A comparison of headfirst and tailfirst microinjection of sperm at intracytoplasmic sperm injection

A comparison of headfirst and tailfirst microinjection of sperm at intracytoplasmic sperm injection Bryan J. Woodward, M.Med.Sci.,a Keith H. S. Campbell, Ph.D.,a and Samuel S. Ramsewak, M.D.b a School of Biosciences, University of Nottingham, Sutton Bonington, Leicestershire, United Kingdom; and b Department of Obstetrics and Gynaecology, University of the West Indies, Saint Augustine, Trinidad and Tobago

Objective: To investigate whether the direction of sperm loading and exit from the injection pipette during intracytoplasmic sperm injection (ICSI) had any bearing on ability to cause fertilization or affect subsequent embryonic development. Design: Prospective randomized trial. Setting: Hospital-based IVF center. Patient(s): Twenty-five couples participating in an intracytoplasmic sperm injection (ICSI) program. Intervention(s): Sperm microinjection was randomly divided into either headfirst injection or tailfirst injection. Main Outcome Measure(s): Fertilization, embryo quality, and implantation rates. Result(s): There were no significant differences in the fertilization rates or the proportion of good-quality embryos, according to the direction of sperm injection. Of the embryos selected for transfer, 41.3% originated from headfirst sperm injection, and 58.7%, from tailfirst sperm injection. After transfer of either two or three embryos into 24 patients, 11 embryos implanted, with an equal probability that these embryos originated from either headfirst or tailfirst sperm injection. However, one dizygotic twin pregnancy was traced to the transfer of two embryos; one resulted from headfirst sperm injection, and one from tailfirst sperm injection. Conclusion(s): The direction of sperm microinjection at ICSI appears to have no effect on fertilization or subsequent development. The preliminary findings of this study have some interesting practical implications for the procedure of ICSI. (Fertil Steril 2008;89:711–4. 2008 by American Society for Reproductive Medicine.) Key Words: Fertilization, ICSI, microinjection, spermatozoa, success rates, sperm direction

Intracytoplasmic sperm injection (ICSI) involves direct microinjection of a single sperm into a metaphase II oocyte. Traditionally, the technique involves aspiration of the immobilized sperm into the injection pipette tailfirst, so that it can be microinjected headfirst into the ooplasm. However, the rationale for this may be questioned because ICSI bypasses the steps of normal fertilization for which sperm direction is clearly necessary. Theoretically, fertilization should occur, provided that the haploid male genetic component is microinjected into the oocyte regardless of direction. More than 175,000 ICSI cycles were undertaken worldwide in 2000 (1). Given the inevitable differences in the individual techniques of ICSI practitioners and the high number of oocytes that are injected worldwide on a daily basis, there is a possibility that some of these oocytes may be injected with the sperm entering the oocyte tailfirst. However, as far as we are aware, there have been no published reports investigating the efficacy of this technique. This study was performed to investigate whether the direction of sperm microinjection during ICSI had any bearing on ability to fertilize an oocyte or to affect subsequent development.

Received November 11, 2006; revised and accepted March 27, 2007. Reprint requests: Bryan Woodward, M.Med.Sci., School of Biosciences, University of Nottingham, Sutton Bonington, Leicestershire, LE12 5RD United Kingdom (FAX: 00-44-115-951-6302; E-mail: theeggman68@ hotmail.com).

0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.03.087

MATERIALS AND METHODS The direction of sperm microinjection was prospectively randomized to be either headfirst (H-ICSI) or tailfirst (T-ICSI) on sibling oocytes (n ¼ 213) for patients (n ¼ 25) who were attending our ICSI-ET program at the Medical Associates Hospital (St. Joseph, Trinidad). All couples were referred for treatment with ICSI as a result of a nonobstructive male infertility factor. Suboptimal sperm parameters were defined as a combination of sperm concentration of <20  106/mL, progressive motility of <50%, and ideal forms of <30%. Oocyte micromanipulation was performed in HEPESbuffered IVF medium (Sperm Preparation Medium; Medicult, Jyllinge, Denmark) in Falcon 1006 dishes (Fahrenheit, Rotherham, South Yorkshire, United Kingdom) by using eight 5-mL droplets surrounding a 5-mL droplet that contained 10% polyvinylpyrrolidone, all overlaid with clinical-grade liquid paraffin. Deliberate randomization to either H-ICSI or T-ICSI was decided by a coin toss immediately before each ICSI procedure: if the coin showed heads, the sperm was microinjected headfirst, and if the coin showed tails, the sperm was microinjected tailfirst. For H-ICSI, the sperm was aspirated into the injection pipette tailfirst and was injected into the oocyte headfirst; for T-ICSI, the sperm was aspirated into the injection pipette headfirst and was injected into the oocyte tailfirst. For each ICSI, the sperm was deposited at the same 9 o’clock position within the ooplasm, with the polar body secured at 6 o’clock.

Fertility and Sterility Vol. 89, No. 3, March 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.

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The ICSI procedure was performed under 400 magnification with an Olympus CK2 inverted microscope (Olympus, London, United Kingdom) that was equipped with Hoffman Modulation Contrast (Modulation Optics Inc., Greenvale, NY) as well as a heated stage set to 37 C. Motile sperm that demonstrated normal morphological features, as determined with light microscopy, were selected for injection. The same ICSI practitioner performed all micromanipulations by using the same micromanipulation station and air syringes (Research Instruments, Cornwall, United Kingdom) to minimize interoperator variation. After either H-ICSI or T-ICSI, the primary outcome parameters measured were whether the oocyte fertilized and how the embryo developed on day 2. Oocytes were co-cultured in either H-ICSI or T-ICSI groups in 20-mL droplets, with up to three oocytes per droplet. Normal fertilization was defined as the presence of two pronuclei (2PN) and two polar bodies at 16–18 hours after ICSI. Fertilized oocytes were transferred to fresh 20-mL droplets, with up to three zygotes per droplet, maintained in their groups according to ICSI sperm direction. The best two or three embryos were selected for day 2 transfer according to quality, regardless of whether they originated from H-ICSI or T-ICSI. Pregnancy was diagnosed by elevated b-hCG levels (>30 mIU/mL) at 12 days after transfer, and by transvaginal ultrasound 4 weeks later that demonstrated an intrauterine gestation sac, a fetal pole, and fetal heart tones. Implantation rates then were assessed according to whether H-ICSI or T-ICSI embryos were transferred. Statistical differences were analyzed by c2 (P<.05). Institutional review board approval was obtained for this study. RESULTS A total of 213 metaphase II oocytes from 25 patients underwent ICSI. All oocytes survived the microinjection procedure. There was an overall fertilization rate of 74.6% displaying 2PN, with 21.6% displaying no pronuclei, and 3.8%, monopronucleate. There was no significant difference (P>.05) in fertilization rates according to whether the oocytes were subjected to H-ICSI or T-ICSI (Table 1). Of the 159 2PN oocytes, 6 were frozen at the 2PN stage, and the remaining 153 were allowed to develop to day 2. Only one 2PN oocyte (0.7%) arrested at the one-cell stage on day 2, with both pronuclei having disappeared by this time. Of the 152 cleaved day 2 embryos, 56.6% were classed as good-quality embryos (grade I or grade II). There were no significant differences (P>.05) in embryo quality according to whether the embryos originated from H-ICSI or T-ICSI (Table 1). Embryos were selected for transfer on the basis of their quality alone; 48.4% (30/62) were considered grade I, 33.9% (21/62) were grade II, and 17.7% (11/62) were grade III. No grade IV embryos were transferred, because higher quality embryos always were available for preferred selection. Of the 62 embryos transferred, 40.3% originated from HICSI, and 59.7%, from T-ICSI (Table 2). Eleven transferred embryos implanted and developed into viable embryos with 712

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fetal hearts and ongoing pregnancy. This resulted in an implantation rate of 17.7% per embryo transferred and in a clinical-pregnancy rate of 33.3% per patient receiving an embryo transfer. Overall, eight patients became pregnant, five with a singleton and three with a dizygotic twin pregnancy. The 11 embryos causing a clinical pregnancy resulted from transfer of nine H-ICSI and nine T-ICSI embryos in total, with three patients receiving embryos originating from an HT combination, one patient, from an HHT combination, and one patient, from an HTT combination. Because all pregnancies resulted from transfer of two or three embryos, the results suggest an equal probability that each viable pregnancy could have originated from either H-ICSI or T-ICSI. However, one dizygotic twin pregnancy resulted from transfer of one H-ICSI embryo and one T-ICSI embryo, one twin pregnancy resulted from an HHH combination, and two singleton pregnancies resulted from a TTT combination. DISCUSSION During normal fertilization, the sperm enters the oocyte headfirst. Historically, this may be why sperm were microinjected headfirst during ICSI. However, because sperm head interactions at the zona pellucida and oolemma are redundant after ICSI, the reasons for continuing with this strict protocol may be questioned. This study showed no significant differences in fertilization rates, embryo development, or pregnancy rates according to the direction of sperm microinjection at ICSI. The overall sperm deposition site relative to the meiotic spindle may be relevant, because this affects the distance between the male and female chromatin (2). During this study, the sperm deposition site was always at 9 o’clock, with the polar body positioned at 6 o’clock. Variations in the ICSI technique, such as microinjection speed, were minimized by having the same ICSI practitioner perform all the micromanipulations on the same equipment, with the same air syringes. Thus, any differences resulting from the microinjection technique could not be attributed to ICSI practitioner variability. The absolute position of the sperm head immediately after ICSI could not be controlled because the sperm was injected into a three-dimensional suspension of ooplasm, and ooplasmic viscosity was thus a factor. However, it is unlikely that the sperm would have changed direction of placement as it entered the ooplasm. Intracytoplasmic sperm injection practitioners may prefer to microinject sperm headfirst for purely technical reasons, rather than biological considerations. The sperm head is larger than the tail and hence more visible under standard microscopy. This may allow for more precise placement of the sperm head at the tip of the injection pipette immediately before ICSI, allowing for greater precision than with tailfirst microinjection. However, omitting the need to aspire the sperm into the injection pipette tailfirst may expedite the ICSI procedure, because the overall time taken for ICSI is reduced. As a consequence, oocytes are exposed to the HEPES-buffered micromanipulation medium for less time.

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TABLE 1 Fertilization rates and day 2 embryo quality resulting from oocytes subjected to ICSI for sperm microinjected headfirst or tailfirst. Fertilization Direction H-ICSI T-ICSI Total

Day 2 embryo grade

n

2PN

1PN

0PN

IDII

IIIDIV

89 124 213

64 (71.9) 95 (76.6) 159 (74.6)

6 (6.7) 2 (1.6) 8 (3.8)

19 (21.3) 27 (21.8) 46 (21.6)

35 (60.3) 51 (54.3) 86 (56.6)

23 (39.7) 43 (45.7) 66 (43.4)

Note: All data are n (%). Values within columns showed no significant differences by c2 (P>.05). Woodward. Comparison of sperm direction at ICSI. Fertil Steril 2008.

Throughout this study, the sperm selection technique was subjective. The best-looking motile sperm were selected, as per our current clinical practice. No grossly abnormal sperm were injected, as assessed by the level of microscopy available. Future animal and human studies may consider the outcome of using grossly abnormal sperm or surgically retrieved sperm and the direction of microinjection. After ICSI, oocytes that appear to be unfertilized may have arrested at various stages of activation before pronuclear formation. In this study, any oocytes that displayed no pronuclei on day 1 and that remained at the one-cell stage by day 2 were classed as unfertilized. There were no significant differences (P>.05) noted in the incidence of monopronucleate oocytes between H-ICSI (6.7%) and T-ICSI (1.6%). Although the fertilization rate was unaffected by the direction of sperm microinjection, embryo quality was evaluated, in case any differences in the time interval for pronuclear alignment may hinder development. However, there was no difference in embryo quality on day 2. Embryo selection for transfer was based on embryo quality, regardless of sperm direction at ICSI. It is possible that preimplantation developmental patterns relating to the direction of sperm microin-

jection may have become more evident at later stages of development (eight-cell stage up to blastocyst). This study used implantation as the next measurable parameter, which necessitated preimplantation development in vivo after day 2. When multiple embryos are transferred, the fate of individual embryos remains uncertain. Embryo transfers in this study included a combination of H-ICSI and T-ICSI embryos, as well as solely H-ICSI or T-ICSI embryos. For those patients who became pregnant, half of the embryos originated from H-ICSI and half from T-ICSI, giving an equal probability that these viable embryos originated from either. However, the dizygotic twin pregnancy resulting from the HT combination clearly indicates that both the H-ICSI and T-ICSI embryos transferred were equally viable. Furthermore, the twin pregnancy resulting from an HHH combination, and the two singleton pregnancies resulting from a TTT combination, confirm H-ICSI and T-ICSI embryos to be equally successful. In conclusion, this study has shown that the direction of sperm microinjection at ICSI appears to have no effect on fertilization or subsequent development, provided that the sperm is successfully deposited into the ooplasm without damage to the oocyte. The presence of the sperm in the

TABLE 2 Pregnancy rates according to whether H-ICSI or T-ICSI embryos were transferred. No. of embryos transferred Embryo origin HH HT TT HHH HHT HTT TTT Total

No. of patients

H

T

Total

No. of pregnancies

1 5 4 2 4 4 4 24

2 5 0 6 8 4 0 25

0 5 8 0 4 8 12 37

2 10 8 6 12 12 12 62

0 3 (2 singleton, 1 twin) 0 1 (twin) 1 (twin) 1 (singleton) 2 (singleton) 8 (5 singleton, 3 twin)

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ooplasm appears to be more critical for fertilization than the sperm’s direction of microinjection. The preliminary findings of this study have some interesting practical implications for expediting the ICSI procedure. Acknowledgments: The authors acknowledge with appreciation both patient cooperation and the help of the team at Medical Associates Hospital.

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REFERENCES 1. International Committee for Monitoring Assisted Reproductive Technology. World collaborative report on in vitro fertilization, 2000. Fertil Steril 2006;85:1586–622. 2. Van Blerkom J, Davis P, Merriam J, Sinclair J. Nuclear and cytoplasmic dynamics of sperm penetration, pronuclear formation and microtubule organization during fertilization and early preimplantation development in the human. Hum Reprod Update 1995;1:429–61.

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