1 Clinica Medica 3, University of Padova, Padova and 2 Centro per la Diagnosi e la Terapia della Sterilità, Divisione di Ostetricia e Ginecologia-Arcispedale S. Maria Nuova, Reggio Emilia, Italy
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Abstract |
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Key words: ATP/fertilization/human spermatozoa/infertility/IVF
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Introduction |
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In previous studies, we have demonstrated that extracellular adenosine triphosphate (ATP), a physiological compound present in all cells, is a rapid and potent inducer of human sperm capacitation and acrosome reaction without affecting cell viability and motility (Foresta et al., 1992Foresta et al., 1996). Furthermore, in a standard hamsteregg penetration assay, we have demonstrated that ATP treated spermatozoa show a high fertilization rate (Foresta et al., 1992). The present study was designed to evaluate the effects of sperm incubation with ATP on oocyte fertilization, embryo quality and pregnancy rate in couples taking part in an IVF programme for male-factor infertility.
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Materials and methods |
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Ovarian stimulation
Ovarian stimulation was carried out by administering a gonadotrophin releasing hormone analogue (triptorelin, Decapeptyl 3.75; Ipsen, Paris, France) at day 21 of the cycle preceding that of ovarian stimulation with follicle stimulating hormone (FSH) (Metrodin; Serono, Milan, Italy) administered from cycle day 3. Follicular growth was monitored by daily vaginal ultrasound and serum oestradiol measurements starting after 9 days of FSH treatment. Human chorionic gonadotrophin (HCG 10 000 IU; Profasi HP, Serono, Milan, Italy) was administered when the cohort of follicles reached a diameter >18 mm. Oocytes were retrieved by transvaginal aspiration 36 h after HCG administration and then transferred to culture wells containing modified human tubal fluid (HTF) medium at 37°C in controlled atmosphere (5% CO2, 5% O2, 95% N2).
Semen collection and preparation
Semen samples were collected at the time of oocyte retrieval by masturbation after 3 days of sexual abstinence. After liquefaction at room temperature, seminal standard parameters following WHO guidelines (World Health Organization, 1992) were determined on all samples. Spermatozoa were selected by discontinuous Percoll gradients utilizing HEPES-buffered HTF medium (Irvine Scientific, Santa Ana, CA, USA). After selection spermatozoa were suspended in HTF medium and divided into two equal aliquots for control and treatment. In the treated samples, ATP (from a 100 mM stock solution in 0.9% NaCl saline) was added at a final concentration of 2.5 mM, whereas in the control samples an equal amount of a 0.9% NaCl solution was added. The amount of ATP stock solution added to the spermatozoa to obtain the final concentration (2.5 mM) depended upon the final volume of sperm suspension obtained after isolation, but it never exceeded 12.5 µl. Sperm samples were then incubated at 37°C in a controlled atmosphere for 60 min. After the incubation, spermatozoa from control and treated samples were washed, resuspended in modified HTF medium (supplemented with 0.5% HSA) and incubated with oocytes previously divided in two groups comparable for oocyte number and quality.
IVF procedure and assessment of fertilization
After sperm incubation in the presence or absence of extracellular ATP, 100 000 motile spermatozoa were added to each one of culture wells containing one oocyte each. Fertilization was evaluated after 1620 h for the presence of pronuclei. Cleavage was checked 4648 h after insemination and embryos were classified for the number of cells and quality (number and shape of blastomeres, percentage of fragments) and scored from 1 to 5, such that embryos scored as 1 and 2 were the `good' embryos and those that scored 35 were the `fair' embryos. Embryo transfer was performed on day 2 after oocyte retrieval, utilizing a Frydman catheter (Laboratoire C.C.D., Paris, France) for uterine transfer.
The luteal phase was supported by administering progesterone (50 mg i.m. daily, Gestone, A.M.S.A., Italy) starting from the day of embryo transfer. Serum ß-HCG test was performed 1115 days after the transfer and was repeated after 4 days in patients with a positive test. Ultrasound examination was performed 5 weeks after the transfer in all patients with a positive test and clinical pregnancy was diagnosed by the presence of a gestational sac with fetal echoes. In pregnant patients, the luteal phase support was stopped at the time of ultrasound examination.
Statistical analysis
Statistical analysis was performed utilizing the Statview 4.0 statistical software package (Abacus Concepts Inc., Berkeley, CA, USA). Data were analysed with Wilcoxon's signed rank test, 2 test or t-test when appropriate and were expressed as means ± SD. A P value <0.05 was considered as statistically significant.
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Results |
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Discussion |
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ATP is a physiological compound present in all living cells, and in recent years it has been demonstrated that it has a role in mediating important physiological functions in different cell types (Dubyak and El-Moatassim, 1993). We have previously demonstrated that in human spermatozoa, a concentration as high as 5 mM ATP possesses an important stimulatory action on fertilizing ability without any toxic or detrimental action on sperm motility and viability, thus indicating the presence of specific functional P2-purinergic receptors on the sperm plasma membrane (Foresta et al., 1992b
, 1996c
). Furthermore, it has been previously demonstrated that, at the time of ovulation, there is a rise in ATP concentration in female genital tract secretions and follicular fluid (Karuhn, 1977
), thus providing a possible physiological role for this nucleotide in in-vivo reproduction. The results of the present study, demonstrating that sperm incubation with extracellular ATP induced a significant increase of fertilization rate during IVF techniques, further support the hypothesis for an important role of this nucleotide in sperm activation and oocyte fertilization.
In conclusion, the present study shows that extracellular ATP can induce an increase in human sperm fertilizing potential and provides a biological rationale for the use of this nucleotide for the in-vitro treatment of human spermatozoa before oocyte insemination during IVF techniques. Further studies with a larger number of couples will be necessary to confirm the beneficial effects of this procedure.
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Acknowledgments |
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Notes |
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References |
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Submitted on July 3, 1998; accepted on November 20, 1998.