embryos, human, laser-assisted hatching, necrotic blastomere removal

In the past decade, cryopreservation of human embryos has become widely used in assisted reproductive technology (1). This procedure allows storage of excess embryos and thus offers the possibility of increasing the cumulative pregnancy rate of a given treatment cycle. It also offers the opportunity to reduce the number of embryos transferred per procedure, thereby reducing the risk for multiple pregnancy. Rates of pregnancy and implantation with frozen-thawed embryos range from 10% to 30% and 5% to 15%, respectively (1–5). Many factors contribute to the success of frozen-thawed embryo transfer: the selection of embryos for freezing (6 – 8), the freezing protocol used (9 –11), hormone supplementation in the frozen-thawed embryo transfer cycle (12), and the ovarian stimulation procedure

used in the oocyte collection cycle (13). Frozen-thawed multicellular embryos are usually considered to have survived if at least half of the blastomeres are intact (14). In practice, both fully intact frozen-thawed embryos and embryos in which the freezing and thawing procedure has caused necrosis of up to half of the blastomeres are commonly used for transfer. It has been reported that the implantation potential of partially damaged embryos after thawing is significantly lower than that obtained with use of fully intact embryos (3.5% vs. 11.4%) (15). The poor implantation rates reported after transfer of damaged embryos may be caused in part by release of toxic metabolites from the necrotic blastomeres and the action of these metabolites on the remaining healthy blas-

tomeres. If this is the case, the viability and developmental potential of partially damaged embryos might be improved by removing the necrotic blastomeres from the embryos before transfer (16). We sough to test this hypothesis.

MATERIALS AND METHODS Patients All patients included in this study had embryos frozen with 1,2-propandiol by performing a slow freezing and rapid thawing protocol on day 3 after ICSI. Ovarian stimulation was done in all patients as described elsewhere (17) by using gonadotropin-releasing hormone agonist (s.c. buserelin acetate, 0.2 mg b.i.d. [Suprefact; Hoechst Marion Roussel, Milan, Italy]) in late luteal phase of the previous cycle, recombinant FSH (75 IU of Gonal-F [Serono, Rome, Italy] or 100 IU of Puregon [Organon, Oss, The Netherlands]), and hCG (Profasi; Serono). Patients were randomly allocated to the treatment or the control group. The study was approved by the ethical committee of the European Hospital.

Assisted Reproduction Procedures The oocyte and ejaculated sperm preparation and the ICSI procedure are extensively described elsewhere (18). Semen density and motility were evaluated according to the 1992 recommendations of the World Health Organization. Strict criteria were used to evaluate sperm morphology (19). Sperm selection was done by centrifugation on a discontinuous gradient (Sperm Grad; Scandinavian IVF Science, Gothenburg, Sweden) or by a swim-up procedure. Cumulus– corona– oocyte complexes were retrieved by vaginal ultrasonography– guided puncture 36 hours after hCG administration. The cells of the cumulus and corona radiata were removed by brief exposure to HEPES-buffered medium (Gamete 100; Scandinavian IVF Science) containing 20 IU/mL hyaluronidase fraction VIII (Hyase-10X; Scandinavian IVF Science) and by aspiration of the cumulus– corona– oocyte complexes in and out of a hand-drawn glass pipette (diameter, 135 ␮m, Sage BioPharma, Bedminster, NJ). The denuded oocytes were rinsed and incubated in pre-equilibrated culture medium (Scandinavian IVF Science). Only metaphase II oocytes were microinjected. A single, motile, normal-appearing spermatozoon was aspirated tailfirst in the injection pipette (Humagen, Charlottesville, VA) and injected into the oocyte cytoplasm. The injected oocytes were then cultured one by one in 35-␮L drops containing IVF medium covered with mineral oil (Ovoil; Scandinavian IVF Science). Fertilization was considered normal if two clearly distinct, equal-sized pronuclei were present. Embryo cleavage of the two-pronuclear oocytes was evaluated twice: after 22–24 hours and after 46 – 48 hours of in vitro culture. For FERTILITY & STERILITY威

each embryo, the number and the size of the blastomeres and the percentage of fragments were recorded. Cleaved embryos with at least six cells on day 3 and less than 10% fragmentation were considered type A. If the embryos had 10%–30% fragmentation or only four to five cells were present on day 3, they were considered type B. If more than 50% fragments were present or fewer than four cells were present on day 3, the embryo was classified as type C. Two or three embryos showing the fastest cleavage and the best morphology were selected for transfer in the oocyte collection cycle. All excess excellent (type A) and good (type B) quality embryos were cryopreserved.

Embryo Freezing and Thawing Protocols The freezing solution consisted of a phosphate-buffered solution with 25 mg/mL of human serum albumin (CryoPBS; Scandinavian IVF Science), a freezing solution containing 1.5 M of 1,2-propandiol in Cryo-PBS (freezing solution 1), or a freezing solution containing 1.5 M of 1,2propandiol plus 0.1 M sucrose in Cryo-PBS (freezing solution 2). A maximum of three embryos were frozen per straw. The freezing procedure was performed at room temperature. Embryos were first equilibrated at 22°C for 10 minutes in Cryo-PBS. They were then transferred in freezing solution 1 for 10 minutes and then in freezing solution 2 and immediately loaded into straws (paillette cristal, Cryo Bio System; IMV Technologies, Aigle, France). The straws were then placed into freezing chamber (CL-863, Cryologic, Mulgrave, Australia) at room temperature. The temperature was decreased to ⫺8°C at a rate of 2°C/min. Seeding was done manually by touching the straw close to the cotton plug with a nitrogen-cooled forceps. After 10 minutes of holding at ⫺8°C, the temperature was decreased at a rate of 0.3°C/ minute to ⫺30°C, followed by rapid cooling (⫺10°C/min) to ⫺110°C. The straws were then submerged in liquid nitrogen and stored until thawing. The straws were rapidly thawed at room temperature, and the retrieved embryos were transferred to a plastic four-well dish. They were then washed from the cryoprotectant by using Thaw Kit 1 (Scandinavian IVF Science) according to the manufacturer’s instructions.

Assessment of Embryo Survival and Removal of Necrotic Blastomeres For all patients, embryos were thawed 1 day before embryo transfer. Embryos were thawed until at least 3 surviving embryos per patient could be selected; the maximum number of embryos thawed was 10. Survival was evaluated immediately after thawing at 200⫻ magnification. The frozen-thawed embryos were considered to have survived if ⱖ50% of the initial blastomeres were intact and if at least three viable cells were present. Surviving embryos were then placed one by one in 10 ␮L-drops containing HEPES-buffered medium (Gamete 1197

FIGURE 1 Removal of necrotic blastomeres. (A), Partially damaged thawed embryo with one necrotic blastomere (arrow). (B) and (C), Aspiration of necrotic blastomere. (D), Embryo after removal of necrotic blastomere.

Rienzi. Necrotic blastomere removal. Fertil Steril 2002.

100) covered with mineral oil (Ovoil) under an inverted microscope.

Embryo Replacement and Assessment of Pregnancy

Laser-assisted hatching was performed in undamaged and partially damaged embryos by drilling a hole about 20 ␮m in diameter in the zona pellucida with a noncontact 1.48 diode laser (Fertilase, Montreux, Switzerland). The undamaged embryos underwent no further manipulation.

The cryopreserved embryos were transferred during the course of a natural cycle. Ovulation was triggered by administration of 10,000 IU of hCG (Profasi HP) when the preovulatory follicle was ⱖ16 mm, the endometrium was ⱖ7 mm in thickness with a type II pattern, the LH level was ⱕ5 IU/mL, and the progesterone concentration was ⱕ1 pg/mL. Progesterone supplementation (Protogest, 100 mg; AMSA, Barberino del Mugello, Italy) was started 48 hours after hCG administration and was continued for at least 2 weeks. The embryo transfer was scheduled for 115 to 120 hours after hCG administration. Patients received prednisolone p.o. (Deltacortene, 5 mg; Lepetit Spa, Anagni, Italy) 10 mg/d, for at least 4 weeks beginning on the first day of the cycle.

In partially damaged embryos, aspiration of necrotic blastomeres was performed as follows (Fig. 1). The embryo was immobilized by the holding pipette, and an assisted hatching micropipette 10 ␮m in diameter (Humagen) was introduced into the perivitelline space through the hole previously drilled in the zona pellucida. All necrotic blastomeres were then gently aspirated. Embryos that survived after thawing were cultured one by one in 35-␮l drops containing IVF medium covered with mineral oil (Ovoil) until transfer. In the control group, laser-assisted hatching was performed on all embryos that survived after thawing. However, embryos were then cultured directly, without additional manipulation. Eighteen to 20 hours after thawing, two to three embryos were transferred performed. Three types of transfer were done: fully survived embryos only, partially damaged embryos only, and both fully and partially survived embryos. 1198 Rienzi et al.

Necrotic blastomere removal

Pregnancy was confirmed by a serial increase in serum hCG concentration on two consecutive occasions at least 12 days after embryo replacement. A clinical pregnancy was determined by fetal cardiac activity on ultrasonography at 7 weeks. The clinical implantation rate was calculated by dividing the number of gestational sacs by the number of embryos transferred. Vol. 77, No. 6, June 2002

TABLE 1 Effects of removal of necrotic blastomeres on pregnancy and implantation rates after transfer of cryopreserved embryos. No. of embryos transferred (%) Patient group Control group (n ⫽ 35) Study group (n ⫽ 35)

No. of pregnancies (%)

Age (y)a

Undamaged

Partially damaged

Total

Ongoing

No. of ongoing implantations (%)

34.3 ⫾ 3.0 33.9 ⫾ 4.0

52 (56.5) 60 (69.6)

40 (43.5) 39 (39.4)

6 (17.1) 16 (45.7)b

4 (11.4) 14 (40.0)b

4 (4.3) 16 (16.2)c

b

b

Mean ⫾ SD. P⬍.05 vs. control group. c P⬍.01 vs. control group. a

b

Rienzi. Necrotic blastomere removal. Fertil Steril 2002.

Statistical Analysis All statistical tests were performed using Statview 4.0 statistical software for Macintosh (Abacus Concepts Inc., Berkeley, CA). A two-tailed test at P⬍.05 was considered significant. The unpaired Student’s t-test was used to compare mean values. A ␹ 2 test was used to compare rates of fertilization, cleavage, implantation, and pregnancy between the groups.

RESULTS According to the terms of the protocol approved by the ethical committee, we discontinued the prospective, randomized pilot study when it became clear that removal of necrotic blastomeres from partially damaged embryos significantly increased the chance of pregnancy after cryopreserved embryo transfer. Thereafter, necrotic blastomeres were always removed from partially damaged cryopreserved embryos before transfer. Accordingly, we report data obtained in the pilot study that demonstrate the superiority of the study protocol over the usual procedure, and we report data on a subsequent uncontrolled clinical series showing that similar success rates are maintained in a larger patient group.

Pilot Study All patients in the control and study groups had embryos that were eligible for transfer after freezing and thawing. The control and study groups did not differ significantly in mean (⫾SD) age (34.3 ⫾ 3.0 vs. 33.9 ⫾ 4.0), mean estradiol level (pg/mL) on the day of hCG administration (2,648 ⫾ 765 vs. 2,465 ⫾ 902), mean ampules of FSH used (27.3 ⫾ 4.8 vs. 28.1 ⫾ 3.1), mean number of oocytes retrieved (17.6 ⫾ 7.1 vs. 16.9 ⫾ 6.4), fertilization rate (72.4% vs. 71.8%), and cleavage rate (91.4% vs. 89.8%). The number and percentage of embryos surviving after thawing which were transferred, and the number and percentage of undamaged and partially damaged embryos, were also similar between groups (Table 1). However, significantly more partially damaged embryos (26 of 39 [74.3%]) in the study group underwent cleavage (at least one blastomere divided) by 18 hours of post-thaw culture compared FERTILITY & STERILITY威

with the control group (11 of 40 embryos [27.5%]) (P⬍.01). In addition, rates of pregnancy, ongoing pregnancy, and ongoing implantation were significantly higher in the study group than in the control group (Table 1). The cleavage rate of undamaged embryos did not differ significantly between the study group (43 of 60 [71.6%]) and the control group (35 of 52 [67.3%]).

Observational Clinical Series One hundred sixty-five frozen-thawed embryo transfers were analyzed. The mean age of the patients was 34.4 ⫾ 4.2 years. All patients had frozen-thawed embryos derived from ICSI cycles using ejaculated sperm. From January 1, 1999 to May 1, 2001, 181 thawing cycles were done, of which 165 (90.2%) resulted in embryo transfer. A total of 420 embryos, of which 247 (58.8%) were undamaged and 173 (41.2%) were partially damaged, were transferred. Removal of necrotic blastomeres was performed consistently in all partially damaged embryos. Rates of clinical pregnancy, ongoing pregnancy, and ongoing implantation rates in this series of patients were 38.2% (63 of 165 patients), 34.5% (57 of 165 patients), and 16.7% (70 of 420 embryos). These data were similar to those in the prospective randomized pilot study (Table 1). Only undamaged embryos were transferred in 62 patients, only partially damaged embryos were obtained after thawing procedure and transferred in 35 patients, and mixed transfer of undamaged and partially damaged embryos was done in 68 patients. These three groups did not differ significantly in any of the variables studied (Table 2). Post-thaw cleavage rates of the embryos were also similar among the groups (75.1% in those receiving undamaged embryos, 64.7% in those receiving partially damaged embryos, and 68.7% in those receiving both types; P⬎.05). Table 3 shows further characteristics of these groups.

DISCUSSION It was reported (15) that the implantation rate obtained by using partially damaged frozen-thawed embryos was three 1199

TABLE 2 Results of transfer of only undamaged embryos (group 1), only partially damaged embryos from which necrotic blastomeres were removed (group 2), and both types (group 3). No. of pregnancies (%) Patient group 1 (n ⫽ 62) 2 (n ⫽ 35) 3 (n ⫽ 68) a

Age (y)a

Total

Ongoing

No. of ongoing implantation (%)

34.7 ⫾ 4.2 34.5 ⫾ 5.5 34.6 ⫾ 3.5

26 (41.9) 10 (28.6) 27 (39.7)

24 (38.7) 9 (25.7) 24 (35.3)

32 (21.0) 11 (14.3) 27 (14.2)

Mean ⫾ SD.

Rienzi. Necrotic blastomere removal. Fertil Steril 2002.

times lower than that obtained by using fully intact embryos. The investigators suggested that degenerated blastomeres negatively affected further development of the intact blastomeres. Experiments on mouse embryos have demonstrated that damaged blastomeres have a toxic effect (20). In that study, destruction of one or two blastomeres of four-cell embryos dramatically reduced the rate of hatching (3.2%). However, embryo viability was restored after microsurgical removal of the degenerating material (hatching rate, 72%) (20). Our study is the first to test the effect of removal of necrotic blastomeres on the developmental potential of

TABLE 3 Characteristics of patients who had transfer of only undamaged frozen-thawed embryos. (Group 1), partially damaged embryos from which necrotic blastomeres were removed (group 2), or both types (group 3). Group Variable Age (y) E2 level on the day of hCG administration No. of ampules of FSH used No. of oocytes retrieved per cycle No. of fertilized oocytes (%)a No. of cleaved embryos (%)b No. of type A and B embryos (%)c No. of fresh embryos transferred (%)c No. of frozen embryos transferred (%)c

1

2

3

34.7 ⫾ 4.2 2,552 ⫾ 914

34.5 ⫾ 5.5 2,653 ⫾ 845

34.6 ⫾ 3.5 2,396 ⫾ 797

27.7 ⫾ 5.1

29.7 ⫾ 5.5

27.9 ⫾ 3.7

19.8 ⫾ 8.5

19.1 ⫾ 6.1

16.8 ⫾ 6.3

678 (71.5)

371 (75.9)

709 (76.1)

640 (94.3)

316 (85.2)

654 (92.2)

528 (82.5)

279 (88.3)

538 (82.2)

135 (21.1)

77 (24.4)

148 (22.6)

393 (61.4)

202 (63.9)

390 (59.6)

Note: Values with the plus/minus sign are means (⫾ SD). a Per injected oocyte. b Per fertilized oocyte. c Per embryo obtained. Rienzi. Necrotic blastomere removal. Fertil Steril 2002.

1200 Rienzi et al.

Necrotic blastomere removal

thawed human embryos in a clinical setting. In the first part of this study, which was randomized, removal of necrotic blastomeres through a laser-drilled opening in the zona pellucida significantly increased the rate of implantation of frozen and thawed human embryos. The removal technique was rapid, easy, and safe. Micromanipulation took slightly more time in the few embryos (⬍10%) that had more than two necrotic blastomeres located in different areas of the embryo. The implantation rate was slightly and nonsignificantly lower in cryopreserved embryo transfer cycles using only partially damaged embryos from which necrotic blastomeres were removed than in cycles using only undamaged embryos. Previously published data have showed a low implantation rate when using partially damaged frozen-thawed embryos whose necrotic blastomeres were left in place (15). Our data are in agreement with those obtained in the mouse model (20). Technically, removal of necrotic blastomeres from frozen-thawed embryos is similar to removal of fragments created during fresh embryo development (21). However, from the biological standpoint, the effect of these types of manipulation probably differs. Fragments are surrounded by an intact plasma membrane that impairs massive release of potentially harmful substances. Moreover, some of the fragments observed in human embryos are dynamic structures that can disappear spontaneously during further embryo development (22). Only certain types of embryo fragmentation reduce developmental potential (23). In contrast, necrotic blastomeres have no physiologic barrier to impede degradation products of nuclear and cytoplasmic components from reaching neighboring healthy cells. Even though the healthy blastomeres may resist the potentially harmful actions of such products, they may have to engage in detoxification and other defense mechanisms that would deplete resources needed for further development and differentiation. It is still not clear how much damage to the zona pellucida (ZP) is caused by the freezing-thawing procedure and how this is involved in the developmental potential of thawed embryos. Because cryopreservation may affect the ZP, it has Vol. 77, No. 6, June 2002

been suggested that partial zona dissection or assisted hatching may improve implantation rates after the transfer of frozen-thawed embryos (24, 25). We did not address this question because all embryos in all patients underwent laser assisted hatching; groups differed only by whether necrotic blastomeres were removed or left in place. Thus, we can be certain that necrotic blastomere removal rather than assisted hatching itself had a beneficial effect. The benefit of removal of necrotic blastomeres is also apparent from the cleavage rates. Significantly more partially damaged embryos in the study group underwent cleavage by 18 hours of post-thaw culture compared with the control group. A previous study found a clear association between blastomere survival and post-thaw intactness of the ZP (26). In that study, the ZP was fractured in only four embryos (0.8%). None of these four embryos had more than 50% viable blastomeres, and all were considered unsuitable for transfer. The proportion of fractured ZP seems to be related to suboptimal cryopreservation procedures (27). The use of mini-straws as storage containers, together with use of slowcooling and rapid-thawing protocols with propanediol as the cryoprotectant, seems to reduce damage to the ZP during embryo freezing and thawing. High rates of preclinical and clinical abortion after transfer of frozen-thawed embryos for ICSI have been reported (28, 29). The preclinical abortion rate in our series of 165 frozen-thawed embryo transfer was 5%, and six first-trimester losses occurred (10%). These results are similar to those after fresh embryo transfer at our center. These data confirm that removal of necrotic blastomeres has a beneficial effect on the viability of thawed embryos. In conclusion, our study confirms that impaired viability of frozen-thawed embryos is related in part to the presence of necrotic blastomeres. Partially damaged embryos have a good potential for implantation after removal of necrotic blastomeres. This procedure has been adopted as routine care in our laboratory. References 1. Mandelbaum J, Belaisch-Allart J, Junca AM, Antoine JM, Plachot M, Alvarez S, et al. Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum Reprod 1998;13(Suppl 3):161–74. 2. Kowalik A, Palermo GD, Barmat L, Veeck L, Rimarachin J, Rosenwaks Z. Comparison of clinical outcome after cryopreservation of embryos obtained from intracytoplasmic sperm injection and in-vitro fertilization. Hum Reprod 1998;13:2848 –51. 3. Van der Elst J, Camus M, Van den Abbeel E, Maes R, Devroey P, Van Steirteghem AC. Prospective randomized study on the cryopreservation of human embryos with dimethylsulfoxide or 1,2-propanediol protocols. Fertil Steril 1995;63:92–100. 4. Van der Elst J, Van den Abbeel E, Vitrier S, Camus M, Devroey P, Van Steirteghem AC. Selective transfer of cryopreserved human embryos with further cleavage after thawing increases delivery and implantation rates. Hum Reprod 1997;12:1513–21. 5. Edgar DH, Bourne H, Speirs AL, McBain JC. A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos. Hum Reprod 2000;15:175–9. 6. Cohen J, Inge KL, Suzman M, Wiker SR, Wright G. Videocinematography of fresh and cryopreserved embryos: a retrospective analysis of embryonic morphology and implantation. Fertil Steril 1989;51:820 –7.

FERTILITY & STERILITY威

7. Hartshorne GM, Wick K, Elder K, Dyson H. Effect of cell number at freezing upon survival and viability of cleaving embryos generated from stimulated IVF cycles. Hum Reprod 1990;5:857– 61. 8. Camus M, Van den Abbeel E, Van Waesberghe L, Wisanto A, Devroey P, Van Steirteghem AC. Human embryo viability after freezing with dimethylsulfoxide as a cryoprotectant. Fertil Steril 1989;51:460 –5. 9. Testart J, Lassalle B, Belaisch-Allart J, Forman R, Hazout A, Volante M, et al. Human embryo viability related to freezing and thawing procedures. Am J Obstet Gynecol 1987;157:168 –71. 10. Testart J, Lassalle B, Forman R, Gazengel A, Belaisch-Allart J, Hazout A, et al. Factors influencing the success rate of human embryo freezing in an in vitro fertilization and embryo transfer program. Fertil Steril 1987;48:107–12. 11. Van den Abbeel E, Camus M, Van Waesberghe L, Devroey P, Van Steirteghem AC. A randomized comparison of the cryopreservation of one-cell human embryos with a slow controlled-rate cooling procedure or a rapid cooling procedure by direct plunging into liquid nitrogen. Hum Reprod 1997;12:1554 – 60. 12. Van der Auwera I, Meuleman C, Koninckx PR. Human menopausal gonadotrophin increases pregnancy rate in comparison with clomiphene citrate during replacement cycles of frozen/thawed pronucleate ova. Hum Reprod 1994;9:1556 – 60. 13. Van der Elst J, Van den Abbeel E, Camus M, Smitz J, Devroey P, Van Steirteghem A. Long-term evaluation of implantation of fresh and cryopreserved human embryos following ovarian stimulation with buserelin acetate-human menopausal gonadotrophin (HMG) or clomiphene citrate-HMG. Hum Reprod 1996;11:2097–106. 14. Van Steirteghem AC, Van den Abbeel E, Camus M, Van Waesberghe L, Braeckmans P, Khan I, et al. Cryopreservation of human embryos obtained after gamete intra-Fallopian transfer and/or in-vitro fertilization. Hum Reprod 1987;2:593– 8. 15. Van den Abbeel E, Camus M, Van Waesberghe L, Devroey P, Van Steirteghem AC. Viability of partially damaged human embryos after cryopreservation. Hum Reprod 1997;12:2006 –10. 16. Nagy ZP, Rienzi L, Iacobelli M, Morgia F, Ubaldi F, Schimberni M, et al. Laser-assisted hatching and removal of degenerated blastomere(s) of frozen-thawed embryos improves pregnancy rate. Fertil Steril 1999;72 Suppl 1:S4. 17. Ubaldi F, Nagy ZP, Rienzi L, Tesarik J, Anniballo R, Franco G, et al. Reproductive capacity of spermatozoa from men with testicular failure. Hum Reprod 1999;14:2796 – 800. 18. Rienzi L, Ubaldi F, Anniballo N, Cerulo G, Greco E. Preincubation of human oocytes may improve fertilization and embryo quality after intracytoplasmic sperm injection. Hum Reprod 1998;13:1014 –9. 19. Kruger TF, Menkveld R, Stander FS, Lombard CJ, Van der Merwe JP, van Zyl JA, et al. Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril 1986;46:1118 –23. 20. Alikani M, Olivennes F, Cohen J. Microsurgical correction of partially degenerate mouse embryos promotes hatching and restores their viability. Hum Reprod 1993;8:1723– 8. 21. Palermo GD, Cohen J, Alikani M, Alder A, Rosenwazs Z. Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil Steril 1995;63:1231– 40. 22. Van Blerkom J, Davis P, Alexander S. A microscopic and biochemical study of fragmentation phenotypes in stage-appropriate human embryos. Hum Reprod 2001;16:719 –29. 23. Alikani M, Cohen J, Tomkin G, Garrisi GJ, Mack C, Scott RT. Human embryo fragmentation in vitro and its implication for pregnancy and implantation. Fertil Steril 1999;71:836 – 42. 24. Tucker MJ, Cohen J, Massey JB, Mayer MP, Wiker SR, Wright G. Partial dissection of the zona pellucida of frozen-thawed human embryos may enhance blastocyst hatching, implantation, and pregnancy rates. Am J Obstet Gynecol 1991;165:341– 4. 25. Check JH, Hoover L, Nazari A, O’Shaughnessy A, Summers D. The effect of assisted hatching on pregnancy rates after frozen embryo transfer. Fertil Steril 1996;65:254 –7. 26. Van Den Abbeel E, Van Steirteghem A. Zona pellucida damage to human embryos after cryopreservation and the consequences for their blastomere survival and in-vitro viability. Hum Reprod 2000; 15:373– 8. 27. Ziebe S, Bech B, Petersen K, Mikkelsen AL, Gabrielsen A, Andersen AN. Resumption of mitosis during post-thaw culture: a key parameter in selecting the right embryos for transfer. Hum Reprod 1998;13:178– 81. 28. VanSteirteghem AC, Van der Elst J, Van den Abbeel E, Joris H, Camus M, Devroey P. Cryopreservation of supernumerary multicellular human embryos obtained after intracytoplasmic sperm injection. Fertil Steril 1994;62:775– 80. 29. Macas E, Imthurn B, Borsos M, Rosselli M, Maurer-Major E, Keller PJ. Impairment of the developmental potential of frozen-thawed human zygotes obtained after intracytoplasmic sperm injection. Fertil Steril 1998;69:630 –5.

1201