Department of Obstetrics and Gynaecology, Fukushima Medical College, Fukushima, Japan
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Abstract |
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Key words: fertilization/intracytoplasmic sperm injection/piezo-micromanipulator/pregnancy
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Introduction |
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Materials and methods |
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Preparation of oocytes and spermatozoa
Gonadotrophin-releasing hormone agonist (GnRHa) (Suprecur: Hoechst Marion Roussel, Tokyo, Japan) was administered at 900 µg per day from day 21 of the previous cycle (long protocol), and stimulation was provided in treatment menstrual cycles using pure follicle stimulating hormone (FSH) and human menopausal gonadotrophin (HMG). Human chorionic gonadotrophin (HCG) 10 000 IU was administered when the maximum diameter of follicles reached 17 mm, and the oocytes were collected 35 h later. The oocytes were cultured in human tubal fluid (HTF: Irvine Scientific Co., Santa Ana, USA) containing 6% plasmanate cutter (PPF: Bayer Yakukin Ltd, Osaka, Japan) for 38 h before ICSI. After that, pipetting was conducted in HEPES-buffered HTF (HEPES-HTF, sperm washing medium, Irvin Scientific Co.) containing 0.025% hyaluronidase (type VIII, Sigma Chemical Co., St Louis, MO, USA) to remove cumulus cells, and spermatozoa were injected into metaphase II oocytes only.
After semen was liquefied for ~30 min, as many motile spermatozoa were recovered as possible by the swim-up method using HTF. When the swim-up method was impossible, the sperm suspensions were prepared by centrifugal washing (250 gx10 min) using HEPES-HTF.
Instruments
An inverted microscope (IX-70: Olympus, Tokyo, Japan) equipped with Hoffman modulation optics (Hoffman Modulation Contrast, Model EP: Olympus) was used. For the microinjector (IM-4B: Narishige, Tokyo, Japan), a gas-tight syringe (#1750-LT: Hamilton, USA) was used, to which a 21 gauge needle with a rounded tip was attached. A polyethylene tube (external diameter 1 mm: PE-90, Clay Adams Intradermic Inc., Sparks, MD, USA) was connected directly to the needle through a needle holder (Figure 1). The tube was filled with distilled water. The needle holder for injection was attached to the drive unit of a piezo-micromanipulator (PMM-MB-A: Prime Tech Ltd, Tsuchiura, Ibaragi, Japan) was used. The drive unit of the piezo-micromanipulator was driven by a controller (PMAS-CT-140: Prime Tech Ltd).
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Method of conventional ICSI
Just before ICSI, air contained at the tip of the injection needle was extruded as much as possible. After that, small amounts of mineral oil were sucked into the needle, then small amounts of HEPES-HTF were sucked in. Next, a motile spermatozoon was taken into the injection needle from drops of the sperm suspension. The needle was transferred into 8% PVP drops. The spermatozoon was immobilized by pipetting, and injected in drops of the HEPES-HTF. Injection of the spermatozoon was performed after suction of a small amount of ooplasm to ensure penetration of the oolemma (Figure 3).
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Luteal support and judgement of pregnancy
Following the embryo transfer, luteal support (progehormone 50 mg/day, Mochida Pharmaceutical Co., Ltd., Japan) was administered for 10 days, and pregnancy was confirmed by detecting an increased urine HCG concentration 14 days after embryo transfer.
Measurement of mercury in the injection drops
We examined whether mercury dissolved in the injection drop. The volume of the injection drop was 510 µl, and was too small for measurement of the mercury concentration. Therefore, larger drops of 100 µl were prepared in the chamber. Fifty unfertilized mouse oocytes were injected with human spermatozoa in the same drop by the same manner used for human piezo-ICSI. Ten injection drops in the chambers were collected to the sampling tube (1.5 ml), with precautions taken to prevent contamination. The mercury concentration in the mHTF was measured by atomic absorption spectrophotometry.
Statistical significance was assessed using the 2 analysis test. At P < 0.05, the difference was considered statistically significant.
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Results |
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Discussion |
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In conventional ICSI, an injection needle penetrates the oolemma through the zona pellucida, which causes considerable deformation of the zona. This deformation may increase the internal pressure of the oocyte, induce the emission of ooplasm from the oocyte after extraction of the needle, and contribute to oocyte death. Since oolemma has high extendibility, the inserted needle may not penetrate the oolemma. Thus, the spermatozoon should be injected after the response of penetration into the oolemma is confirmed. This response has been confirmed by observation of the oolemma, which is pushed into the oocyte as the needle is inserted, then returns to its original position upon penetration. When this response cannot be obtained, the spermatozoon is injected after small amounts of ooplasm are sucked into the needle. On the other hand, the first report on piezo-ICSI was published by Kimura and Yanagimachi (1995), who used mice as subjects. By their method, oolema deformation by change in voltage of piezoelectric elements is achieved by the vibration of piezoelectric elements, and the zona pellucida can be penetrated without deformation by vibrating the injection needle fixed on the piezodriver. Similarly, after the oolemma is extended by inserting the needle, the oolemma can be punctured by applying one piezo pulse. In mice, the plasma membrane has high extendibility and ooplasm has low viscosity. As a result, the secure injection of the spermatozoon into the oocyte was considered difficult, and Kimura and Yamagimachi (1995) reported an 80% survival rate and 78% fertilization rate to injected oocytes using their method. We applied their method to human ICSI, and observed a significant increase in survival and fertilization rates. Improvements in survival rate were, according to Kimura and Yamagimachi (1995), due to the fact that the extended oolemma agglutinates more securely after extraction of the needle, and the internal pressure of the ooplasm is lowered due to less deformation. In addition, oocytes are sufficiently activated by sperm factor brought in by an injected spermatozoon (Homa et al., 1994; Tesarik et al., 1994; Parrington et al., 1996
; Palermo et al., 1997
). Thus, no suction of ooplasm is necessary, which makes it possible to conduct ICSI while minimizing damage to oocytes. Improvements in the fertilization rate were considered due to the elimination of failures in the injection of spermatozoa, as spermatozoa were injected only after the puncture of oolemma by the needle was confirmed. An injection needle with a flat tip is used in piezo-ICSI. The mechanism of smooth puncture of the zona pellucida and oolemma by the needle seems to be as follows: the needle vibrates back and forth by taking piezo pulses. At this point, since inertia of mercury at the tip of the needle is high, rapid variation in internal pressure is caused in the lumen from mercury to the tip, and by pressing the flat tip of the needle against the zona pellucida, the zona is perforated in the shape of the tip lumen. Deep insertion of the needle into the ooplasm by hand causes no rupture of the oolemma in ~82% of metaphase II oocytes (unpublished data), but applying one piezo pulse to such oocytes causes rupture of the oolemma by variation in the internal pressure at the tip of the needle. It is at the time when a needle breaks the oolemma that vibration is transmitted to an oocyte. Because the vibration added is one piezo pulse and no suction of ooplasm is conducted at the sperm injection, it is thought that the damage caused by piezo-ICSI is much less than that by conventional ICSI. Grinding and spiking of the tip of the needle are not necessary in piezo-ICSI, which is considered an advantage because it reduces the amount of preparation required by researchers. In conventional ICSI, immobilization of motile spermatozoa to be injected is conducted by abrasion of sperm tails or pipetting, while in piezo-ICSI it can be conducted relatively easily by applying several piezo pulses to sperm tails. Huang et al. (1996) first reported the application of the piezo-manipulator to humans. In this method, an injection needle having a unbevelled tip was also used, and they concluded that the fertilization rate and pregnancy rate were comparable to those for other successful techniques. However, they did not report any results comparing piezo-ICSI with conventional ICSI in their hospital.
With regard to embryonic development after ICSI, since no suction of ooplasm is conducted in piezo-ICSI, it was presumed that the influence on the cytoskeleton would be slight and a higher rate of embryos with good morphology could be expected. However, no difference was recognized between the two groups by grade classification of the embryos after injection. On the other hand, pregnancy rates were improved by piezo-ICSI, which is considered due to the higher number of transferred oocytes per embryo transfer that were obtained from higher survival and fertilization rates in piezo-ICSI.
We applied piezo-ICSI to human ICSI. This method was the same as that reported by Kimura and Yanagimachi (1995) in mice. In their report, the oocytes fertilized by piezo-ICSI developed into blastocysts better than those by conventional ICSI (68 versus 33%). All eight foster mothers receiving sperm-injected oocytes (by piezo-ICSI) delivered their own and foster offspring, and all pups grew into normal adults. It is thought that the mercury contained in the injection needle does not influence development of the embryo. In our experiment, mercury (0.6 µg/l) was detected in the injection drop. The mercury concentration of blood in healthy humans was reported to be 0.31.05 µg/l (Abraham et al., 1984 ; Snapp et al., 1989
). In our data (unpublished), the mean concentration of mercury in the cervical mucus of women was 220 µg/l and the concentration in the seminal plasma of men was 52.4 µg/l. Therefore, the value of mercury concentration in the injection drop is thought to be comparatively low and safe.
In conclusion, stable and favourable fertilization results can be obtained by piezo-ICSI, which is a promising method for obtaining improved ICSI results.
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Acknowledgments |
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Notes |
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References |
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Submitted on April 7, 1998; accepted on November 18, 1998.