Human Reproduction, Vol. 14, No. 3, 694-697,
March 1999
© 1999 European Society of Human Reproduction and Embryology
Sperm treatment with extracellular ATP increases fertilization rates in in-vitro fertilization for male factor infertility
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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Previous work from our laboratory has revealed that extracellular ATP is a rapid and potent activator of human sperm acrosome reaction and fertilizing ability. In the present study, we assessed the effects of in-vitro sperm incubation with ATP on fertilization and embryo development in couples undergoing in-vitro fertilization (IVF) for male factor infertility. Oocytes from 22 women undergoing ovulation induction were divided in two groups and inseminated in vitro either with selected spermatozoa from the corresponding partner suffering from male factor infertility pre-incubated with ATP (2.5 mM) for 1 h, or with spermatozoa incubated with 0.9% NaCl solution (control group). After insemination, fertilization was assessed by the presence of pronuclei and then by embryo cleavage. The fertilization rate in the group of oocytes inseminated with ATP-treated spermatozoa improved significantly with respect to the control group (65.7 versus 42.5%, P < 0.01). No significant differences were observed in embryo cleavage and embryo quality. Embryos from both treated and control groups were transferred together in 20 transfer procedures, and in two couples fertilization was not obtained. Nine pregnancies occurred: one biochemical, one miscarriage, and seven patients delivered 9 healthy babies. Two pregnancies were twin with an overall pregnancy rate of 40.9% per cycle and of 45% per transfer. In conclusion, the results of the present study demonstrate that, in humans, extracellular ATP induces a significant increase of sperm fertilizing potential, as these findings are a rationale for the use of ATP for in-vitro treatment of human spermatozoa during IVF.
Key words: ATP/fertilization/human spermatozoa/infertility/IVF
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Introduction |
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Among infertile couples undergoing in-vitro fertilization (IVF) for male factor infertility, ~40% will have failed fertilization (Tournaye et al., 1992
). Fertilization is a complex process of sperm motility hyperactivation, capacitation and acrosome reaction triggered by factors from the egg and its surrounding structures, such as progesterone and ZP3 (Foresta et al., 1992a, 1993; Arnoult et al., 1996). Fertilization failure may be due to alterations of these complex processes or to general methodological problems related to IVF procedures (Tournaye et al., 1994). At present, the majority of spermatozoa and oocyte functions cannot be investigated and in many cases IVF represents the only way to resolve infertility problems (Oehninger et al., 1991). In recent years, the introduction of intracytoplasmic sperm injection (ICSI) has improved the results of IVF in severe male factor infertility and in couples with previous IVF failure (Van Steirteghem et al., 1993). Although the results of this technique are promising, it requires highly specialized personnel and equipment and it is not available in all IVF units. Furthermore, ICSI is not without risks, including possible damage to the oocyte or transmission of some genetic abnormalities such as cystic fibrosis transmembrane receptor (CFTR) gene alterations or deletions of the long arm of Y chromosome (Foresta et al., 1996a,b). A strong debate regarding the role of ICSI in human reproduction has been opened recently (Foresta et al., 1996a,b). IVF techniques that utilize autonomous sperm penetration of oocyte seem to be preferred to ICSI, since it can be argued that in IVF the penetrated spermatozoon has undergone normal capacitation, acrosome reaction and other physiological processes related to oocyte fertilization (Foresta et al., 1996b). In this regard, previous studies have reported the use of pharmacological agents to stimulate sperm function in order to improve the fertilization rates in standard IVF techniques (Aitken et al., 1986; Rees et al., 1990; Yovich et al., 1990; Tesarik et al., 1992a,b). Among the different agents proposed so far and known to influence sperm functions, methylxanthines have been extensively utilized both in vitro and in vivo (Yovich et al., 1990, 1993; Tesarik et al., 1992a,b). It has been demonstrated that methylxanthines increase sperm motility hyperactivation (Tesarik et al., 1992a) and the acrosome reaction (Tesarik et al., 1992b). Recent studies have reported higher fertilization rates with the use of pentoxifylline in IVF in couples with male factor infertility (Yovich et al., 1988; Tesarik et al., 1993; Tarlatzis et al., 1995), but conflicting results have been observed (Yovich et al., 1993; Tournaye et al., 1993, 1994; Lacham-Kaplan et al., 1994; Tarlatzis et al., 1995). 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 hamster–egg 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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Subjects
The study was approved by the ethics committee at the hospital and all couples were informed about the use of a sperm activator in the treatment of spermatozoa for IVF and gave their informed consent. From November 1996 to January 1997, 22 couples were enrolled in the study. The mean age of the female partners was 34.6 ± 4.5 (SD) while that of male partners was 36.2 ± 3.7 years. Long-standing male-factor infertility was diagnosed on the basis of three seminal analyses performed on three different occasions separated by a 3 week interval. The mean duration of infertility was 4.4 ± 2.0 years. All female partners were completely normal after their infertility work-up, which included endocrinological and immunological investigations as well as hysterosalpingography.
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 16–20 h for the presence of pronuclei. Cleavage was checked 46–48 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 3–5 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 11–15 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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In Table I
, the seminal characteristics of sperm samples obtained from patients on the day of oocyte retrieval before and after sperm treatment with ATP are reported. No differences in motility and viability were observed before and after sperm treatment with extracellular ATP. Table II summarizes the main data regarding inseminated oocytes, fertilization rates and embryo quality. Overall, 205 oocytes were inseminated (9.3 ± 3.7/patient) with a fertilization rate of 53.7%. In particular, 106 oocytes were inseminated with control spermatozoa (4.8 ± 1.8 per patient) with a fertilization rate of 42.5%, and 99 oocytes were inseminated with ATP-treated spermatozoa (4.5 ± 2.0 per patient, not significantly different from control group) with a fertilization rate of 65.7%, significantly different from that of control spermatozoa (P < 0.01). In one couple fertilization was obtained only in the control group and not in the treated group. In two couples, fertilization was not obtained either in control or in the treated group. No significant differences were observed in embryo cleavage and embryo quality between the control and the treated group. Overall, 106 embryos from both treated and control groups were transferred together in 20 transfer procedures with a mean of 4.8 ± 2.9 embryos per patient. Nine pregnancies occurred: one biochemical, one patient miscarried before the 8th week of pregnancy and seven patients delivered nine healthy babies, of which two pregnancies were twin (pregnancy rate/cycle = 40.9%, pregnancy rate/transfer = 45.0%). In all patients, no obstetric problems have been reported during pregnancy and delivery and nine healthy babies were born with normal karyotypes and normal phenotypes.
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Discussion |
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The main goal of IVF techniques is to improve the fertilization rate and to obtain embryos of good quality. It has been reported that with the modern IVF techniques in male factor infertility there is a fertilization rate of ~60% (Tournaye et al., 1992
). Therefore, different methods to increase sperm fertilizing potential during IVF procedures have been proposed (Aitken et al., 1986; Rees et al., 1990; Yovich et al., 1990; Tesarik et al., 1992a,b). Most studies have utilized in-vitro sperm incubation with pentoxifylline, an inhibitor of phosphodiesterase (Tash and Means, 1983) that induces accumulation of cAMP that in turn activates still unknown processes influencing sperm motility and acrosome reaction (De Jonge et al., 1991). However, the results of these studies have been contradictory and no definitive data have been reported (Tournaye et al., 1993, 1994; Yovich et al., 1993; Lacham-Kaplan et al., 1994; Tarlatzis et al., 1995). Other authors have reported that another pharmacological agent, 2-deoxyadenosine, possesses stimulating effects on sperm motility and fertilizing ability when utilized for IVF (Aitken et al., 1986; Imoedemhe et al., 1992), although the effects of this agent have also been questioned (Tournaye et al., 1994). Furthermore, both pentoxifylline and 2-deoxyadenosine exert detrimental effects on oocytes and embryos (Scott and Smith, 1995), thus limiting the use of these agents. The results obtained in our previous studies demonstrated that extracellular ATP is a non-toxic potent and rapid activator of human sperm fertilizing potential (Foresta et al., 1992b) and prompted us to verify these effects in a clinical study with human oocytes. The results of this study show that fertilization rates obtained with the use of ATP-treated spermatozoa were significantly higher than those of a control group, with comparable embryo development in terms of cleavage percentages and embryo quality. The similar sperm motility and viability, cleavage rates and embryo quality between treated and control group demonstrate that sperm treatment with ATP has no detrimental effects on both sperm viability, oocyte survival and maturation and embryo development.
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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The authors wish to thank Drs Cinzia Campari, Leda Sconcerti and Barbara Valli for their skilful work in performing sperm isolation, oocyte insemination and embryo culture.
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Notes |
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3 To whom correspondence should be addressed at: University of Padova, Clinica Medica 3, Via Ospedale 105, 35128 Padova, Italy
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Submitted on July 3, 1998; accepted on November 20, 1998.
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