1 Department of Obstetrics and Gynecology, Jichi Medical School, Minamikawachimachi, Tochigi, 329-0498 and 2 Institute for central clinic, Minamikawachimachi, Tochigi, 329-0431 Japan
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Abstract |
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Key words: ICSI/karyotype/oligoasthenozoospermia/Robertsonian translocation/spermatozoa
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Introduction |
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Materials and methods |
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Preparation of oocytes
B6D2F1 female mice, 812 weeks old, were induced to superovulate by s.c. injection of 8 IU of pregnant mare's serum gonadotrophin followed by i.p. injection of 8 IU human chorionic gonadotrophin (HCG) 48 h later. Oocytes were collected from the oviducts about 16 h after HCG injection. They were freed from cumulus cells by 5 min incubation in M2 medium containing 100 IU/ml hyaluronidase (Sigma). The oocytes were rinsed thoroughly in M2 medium and kept in M16 medium (Sigma) for up to 1 h at 37°C under 5% CO2 in air.
Sperm injection into oocytes
The injection of mouse oocytes with human spermatozoa was performed at 1723 h post-HCG administration. Oocytes that had extruded their polar body (metaphase II) were transferred into a small drop of M2 medium under mineral oil at 17°C for 5 min before injection by ICSI at 17°C. The injection pipette had an inner diameter of 4.55.0 µm, and the holding pipette had an inner and outer diameter of 8 and 130 µm respectively. The patient's motile spermatozoa were immobilized by two to three piezo-pulses (Kimura and Yanagimachi, 1995) before injection into mouse oocytes using a Piezo-driven micromanipulator (Prima Meat Packers, Tsuchiura-city, Ibaraki-ken, Japan). After injection, the oocytes were kept in M2 medium at room temperature (2425°C) for ~20 min before transfer to M16 medium at 37°C under 5% CO2 in air.
Cytological analysis
Following culture, the oocytes with one pronucleus or two pronuclei were transferred in M2 medium with 0.2 µg/ml nocodazole (Sigma) to prevent mouse and human pronuclei fusing and forming the common metaphase plate. They were checked every 30 min and were incubated until 1 h after the observation of pronuclear disappearance. The oocytes were fixed using a modification of the air-drying method for chromosome preparation from mouse eggs described by Tarkowski (1966). Fertilized oocytes were placed in a 1% sodium citrate hypotonic solution for 90 s. The swollen zygotes were transferred into a solution of 5:1:4 methanol:acetic acid:distilled water for a few seconds and transferred to the centre of a slide. Two to three drops of 3:3:1 methanol:acetic acid:distilled water solution were dropped onto the swollen zygotes until the oolemma was broken and the oocyte cytoplasm was dispersed. These procedures were done under direct visual control using a dissecting microscope. As soon as the ooplasm dispersed, the slide was dehydrated. Unfertilized oocytes were also fixed at 30 h after ICSI.
G-banding of chromosomes
Fresh metaphase spreads were incubated at 50°C in air for 48 h to age the chromosomes. The slides were treated with a 0.125% trypsin solution in a Ca2+- and Mg2+-free phosphate buffer solution at pH 6.8 for 40 s at room temperature (2425°C). The slides were washed with a Ca2+- and Mg2+-free phosphate buffer solution in 50% ethanol, and treated with 5% Gimsa stain solution for 4 min.
Statistics
The differences in survival and activation rates between the two categories of spermatozoa injected were statistically analysed using the 2-test.
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Results |
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Discussion |
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FISH is performed easier and faster for large numbers of spermatozoa. However, it is difficult to reveal the details of some chromosomal abnormalities, i.e. reciprocal carriers, inversions etc. The present method is certainly worth applying to these patients because of yielding G-banding of chromosomes without using any specific probes. The majority of spermatozoa displayed alternate chromosome segregation. Similarly, analysis of meiotic prophase cells in heterozygous carriers of different Robertsonian translocations have a preferential cis-configuration of the meiotic trivalent structure (Vidal et al., 1982; Luciani et al., 1984
; Templado et al., 1984
; Rosenmann et al., 1985
). This predominance of cis-configuration supports alternate segregation. In the present study, the incidence of adjacent chromosome segregation was observed in 8.9% of the spermatozoa, and that of abnormalities unrelated to the translocation was 4.4%. These findings agree with those of meiotic and epidemiological studies. The incidence of unbalanced constellations determined by sperm chromosome analyses is distinctly higher than that observed in prenatal diagnosis and neonates (Daniel et al., 1980
; Boue and Gallano, 1984
). The hypothesis of a 1:1 segregation ratio of normal and balanced gametes was based on family studies of carriers for Robertsonian translocations between D-group chromosomes der (D;D) (Evans et al., 1978
). With one exception (Balkman and Martin, 1983), normal and balanced sperm chromosome complements were found in almost equal numbers in all studies, utilizing the hamster egghuman sperm fusion assay. However, in couples with paternal carriers for a (13;14) Robertsonian translocation there is an excess of offspring with balanced karyotypes compared to those with normal karyotypes (Boue and Gallano, 1984
). In the present study, an excess of balanced spermatozoa was observed, but this difference was not a significant deviation from a 1:1 segregation ratio (
2 = 0.9, P > 0.3). Therefore, the present study suggests that normal and balanced sperm chromosome complements may not always be equal. After consultation with the patients, ICSI treatment was performed. Five oocytes were retrieved and four of them fertilized and cleaved. The three highest quality embryos were transferred and two implanted. Both fetuses were carriers of a balanced Robertsonian translocation detected by prenatal diagnosis at 16 weeks gestation. The pregnancy was uneventful and a healthy female infant with birthweight of 2335 g and a healthy male infant with birthweight of 2420 g were delivered at 36 weeks gestation by Caesarean section. In specific patients chromosomal abnormalities are thought to be a major contributor to the genetic risks of infertility treatment by ICSI (Mau et al., 1997
; Yoshida et al., 1997
; Meschede et al., 1998
). In terms of genetic counselling prior to ICSI treatment, the chromosomal analysis of human spermatozoa is thought to be feasible for such patients.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 24, 1999; accepted on February 7, 2000.