Comparison of development and implantation of human embryos biopsied with two different methods: aspiration and displacement

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1 Comparison of development and implantation of human embryos biopsied with two different methods: aspiration and displacement Wei-Hua Wang, Ph.D., Khalied Kaskar, M.S., Yuhong Ren, M.S., Jimmy Gill, M.D., Traci DeSplinter, M.D., Ghassan Haddad, M.D., and Manvinder Singh, M.D. Houston Fertility Institute/Tomball Regional Hospital, Tomball, Texas Objective: To compare the development and implantation of human embryos biopsied with two different methods for preimplantation genetic diagnosis (PGD). Design: Technique and method. Setting: A regional hospital IVF laboratory and private reproductive medicine clinic. Patient(s): Women undergoing IVF and PGD. Intervention(s): Day 3 embryos were biopsied with aspiration and displacement; the embryos were cultured to blastocyst stage and then transferred. Main Outcome Measure(s): Blastocyst rate, pregnancy rate, and implantation rate. Result(s): One hundred fifty-one embryos from 14 patients were biopsied with the blastomere displacement method and 51 embryos from 5 patients were biopsied with the aspiration method. Displacement used less time than aspiration; thus, the time of embryos exposed to biopsy solution was shorter when displacement was used. Blastocyst formation (55.6% 56.8%) and ongoing pregnancy rate (50%) were not different between the two biopsy methods. However, the implantation rate was significantly higher in patients with embryos biopsied using the displacement method (64.7%) than with the aspiration method (25%). Conclusion(s): Blastomere displacement uses less time and is an easy and simple method for embryo biopsy and could be used as an alternative method for embryo biopsy. Our results indicate that the displacement method minimizes embryo damage during biopsy that was indicated by a higher implantation rate. (Fertil Steril Ò 2009;92: Ó2009 by American Society for Reproductive Medicine.) Key Words: Biopsy, method, human embryos Since preimplantation genetic diagnosis (PGD) was introduced in human in vitro fertilization (IVF), it has become a routine method for examination of embryo aneuploidy, sex selection, and genetic disorders of embryos in humanassisted reproduction programs (1). It has been indicated that PGD significantly reduced pregnancy loss in IVF patients (2) and improved the pregnancy outcome of some patients, such as translocation carriers and in women of advanced maternal age (2 4). One of the most important factors affecting embryo development and implantation for PGD patients is the successful removal of one blastomere with limited adverse effects on subsequent embryo quality (5). With the advent of PGD, various methods have evolved for the successful isolation of a blastomere for testing of chromosomal abnormalities. Many factors that may influence development of embryos undergoing biopsy include extended exposure to acid Tyrodes Received March 20, 2008; revised May 14, 2008; accepted May 30, 2008; published online August 11, W.-H.W. has nothing to disclose. K.K. has nothing to disclose. Y.R. has nothing to disclose. J.G. has nothing to disclose. T.D. has nothing to disclose. G.H. has nothing to disclose. M.S. has nothing to disclose. Reprint requests: Wei-Hua Wang, Ph.D., IVF Laboratory, Tomball Regional Hospital, 605 Holderrieth, Tomball, TX (FAX: ; wangweihua11@yahoo.com). or Ca þþ /Mg þþ free medium, physical damage, and the removal of more than one blastomere (5). A recent study showed that PGD did not increase but significantly reduced the pregnancies and live birth rate in human IVF (6), and the investigators concluded that this reduction in implantation was because of the removal of a blastomere from the embryo. Blastomere biopsy is traditionally accomplished by direct aspiration of the blastomere through an opening in the zona pellucida. However, this method is difficult to learn, and can cause severe damage to the embryo by aspirating too many blastomeres, and may also rupture the biopsied blastomere, which would necessitate the removal of another blastomere, thereby reducing the developmental potential of the embryo (5). A less traumatic displacement method has been developed for biopsy of human day 3 embryos, which involves a zona opening and the introduction of medium into the perivitelline space (PVS) causing the blastomere to be extruded through the opened zona (7, 8). This displacement method of obtaining blastomeres is simple and easy to learn. Most importantly, this method significantly reduces the biopsy time and does not negatively impact the blastomere integrity or the subsequent development of the embryo to the blastocyst stage (8). However, a direct comparison has not been done between the two methods in terms of embryo development, pregnancy, 536 Fertility and Sterility â Vol. 92, No. 2, August /09/$36.00 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

and implantation. Therefore, in this study, we compared the two embryo biopsy methods in relation to subsequent embryo development and clinical pregnancy outcome.

2 and implantation. Therefore, in this study, we compared the two embryo biopsy methods in relation to subsequent embryo development and clinical pregnancy outcome. MATERIALS AND METHODS Source of Embryos Embryos were obtained from patients undergoing IVF treatment with PGD. The biopsy of embryos for PGD was approved by the institutional review board of the Houston Fertility Institute and Tomball Regional Hospital. FIGURE 1 Photographs of simplified displacement method for embryo biopsy. (A) An embryo being held using a holding pipette (left side), an opened zona by laser (arrowhead), and an assisted hatching pipette (arrow) being inserted into the zona. (B) A blastomere (arrow) being displaced and the assisted hatching pipette still inside the zona (out of focus). Bar ¼ 70 mm. Egg Collection and Fertilization Eggs were retrieved 35 to 36 hours after hcg injection. Four to five hours after egg retrieval, oocyte cumulus complexes were removed in HEPES-buffered HTF with 40 IU/mL hyaluronidase. Intracytoplasmic sperm injection (ICSI) was used to fertilize all metaphase II eggs. Fertilization was examined 16 to 20 hours after ICSI. Fertilized eggs were cultured in Cleavage Medium (Cooper Surgical, Trumball, CT) supplemented with 10% serum protein substitute (SPS). Blastomere Biopsy Embryos were cultured up to day 3 in Cleavage Medium. On day 3, embryos were placed in HEPES-buffered Ca þþ /Mg þþ free biopsy medium (Cooper Surgical) supplemented with 10% SPS for a period of 2 to 5 minutes before biopsy to facilitate the dissociation of the blastomeres from each other. Blastomeres were biopsied either via displacement or direct aspiration techniques. For the displacement method, embryos were held in place by gentle suction using a holding micropipette (Humagen Fertility Diagnostics, Charlottesville, VA). The embryo was rotated until the desired blastomere for biopsy was at the right side of the embryo. A laser was used to create the opening in the zona of the embryo where the PVS was relatively large or fragmentation was present. An assisted hatching micropipette (7 mm in diameter) filled with medium was placed through the opening in the zona (Fig. 1A). The media was gently expelled using a 1-mL syringe or mouthpiece pipette into the space within the embryo, forcing the blastomere closest to the opening to be extruded by the positive pressure created (Fig. 1B). Special care was taken to expel just enough medium to extrude only one blastomere. Once the blastomere was completely out of the embryo, the blastomere was moved away from the embryo using the same assisted hatching micropipette. In some cases, the blastomere was held in place by gentle suction on the assisted hatching micropipette and moved away from the embryo. For the direct aspiration method, embryos were held in place with a holding pipette with the blastomere to be aspirated at the 3 o clock position. An opening in the zona was created using a laser. A blastomere biopsy pipette with an inner diameter of 35 mm was place at the opening of the zona. With gentle suction using a 1-mL syringe, the blastomere was Wang. Biopsy of human embryos for PGD. Fertil Steril aspirated into the pipette. The micropipette was moved away from the embryo and the blastomere was gently expelled. The biopsied embryos were then washed and cultured in blastocyst medium (Cooper Surgical) supplemented with 10% SPS. On day 5, the biopsied embryos were evaluated for blastocyst formation and morphology. Blastocyst formation rates were calculated from blastocysts obtained on both days 5 and 6. All blastocysts were either transferred (day 5) or cryopreserved (days 5 and 6) for future use except that abnormal blastocysts were discarded as requested by the patients. Pregnancy and implantation outcome were determined by ultrasound gestational sac documentation. Fertility and Sterility â 537

3 Statistical Analysis Categoric variables (oocyte maturation, fertilization, cleavage, blastocyst development, live birth/ongoing pregnancy, and implantation rates) were analyzed with c 2. A value of P<.05 was considered to be statistically significant. RESULTS A total of 19 fresh (17 nondonor and 2 donor) cycles underwent IVF/PGD were performed in our clinic in 2007 and was included in this study. As shown in Table 1, 151 embryos were biopsied from 14 patients using the displacement method and 51 embryos were biopsied from 5 patients with the aspiration method. No significant differences were observed in the patient ages, average number of oocytes retrieved, or oocyte maturation rates. No differences were observed in the fertilization rates, day 3 embryo cleavage rates, and the blastocyst development rates. The time for zona opening was different between embryos depending on zona thickness and hardening, so we did not calculate for each embryo. After zona opening, we calculated the time for isolating a blastomere from embryo and found that displacement took 5 to 10 seconds, but aspiration took >60 seconds to isolate one blastomere from each embryo (sometimes it even took a few minutes with aspiration). Biopsy of four embryos with displacement (we placed four embryos in the dishes each time) can be completed within 10 minutes from placing embryos in biopsy solution to placing embryos back to the incubator after biopsy and washing. However, when the aspiration method was used, the time was about 30 minutes. Thus, the time of embryos exposed to biopsy solution was significantly reduced when displacement was used. In this study, 87.6% (displacement) and 86.3% (aspiration) of embryos were 5 to 10 cell stages at biopsy. After culture, 61.2% and 63.6% of embryos developed to blastocysts, respectively. Other embryos (12.4% and 13.7% for displacement and aspiration, respectively) were equal to or less than the four-cell stage, and 10.5% and 14.3% developed to blastocysts, respectively. The overall blastocyst rates were the same (55.6 vs 56.8%) between biopsy methods (Table 1). Most patients had a good quality (both trophoblast and inner cell mass) of blastocysts for transfer except that one patient with translocation had only one normal morula for transfer (all blastocysts were abnormal) and another patient had one early blastocyst and one morula for transfer. Embryo biopsy from both cases was performed with the displacement method, and both patients were not pregnant. In our clinic, there are two different methods for embryo transfer. One is to use a Frydman Echo tip trial catheter before embryo transfer to check if it is easy to pass the catheter to the cervix. If it is easy, we use a Frydman soft catheter to load and transfer embryos. Another is to use a TDT catheter for transfer. If it is difficult to pass a trial catheter to the cervix, a TDT catheter is used to pass to the cervix first and then embryos are loaded to the inner catheter for embryo transfer. All embryo transfers included in this study were done with a Frydman soft catheter, indicating that all transfers were easy. Therefore, there were no obvious differences in the embryo quality and ease of embryo transfer between two groups. TABLE 1 Comparison of embryo development and implantation of human embryos biopsied with two different methods. Biopsy methods Aspiration Displacement No. of cases 5 14 Age of patients Total No. of eggs retrieved Mean No. of eggs No. (%) of M-II 59 (85.5%) 171 (86.8%) No. (%) of 2PN 51 (86.4%) 156 (91.2%) No. (%) of embryos biopsied 51 (100%) 151 (96.8%) No. (%) of blastocysts 29 (56.8%) 84 (55.6%) No. of patients with transfer 4 a 10 a No. of live birth/ongoing pregnant 2 (50%) 5 (50%) Total No. of embryo transferred 8 17 Mean No. of embryos for transfer Total of No. embryo implanted 2 11 Implantation rate (%) 25 b 64.7 c a Patients without normal embryos or blastocysts for transfer were not included. b,c P<.05. Wang. Biopsy of human embryos for PGD. Fertil Steril Wang et al. Biopsy of human embryos for PGD Vol. 92, No. 2, August 2009

4 When the live birth/ongoing pregnancy and implantation rates were compared between the two groups, similar live birth/ongoing pregnancy rates (50%) were observed in the two groups. However, implantation was significantly higher (P<.05) in the embryos transfers after biopsy with displacement (64.7%) than with aspiration (25%). DISCUSSION There are three events in PGD that are different from regular IVF/ICSI: embryo biopsy, blastomere fixation, and fluorescence in situ hybridization or PCR. Although all affect the final diagnosis results, only embryo biopsy affects the subsequent embryo development. It is believed that biopsy of an intact blastomere without any damage to the embryo is the most important technical key for PGD (9). Various methods exist to achieve this goal, most of which require lots of practice and experience (5, 9). We have found that the displacement method is very easy to learn and does not require a lot of additional training (8). In addition, this method allows for the rapid removal of the blastomere, thereby limiting the exposure of the embryo to Ca/Mg free biopsy medium. For embryo biopsy, creating an opening in the zona is one of the factors that is important in affecting the embryo development. Traditionally, most centers have used acidified Tyrode s solution. However, prolonged exposure of the embryo to acidified solution is detrimental to further embryo development, and requires extensive training before performing this procedure. In contrast, laser zona drilling is a simpler and faster way to open the zona, thereby reducing the time for the biopsy (10, 11). Laser zona drilling is also an easy technique to learn, requiring a shorter training time, shorter amount of time to perform, and most importantly, it is more precise (11). Hence, the high success rates obtained in the present study may also be attributed to the use of laser zona opening. Furthermore, the size of the opening in the zona needed to extrude one blastomere is much smaller in the displacement method than in aspiration. The opening would only need to be approximately two-thirds of blastomere size to allow for the insertion of the assisted pipette and extrusion of the blastomere. However, during aspiration, the size of the opening is determined by the inner diameter of the biopsy pipette (usually 35 mm). Whether the zona opening is created using a laser,mechanical cutting, or acid Tyrodes, the extended time and exposure to these methods may possibly have detrimental effects to the embryo itself (5). The displacement method also allows for the biopsy of intact blastomeres because there is little or no direct contact between the blastomere and the pipette, as opposed to the aspiration method where the blastomere is squeezed into the biopsy pipette. Biopsy pipettes with a very small diameter or with jagged edges (unpolished) may increase the likelihood of rupturing the biopsied blastomere making it difficult for fixing onto a slide and further analysis of the nucleus. In the event of a blastomere being ruptured, a second blastomere is usually biopsied, thereby reducing the developmental potential of the embryo (5). Several studies from centers performing large numbers of PGD cases indicated that embryo biopsy did not affect embryo development (blastocyst formation), and PGD increased implantation rates (1 4). We also found that the blastocyst development rate of biopsied embryos was not different from nonbiopsied embryos (data not shown). In the present study, no difference in the blastocyst development rates was observed after two different embryo biopsies. However, the implantation was significantly higher (P<.05) in patients with embryos biopsied using the displacement method than using the aspiration method. The high implantation rate is reflective of good blastocyst quality, and clearly shows that the displacement method is atraumatic and less invasive to the developing embryo. Even though direct aspiration of blastomeres may not show any adverse effects on blastocyst development and pregnancy rate, this method may cause some inherent damage to the blastocyst that prevents it from proper implantation. Mastenbroek et al. (6) recently collected PGD data from multiple IVF centers based on large case numbers, and showed that PGD did not increase but significantly reduced the pregnancies and live birth rate in humans after IVF (5). The investigators pointed out that the biopsy of a blastomere on embryos prevented the embryo implantation, thus reducing the pregnancy and live birth rate. These results clearly indicate that embryo biopsy is the most important event in the PGD. Technicians in the IVF laboratory must have enough training and wide experience to perform an expert biopsy, as pointed out by Munne et al. (5). This training needs time and extra embryos. Thus, most small IVF centers require outside experienced technicians to perform embryo biopsy. However, this increases patients cost and is inconvenient for both the host laboratory and the outside technician. By using the current displacement biopsy method, the technician can use less time and fewer embryos for practice before performing biopsy on patient s embryos (8). Embryo implantation is affected by many factors, such as embryo quality and transfer procedures. In the present study, we did not find differences on the quality of blastocysts transferred and the ease of embryo transfer. In most cases, one or two good-quality embryos were transferred and the transfers were easy. The only differences between the two biopsy methods were the time the embryos were exposed to the biopsy solution and the ease of biopsy procedures. Displacement used a shorter time and was easier to isolate a blastomere from embryos; thus, the time the embryos were exposed to the biopsy solution is shorter. From these data, it would appear that higher implantation rates observed in the displacement group are because of the biopsy procedure. In conclusion, the limited data obtained in this study show that the displacement method is an efficient way of obtaining Fertility and Sterility â 539

5 blastomeres for PGD, and maintains the blastomere competence and subsequent clinical pregnancy. Furthermore, the resulting blastocysts show good implantation potential when compared with those blastocysts after direct aspiration. Because of its speed, ease of use, and positive clinical outcome, we recommend that the displacement method be used for biopsy purposes, especially for new technicians who are entering the field of PGD. REFERENCES 1. Verlinsky Y, Cohen J, Munne S, Gianaroli L, Simpson JL, Ferraretti AP, et al. Over a decade of experience with preimplantation genetic diagnosis: a multicenter report. Fertil Steril 2004;82: Munne S, Fischer J, Warner A, Chen S, Zouves C, Cohen J. Preimplantation genetic diagnosis significantly reduces pregnancy loss in infertile couples: a multicenter study. Fertil Steril 2006;85: Munne S, Sandalinas M, Escudero T, Velilla E, Walmsley R, Sadowy S, et al. Improved implantation after implantation genetic diagnosis of aneuploidy. Reprod Biomed Online 2003;7: Otani T, Roche M, Mizuike M, Colls P, Escudero T, Munne S. Preimplantation genetic diagnosis significantly improves the pregnancy outcome of translocation carriers with a history of recurrent miscarriage and unsuccessful pregnancies. Reprod Biomed Online 2006;13: Munne S, Gianaroli L, Tur-Kaspa I, Magli C, Sandalinas M, Grifo J, et al. Substandard application of preimplantation genetic screening may interfere with its clinical success. Fertil Steril 2007;88: Mastenbroek S, Twisk M, van Echten-Arends J, Sikkema-Raddatz B, Korevaa JC, Verhoeve HR. In vitro fertilization with preimplantation genetic screening. N Engl J Med 2007;357: Pierce KE, Michalopoulos J, Kiessling AA, Seibel MM, Zilberstein M. Preimplantation development of mouse and human embryos biopsied at cleavage stages using a modified displacement technique. Hum Reprod 1997;12: Wang WH, Kaskar K, Gill J, DeSplinter T. A simplified technique for embryo biopsy for preimplantation genetic diagnosis. Fertil Steril 2007 [Epub ahead of print]. 9. De Vos A, Van Steirteghem A. Aspects of biopsy procedures prior to implantation genetic diagnosis. Prenat Diagn 2001;21: Joris H, De Vos A, Devroey P, Liebaers I, Van Steirteghem A. Comparison of the results of human embryo biopsy and outcome of PGD after zona drilling using acid Tyrode medium or a laser. Hum Reprod 2003;18: Jones AE, Wright G, Kort HI, Straub RJ, Nagy ZP. Comparison of laserassisted hatching and acidified Tyrode s hatching by evaluation of blastocyst development rates in sibling embryos: a prospective randomized trial. Fertil Steril 2006;85: Wang et al. Biopsy of human embryos for PGD Vol. 92, No. 2, August 2009

Preimplantation genetic diagnosis: polar body and embryo biopsy

Human Reproduction, Vol. 15, (Suppl. 4), pp. 69-75, 2000 Preimplantation genetic diagnosis: polar body and embryo biopsy Luca Gianaroli SISMER, Via Mazzini 12, 40138 Bologna, Italy Scientific Director