Absence of glucose decreases human fertilization and sperm movement characteristics in vitro

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1 hrep$$0107 Human Reproduction vol.12 no.1 pp , 1997 Absence of glucose decreases human fertilization and sperm movement characteristics in vitro Maha M.Mahadevan 1, Michael M.Miller and (Edwards, 1981). Others are complex media such as Ham s Dean M.Moutos F10 which are suitable for long-term serum-free cultures and contain nutrients such as vitamins, amino acids, and other Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA metabolites (Ham, 1963; Lopata, 1982). Quinn (1995) reported that absence of glucose in a simple medium decreased human 1 To whom correspondence should be addressed at: Arkansas IVF rates but that normal fertilization could be achieved Reproductive Technology Laboratory, 5800 W. 10th, Suite 702, Little Rock, Arkansas USA by increasing the number of spermatozoa inseminated. In agreement with animal studies, he reported enhanced pregnancy The effect of glucose in a modified Ham s F10 medium rates when human zygotes were cultured in the absence of (MM) without hypoxanthine, phosphate and transition glucose (Quinn, 1995; Quinn et al. 1995). Recently, we also metals on human fertilization and sperm survival in vitro reported that culturing zygotes in a modified Ham s F10 was determined. Mature human oocytes from in-vitro medium lacking glucose, transition metals, hypoxanthine and fertilization (IVF) patients or Percoll-washed human sper- phosphates (MM) resulted in better human embryo growth matozoa were randomly allocated to one of the treatment and pregnancy rate in comparison with culture in normal groups: normal Ham s F10, MM, MM with 5 mm glucose Ham s F10 (Mahadevan et al., 1996). It is clinically important (HGMM) and MM with 0.5 mm glucose (LGMM). Oocytes to determine whether MM with no or low concentrations of were inseminated in one of the four media for h and glucose is able to support normal fertilization and subsequently checked for fertilization. Sperm were incubated likewise produce better quality embryos and higher clinical pregnancy for 4 and 24 h, and sperm motility and sperm movement rates. Another rationale for assessing medium with low glucose characteristics including average path velocity (VAP), cur- is provided by the observation that, at the time of ovulation, vilinear velocity (VCL), straight line velocity (VSL), ampli- glucose concentration in tubal fluid decreased from 5 to tude of lateral head displacement (ALH), beat cross 0.5 mm in the human (Gardner et al., 1996). frequency (BCF), straightness (STR), and linearity (LIN) The energy for sperm motility and movement characteristics were determined using computer-assisted semen analysis. necessary for fertilization is produced by mitochondria in the Fertilization rates were significantly lower in oocytes cul- mid-piece of the spermatozoon (Mitchell et al., 1976). ATP is tured in MM (23.8%) compared to LGMM (75.5%), generated by glycolysis of sugars and mitochondrial respiration. HGMM (73.6%) or Ham s F10 (71.1%). Sperm character- Energy substrates have various effects on fertilization in vitro istics after 4 h incubation in all four media were similar, in different mammals and the effects are species specific. except VAP, VSL, VCL and ALH were significantly lower Glucose appears to be necessary or beneficial for fertilization in MM (no glucose) in comparison with the other three in mice (Hoppe, 1976; Fraser and Quinn, 1981), and rat media. After 24 h VAP, VSL, VCL, ALH, LIN and percent- (Tsunoda and Chang, 1975; Niwa and Iritani, 1978), whereas age rapid spermatozoa were significantly higher in sperma- in bovine (Parrish et al., 1989) and guinea pig (Rogers tozoa incubated in HGMM or Ham s F10 compared with and Yanagimachi, 1975) capacitation and/or fertilization is MM or LGMM. Also after 24 h, the percentage of spermato- decreased in the presence of glucose. zoa which were highly motile was greater in HGMM than The purpose of this preliminary investigation was to compare in Ham s F10. Absence of glucose significantly lowered human fertilization rates and sperm survival in vitro in either fertilization rates and sperm movement characteristics Ham s F10 (6 mm glucose) or in MM medium with 0, 0.5 in vitro. (LGMM), or 5 mm glucose (HGMM). Key words: culture medium/fertilization/glucose/movement characteristics/spermatozoa Materials and methods Introduction At present, in-vitro fertilization (IVF) programmes are using several different types of media based on animal and human studies (Bavister, 1995). Some are based on simple salt solutions formulated by Ringer, Earle, and Eagle with the addition of pyruvate, lactate, glucose and albumin, e.g. Earle s Culture medium Both Ham s F10 and MM were purchased (Gibco BRL, Grand Island, NY, USA) as powder and prepared in our laboratory. HGMM and LGMM were made by adding 1 ml and 0.1 ml of 50 mm stock glucose (Sigma Chemical Co, St Louis, MO, USA) to 9 and 9.9 ml of MM respectively. Osmolarity was adjusted to ~280 mosm by addition of water. MM contained all chemicals in Ham s F10 except for glucose, hypoxanthine, CuSO 4, FeSO 4, ZnSO 4, Na 2 HPO 4 and European Society for Human Reproduction and Embryology 119

2 M.M.Mahadevan et al. KH 2 PO 4. In addition, all media contained 25 mm NaHCO 3, 2.23 mm Ca lactate and 5 mg/ml bovine serum albumin (BSA). Table I. Effect of glucose on fertilization of human oocytes in vitro Fertilization of oocytes Modified media (MM) Standard protocols for ovarian stimulation were used for IVF (Droesch Ham s F10 5 mm Glu 0.5 mm Glu 0 mm Glu et al., 1989). Briefly, patients were down-regulated with leuprolide acetate (Lupron; TAP Pharmaceuticals, Dearborn, IL, USA) beginning No. of patients No. with at least one 17 (94.4) 17 (94.4) 17 (94.4) 4 (36.4) in the mid-luteal phase. Once ovarian suppression was achieved, a oocyte fertilized (%) human menopausal gonadotrophins (HMG) were begun. Oocytes No. oocytes were retrieved h after human chorionic gonadotrophin (HCG) inseminated injection, washed in MM medium and pooled in one or two dishes. No. oocytes fertilized 43 (71.7) 39 (73.6) 37 (75.5) c 5 (23.8) b (%) Patients with male factor infertility, aged 38 years and/or fewer Fertilization rate than eight oocytes retrieved were excluded from the study, which range (%) consisted of a total of 18 patients. Oocytes were classified subjectively Inter-coefficient of as mature, moderately mature, immature, over-mature (luteinizing) variation (%) or atretic based on the appearance of oocyte, corona and cumulus No. polyspermy ( 3PN) under dissecting microscope (Mahadevan and Fleetham, 1990). Pooled mature oocytes were selected randomly and placed in groups of one No. with 1PN to three oocytes in an organ culture dish containing one of the four media supplemented with 5 mg/ml BSA (Sigma) in the following PN pronuclear; Glu glucose. Randomization (Monte Carlo) test comparing all four groups, P order: Ham s F10, HGLM, LGMM and MM. All other non-mature oocytes were inseminated in Ham s F10 and not included in the b χ 2 test comparing all four groups, P study. In all, ~ motile mini-percoll (Sigma)-washed spermato- c χ 2 test comparing all groups except 0 mm Glu MM, P zoa (Ord et al., 1990) in MM were added to each dish containing mature oocytes. The contact-time between the spermatozoa and MM was ~2 3 h prior to mixing with oocytes. Oocytes were examined , non-motile head size 4, non-motile head intensity 90, medium for fertilization ~12 20 h after insemination. Oocytes and embryos VAP 25 µm/s, straitness (STR) threshold 80% and slow motile cells were incubated in humidified air of 5% CO 2,5%O 2 and 90% N 2 yes. The following sperm movement parameters were determined: at 37 C. average path velocity (VAP), curvilinear velocity (VCL), straight line Four zygotes were left in culture for transfer and the remainder of velocity (VSL), amplitude of lateral head displacement (ALH), beat the embryos were frozen at the two pronuclear (2PN) stage. Whenever cross frequency (BCF), straightness (STR), and linearity (LIN). When possible, at least two embryos were cultured together in an organ the VAP was 25 µm/s, the spermatozoa were considered highly culture dish containing 2 ml of culture medium. Therefore, the effect motile (%RAPID). Coefficients of variation for all parameters examof the fertilization media on the embryo quality or pregnancy rates ined were 10% as assessed by using a quality control video tape was not investigated in this study. Once it was obvious that media on each day of analysis. When fresh samples were used intra-assay without glucose (MM) had a significantly lower fertilization rate, that coefficients of variation were 15% except BCF (17.2%). part of the trial was discontinued (after 11 patients in each group) and the trial continued with the other three media (in 18 patients). Statistical analysis Comparisons were done within each patient. The media in which the oocytes were inseminated and cultured were statistically compared in terms of number of patients with at least one Sperm analysis oocyte fertilized (Randomization test; Monte Carlo) and proportion of Sperm survival and movement characteristics were studied using oocytes fertilized (χ 2 test). The sperm motility and movement semen samples from five different donors undergoing investigation characteristics in different media were compared by Kruskal Wallis for IVF, who had normal semen analysis parameters based on World analysis of variance with Duncan multiple comparison. The study Health Organization (WHO, 1992) criteria and normal fertilization was approved by the Human Research Advisory Committee at the rates in vitro. Spermatozoa were washed with the mini-percoll University of Arkansas for Medical Sciences, USA. technique as for IVF using MM containing 5 mg/ml BSA (Sigma). Manual semen analysis was performed with a Makler chamber using phase contrast 20 optics and the necessary volume of sperm Results suspension was added to 1 ml of appropriate medium (Ham s F10, The outcome of insemination of oocytes in four different HGLM, LGMM and MM) in a5mltissue culture tube which was modified Ham s F10 media are given in Table I. The fertilization equilibrated in a humidified incubator at 37 C and 5% CO 2,5% rate in the modified Ham s F10 medium without glucose (MM) O 2, and 90% N 2. Computer-assisted sperm analysis (CASA) was was significantly lower than that in the MM media with performed after 4 and 24 h (HTMA, version 10.6; Hamilton Thorn 0.5 mm glucose (LGMM) or normal Ham s F10 medium Research Inc., Danvers, MA, USA). A well-mixed sample of 7 µl (P ). Similarly, the number of patients with at least was placed in a 20 µm depth glass chamber (Cell-vu; Fertility one oocyte fertilized was also significantly lower in MM when Technologies, Natick, MA, USA) which was warmed on a 37 C slide compared with the other three media (P ). There warmer. The definitions of the sperm movement characteristics are described by Mortimer (1989). The HTMA settings were as follows: were no significant differences between the other three groups internal optics ( 10), 37 C, 3 10 fields acquired to obtain minimum in terms of fertilization. We observed that, at the time of of 100 spermatozoa, acquire manual, magnification 1.96, frame rate fertilization check, oocyte corona complexes were not attached 60/s, frames acquired 30, minimum static contrast 30, minimum cell to the dish in the MM dishes compared to other media dishes size 2 pixels, low/high size gate , low/high intensity gates and it was easier to remove the corona cells from the zona 120

3 Glucose improves human fertilization Table II. Human sperm motility and movement characteristics (mean SEM )in various media Modified media (MM) Significance P Ham s F10 5 mm Glu 0.5 mm Glu 0 mm Glu P Sperm motility (%) 4 h h % RAPID 4 h h c b a a VAP 4 h b b b a h b b a a VSL 4 h b b b a h c c b a VCL 4 h b b b a h b b a a ALH 4 h b b b a h b b a a BCF 4 h h STR 4 h h b b b a LIN 4 h h b b a a %RAPID highly motile spermatozoa (VAP 25 µm/s); VAP average path velocity; VCL curvilinear velocity; VSL straight line velocity; ALH amplitude of lateral head displacement; BCF beat cross frequency; STR straightness; LIN linearity (LIN). a,b,c Kruskal Wallis analysis of variance; Duncan multiple comparison. Values with different superscripts in a row are significantly different (P 0.05). pellucida. This suggests that glucose is important for healthy Discussion growth, attachment or survival of corona and cumulus cells. Energy substrates such as glucose, lactate and/or pyruvate are Some of the unfertilized oocytes in MM media were examined present in most culture media used for IVF. These substrates under the inverted microscope at 37 C: 5 25 spermatozoa are needed for sperm (Mitchell et al., 1976; Fraser and Quinn, were bound to the zona pellucida (Hoffman modulation contrast 1981) or oocyte function (Tsunoda and Chang, 1975). Of optics 400) and the sperm movement was sluggish (slow these, glucose is essential for fertilization in the mouse (Hoppe, moving). Therefore, failure of fertilization in these oocytes 1976; Fraser and Quinn, 1981) and rat (Tsunoda and Chang, was not because spermatozoa failed to bind to the zona but 1975; Niwa and Iritani, 1978) in vitro. However, in bovine perhaps because spermatozoa did not have the vigour to (Parrish et al., 1989) and in guinea pig (Rogers and penetrate the zona. Yanagimachi), glucose retards capacitation under certain condi- The sperm motility and sperm movement characteristics in tions and/or reduces fertilization. The effect of glucose on the four media are given in Table II. Sperm motility (%) was zona-free hamster egg penetration by human spermatozoa is not significantly different between all four media at 4 and 24 h controversial (Mitchell, et al. 1976; Hoshi et al., 1982; Rogers of incubation. However, sperm movement was sluggish in and Perreault, 1990). However, Urner and Sakkas (1996) MM and LGMM at 24 h and sperm motility was highly reported recently that glucose is essential for sperm penetration variable between donors in comparison with the other two into zona-free oocytes in mice. Pyruvate appears to promote media. This may be why there was no significant difference mammalian fertilization by maintaining the viability of the in percentage sperm motility between the groups. When only mouse and rat oocytes (Tsunoda and Cheng, 1975). Lactate %RAPID spermatozoa were considered, both HGMM and does not play an important role in fertilization in the mouse Ham s F10 were more able to support motility than MM or (Hoppe, 1976; Fraser and Quinn, 1981). LGMM. Moreover, %RAPID spermatozoa was significantly In our study glucose appears to be the most important energy higher in HGMM than in Ham s F10 at 24 h (68.8 versus substrate for human fertilization. Although the fertilization rate 60.6%). was significantly lower in the absence of glucose, fertilization Sperm VAP, VSL, VCL and ALH were significantly lower was obtained in some patients. This may be due to the intrinsic in MM after 4 h incubation in comparison with the other three level of energy substrates in the spermatozoa or cumulus mass media (Table II). BCF, STR and LIN were not significantly of these patients. Quinn et al. (1995) also reported that glucosedifferent in the four media tested at 4 h. At 24 h, spermatozoa and phosphate-free modified human tubal fluid (HTF) had a incubated in MM and LGMM had significantly lower VAP, significantly lower but still acceptable fertilization rate (73 VSL, VCL, ALH and LIN compared with HGMM and Ham s versus 84%) in humans compared with HTF containing glucose. F10 (Table II). There was no significant difference in the However, Quinn (1995) was able to achieve a normal fertilizasperm movement characteristics of spermatozoa incubated in tion rate (86%) in glucose-free HTF medium by increasing Ham s F10 and HGMM at 4 or 24 h. After incubation for 4 h, the motile sperm number from to in 30 µl sperm movement parameters VAP, VSL, VCL, ALH were droplets. This suggests that in the absence of glucose, a significantly higher in LGMM compared with MM. reduced number of spermatozoa are undergoing capacitation 121

4 M.M.Mahadevan et al. or acrosome changes necessary for normal fertilization. By Glucose and/or phosphate inhibit 2-cell hamster embryos when increasing the sperm concentration in the insemination medium exposed just prior to cleavage (Barnett and Bavister, 1996). ( versus in our study) normal fertilization was These authors postulated that disruption of mitochondrial obtained, possibly by increasing sperm oocyte interactions. organization decreases the energy production necessary for Another possible reason why Quinn (1995) was able to achieve normal cleavage and development. The fact that the inhibitory normal fertilization in a droplet with high sperm numbers is effect may not be evident in hamsters until the morulae/ the leakage of glucose or other glycolysis byproduct(s) to a blastocyst stage has implications for human IVF where embryos concentration that supports normal fertilization. Although lactate are routinely transferred at the 4- to 8-cell stage (Barnett and is not essential for fertilization in the mouse (Hoppe, 1976; Bavister, 1996). This might be true in humans because neither Fraser and Quinn, 1981), the modified HTF medium used by Quinn et al. (1995) nor Mahadevan et al. (1996) observed a Quinn (1995) had almost a 10-fold higher lactate concentration significant effect of glucose on early zygote development, but in comparison with MM (2.23 versus 21.4 mm). Unlike HTF, found that the patients whose embryos were cultured in a Ham s F10 is a complex medium developed for long term glucose-free medium had high pregnancy rates. This supports culture of mammalian cells and the chemical composition is the notion that human IVF and early embryo culture may need different in many respects (Ham, 1963). Rogers and Perreault different types of media for optimal development. (1990) reported that glucose is essential, whereas Hoshi et al. (1982) found no effect on hamster egg penetration by human Acknowledgements sperm. However, Hoshi et al. (1982) using a limited number The authors thank Mahlon O.Maris and Dion L.Smith for their of salt-stored human eggs showed that in the absence of energy technical assistance, and Ms. Becky Pillert for her help in the substrates (glucose in particular) few spermatozoa bound or preparation of this manuscript. penetrated the zona. De Jonge (1989) in the human, and Vandevoort and Overstreet (1995) in macaque, showed that glucose is not essential for capacitation or acrosome reaction. References Adeoya-Osiguwa, S.A. and Fraser, L.R. (1993) A diphasic pattern of 45 Ca Our study confirms other studies in animal and in human 2 uptake by mouse spermatozoa in vitro correlates with changing functional that glucose is important for sperm activation and fertilization. potential. J. Reprod. Fertil., 99, A very low concentration of glucose (µm range) is sufficient Barnett, D.K. and Bavister, B.D. (1996) Inhibitory effects of glucose and for capacitation to occur, but mm amounts of glucose are phosphate on the second cleavage division of hamster embryos: is it linked to metabolism? Hum. Reprod., 11, needed for hyperactivation and fertilization in the mouse Bavister, B.D. (1995) Culture of preimplantation embryos: facts and artifacts. (Adeoya-Osiguwa and Fraser, 1993). Quinn (1995) showed Hum. Reprod. Update, 1, that VAP decreased within 30 min in a simple modified HTF Edwards R.G. (1981) Test-tube babies. Nature, 293, L when glucose was absent (67 versus 56 µm/s). Our study De Jonge, C.J., Mack, S.R. and Zaneveld, L.J.D. (1989) Synchronous assay for human sperm capacitation and the acrosome reaction J. Androl., 10, confirms this with the additional finding that movement charac teristics of spermatozoa in MM were reduced in comparison Droesch, H., Muasher, S.J., Brzyski, R.G. et al. (1989) Value of suppression with HGMM after 4 h. The higher VAP of spermatozoa in with a gonadotropin-releasing hormone agonist prior to gonadotropin stimulation for in vitro fertilization. Fertil. Steril., 51, HGMM compared to modified HTF (Quinn s study) may be Fraser, L.R. and Quinn, P.J. (1981) A glycolytic product is obligatory for due to different glucose concentrations in the two media initiation of the sperm acrosome reaction and whiplash motility required (5 versus 2.8 mm glucose respectively) or other unknown for fertilization in the mouse. J. Reprod. Fertil., 61, differences in the methods. Sperm motility depends on the Gardner, D.K., Lane, M., Calderon, I. et al. (1996) Environment of the preimplantation human embryo in vivo: metabolite analysis of oviduct and availability of energy substrates and the capacity of the uterine fluids and metabolism of cumulus cells. Fertil. Steril., 65, spermatozoon to utilize them in the mitochondria to generate Halangk, W., Bohnensack, K. and Kunz, W. (1985) Effect of various substrates ATP and camp (Mitchell et al., 1976; Halangk et al., 1985). on mitochondrial and cellular energy state of intact spermatozoa. Biomed. Biochim. Acta, 44, Glucose or its glycolysis byproduct increases whiplash-like Ham, R.G. (1963) An improved nutrient solution for diploid chinese hamster hyperactivity of sperm which enhances fertilization. Glucose and human cell lines. Exp. Cell. Res., 29, can be metabolized to lactate and pyruvate and enter the Hoppe, P.C. (1976) Glucose requirement for mouse sperm capacitation in vitro. respiratory chain. Glutamate, succinate, citrate and acetate can Biol. Reprod., 15, Hoshi, K., Saito, A., Suzuki, M. et al. (1982) Effects of substrates on also play a role. This study provides further evidence that penetration of human spermatozoa into the zona pellucida of human eggs glucose may be necessary for the processes involved in human and the zona free hamster eggs. Jap. J. Fertil. Steril., 27, fertilization. Therefore the role of energy substrates (glucose Lopata, A. (1992) The neglected human blastocyst. J. Assist. Reprod. Genet., 9, in particular) in human sperm survival and fertilizing capacity Mahadevan, M.M. and Fleetham, J. (1990) Relationship of a human oocyte needs further investigation. scoring system to oocyte maturity and fertilizing capacity. Int. J. Fertil., This knowledge is particularly important because our data 35, suggest that different concentrations of glucose appear optimal Mahadevan M.M., Miller, M.D. and Moutos, D.M. (1996) Improved human zygote development in a modified Ham s F10 medium in vitro. J. Assist. for oocyte fertilization and embryo culture. Glucose in modified Reprod. Genet., 13, Ham s F10 is important for human fertilization in vitro but Mitchell, J.A., Nelson, L. and Hafez, E.S.E. (1976) Motility of spermatozoa. even low concentrations of glucose appear to be deleterious In Hafez, E.S.E., (ed.), Human Semen and Fertility Regulation in Men. C.V.Mosby Co., St. Louis, USA, p. 83. for human zygote culture (Mahadevan et al., 1996). Glucose Mortimer, D. (1989) Objective analysis of sperm motility and kinematics. In and/or phosphate may inhibit embryo development in several Keel, B.A. and Webster, B.W. (eds) Handbook of the Laboratory Diagnosis species by the Crabtree effect of glycolysis (Bavister, 1995). and Treatment of Infertility. CRC Press, Boca Raton, USA, p

5 Glucose improves human fertilization Niwa, K. and Iritani, A. (1978) Effect of various hexoses on sperm capacitation and penetration of rat eggs in vitro. J. Reprod. Fertil., 53, Ord, T., Patrizio, P., Marello, E. et al. (1990) Mini-Percoll: a new method of semen preparation for IVF in severe male factor infertility. Hum. Reprod., 5, Parrish, J.J., Susko-Parrish, J.L. and First, N.L. (1989) Capacitation of bovine sperm by heparin: inhibitory effect of glucose and role of intracellular ph. Biol. Reprod., 41, Quinn, P. (1995) Enhanced results in mouse and human embryo culture using a modified human tubal fluid medium lacking glucose and phosphate. J. Assist. Reprod. Genet., 12, Quinn, P., Moinipanah, R., Steinberg, J.M. and Weathersbee, P.S. (1995) Successful human in vitro fertilization using a modified human tubal medium lacking glucose and phosphate ions. Fertil. Steril., 63, Rogers, B.J. and Perreault, S.D. (1990) Importance of glycolysable substrates for in vitro capacitation of human spermatozoa. Biol. Reprod., 43, Rogers, B.J. and Yanagimachi, R. (1975) Retardation of guinea pig sperm acrosome reaction by glucose: the possible importance of pyruvate and lactate metabolism in capacitation and acrosome reaction. Biol. Reprod., 13, Tsunoda, Y. and Chang, M.C. (1975) In vitro fertilization of rat and mouse eggs by ejaculated sperm and the effect of energy sources on the in vitro fertilization of rat eggs. J. Exp. Zool., 193, Urner, F. and Sakkas, D. (1996) Glucose participates in sperm oocyte fusion in the mouse. Biol. Reprod., 55, Vandevoort, C.A. and Overstreet, J.W. (1995) Effects of glucose and other energy substrates on the hyperactivated motility of Macaque sperm and the zona pellucida-induced acrosome reaction. J. Androl., 16, World Health Organization (1992) Laboratory Manual for the Examination of Human Semen and Semen Cervical Mucus Interaction. 3rd edn. Cambridge University Press, Cambridge, UK. Received on August 2, 1996; accepted on October 29,