Effects of relaxin and IGF-I on capacitation, acrosome reaction, cholesterol efflux and utilization of labeled and unlabeled glucose in porcine spermatozoa

Aim:  Relaxin and insulin-like growth factor (IGF)-I have pronounced effects on the male and female reproductive tracts. The aim of this study was to investigate the effects of relaxin and IGF-I on the motility, capacitation, acrosome reaction, cholesterol efflux and utilization of glucose in porcine spermatozoa.
Methods:  Swim-up separated spermatozoa that had been washed twice were incubated at 37°C for 1 or 4 h in modified Tyrode's albumin lactate pyruvate (mTALP) medium supplemented without (control) or with relaxin (20 ng/mL) or IGF-I (20 ng/mL) or both (10 + 10 ng/mL).
Results:  Progressive motility and the induction rate of capacitation and acrosome reaction were increased ( P <  0.05) by relaxin and IGF-I alone or in combination, especially after 4 h of incubation. Relaxin alone or combined with IGF-I enhanced ( P  < 0.05) the cholesterol efflux after 4 h, whereas IGF-I alone did not show any significant effect on the cholesterol efflux compared with the control at any time point. The utilization rates of labeled and unlabeled glucose increased ( P <  0.05) in spermatozoa incubated with relaxin or IGF-I alone or in combination compared with the control.
Conclusion:  Thus, supplementation of relaxin alone or combined with IGF-I into the medium possibly plays a beneficial role in porcine spermatozoal prefertilization events in vitro . (Reprod Med Biol 2008; 7 : 29–36)     

Effect of amino acids and dipeptides on the acrosome reaction and accumulation of ammonia in porcine spermatozoa

Aim :  The present study was designed to investigate the effect of amino acids and their dipeptides in the medium related to the urea cycle on the motility, viability, acrosome reaction (AR) and accumulation of ammonia in the medium over different incubation periods in porcine spermatozoa and to assess the utilization of glucose.
Methods :  Porcine spermatozoa were washed, swim-up and incubated at 37°C for 0–4 h in mTALP medium supplemented with 75–600 µmol/L ammonia. Amino acids (1.0 mmol) or their dipeptides (2.0 mmol) were added individually to the mTALP medium containing either no ammonia or 300 µmol/L of ammonia. The viability and AR of porcine spermatozoa were assessed using the triple-staining technique and the accumulation of ammonia in the medium was measured using the indophenol method.
Results :  The motility, viability and AR were adversely affected ( P  < 0.05) by concentrations of ammonia ≥300 µmol/L compared with the control. Supplementation of l -alanyl- l -glutamine (AlaGln), l -glycyl- l -glutamine (GlyGln) and AlaGln + GlyGln in the presence of 300 µmol/L ammonia significantly increase ( P  < 0.05) the rate of motility, viability, AR, incorporation, accumulation of ammonia and oxidation of ¹⁴C(U)-glucose compared with the ammonia supplement control.
Conclusion :  AlaGln and GlyGln in mTALP medium were more stable and effective than the individual amino acids in reducing the accumulation of ammonia, and subsequently increasing the rate of AR and the utilization of glucose in porcine spermatozoa. (Reprod Med Biol 2008; 7 : 123–131)     

Effect of relaxin on motility, acrosome reaction and viability of cryopreserved boar spermatozoa

Background and Aims:   Relaxin has an important role in stimulating motility and the acrosome reaction (AR) of fresh boar spermatozoa. The objective of the present study was to determine whether relaxin can improve the motility, AR and viability of cryopreserved boar spermatozoa.
Methods:   Cryopreserved boar spermatozoa were thawed, washed and incubated at 37°C for 4 h in modified Beltsville thawing solution supplemented with 0, 20 or 40 ng/mL relaxin. Sperm motility, AR, viability, and incorporation and oxidation of ¹⁴C-glucose were evaluated during 0–4 h of incubation.
Results:  The results show that the supplementation of relaxin (especially at 20 ng/mL) in the thawing solution improved sperm motility significantly ( P  < 0.05) at 1–3 h of incubation. The percentage of acrosome reacted live spermatozoa was improved significantly ( P  < 0.05) when the spermatozoa were treated with 20 ng/mL relaxin. Viability was not significantly ( P  > 0.05) improved by supplementation with relaxin. The rates of incorporation and oxidation of ¹⁴C-glucose were increased in correlation with AR up to 4 h of incubation.
Conclusion:  We conclude that relaxin can improve the sperm motility and AR, and enhance the glucose metabolism of cryopreserved boar spermatozoa. (Reprod Med Biol 2006; 5 : 215–220)     

Effect of fatty acids bound to bovine serum albumin-V on acrosome reaction and utilization of glucose in boar spermatozoa

Aim:  The present study has been designed with the objective of determining if fatty acids bound to bovine serum albumin-V (BSA-V) can improve motility, viability, and increase acrosome reaction (AR) and utilization of glucose in boar spermatozoa.
Methods:  Boar spermatozoa were washed, swum-up and incubated at 37°C for 6 h in TALP medium supplemented with fatty acids bound to bovine serum albumin fraction V (BSA-V), fatty acid free BSA (BSA-FAF), polyvinyl alcohol + main fatty acids bound to BSA-V (PVA + FA) and PVA. Sperm motility, viability, AR, and the incorporation and oxidation of ¹⁴C-glucose were evaluated during 6 h of incubation.
Results:  The results show that the BSA-V was superior to BSA-FAF and PVA in improving motility and AR. Viability was significantly increased ( P  < 0.05) by only BSA-V compared with PVA. When the main fatty acids compound of BSA-V were added to PVA, the sperm motility, viability and AR became almost the same as with BSA-V. The rate of incorporation and oxidation of ¹⁴C-glucose were significantly increased ( P  < 0.05) by BSA-V compared with BSA-FAF and PVA. Fatty acids bound to BSA-V are important for improvement of sperm functions.
Conclusions:  The present study postulates that fatty acids bound to BSA-V are important to acrosome reaction and the utilization of glucose in boar spermatozoa.     

Regulation of hyperactivation of hamster spermatozoa by progesterone

Aim:  Although it is accepted that progesterone (P) induces acrosome reaction through non-genomic regulation, it is not well known if P also affects hyperactivation of sperm.
Methods:  Hamster spermatozoa were hyperactivated by incubation for 4 h on modified Tyrode's albumin lactate pyruvate medium and recorded on a DVD via a charge-coupled device camera attached to a microscope with phase-contrast illumination and a small CO₂ incubator. Phosphorylation of proteins was detected by western blotting using antiphosphotyrosine antibodies.
Results:  Sperm hyperactivation was significantly increased and accelerated by a non-genomic signal of P. Although acceleration of motility of hyperactivated sperm occurred with 10, 20 and 40 ng/mL P, the most effective concentration was 20 ng/mL. Progesterone also significantly increased 80-kDa tyrosine phosphorylation of sperm proteins. Both extracellular Ca²⁺ and albumin were essential for sperm hyperactivation, and the former was also essential for maintaining sperm flagellar movement. Moreover, phospholipase C (PLC) was associated with the regulation of hyperactivation by P.
Conclusion:  It is likely that P regulates sperm hyperactivation by a non-genomic signal in relation to tyrosine phosphorylation and PLC. (Reprod Med Biol 2008; 7 : 63–74)     

Effect of amino acids and dipeptides on accumulation of ammonia in the medium during <Emphasis Type="Italic">in vitro</Emphasis> maturation and fertilization of porcine oocytes

Aim:  The present study was designed to investigate the effect of amino acids and their dipeptides on the accumulation of ammonia in the medium during in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes.
Methods:  The IVM and IVF media were modified North Carolina State University-37 solution and modified Tyrode's albumin lactate pyruvate, respectively. Porcine oocytes were matured in IVM medium containing 75–2400 µmol ammonia. Amino acids (1.0 mmol) or their dipeptides (2.0 mmol) related to the urea cycle were added individually to the IVM and IVF media containing 300 µmol ammonia. Oocyte maturation and fertilization were assessed using acetic–orcein staining, and the accumulation of ammonia in the media was measured using the indophenol method.
Results:  Percentages of metaphase II (MII) were adversely affected ( P <  0.05) by ≥300 µmol concentrations of ammonia in the IVM medium. In the presence of 300 µmol ammonia in the IVM and IVF media, glutamic acid, l -alanyl-L-glutamine (AlaGln), l -glycyl-L-glutamine (GlyGln) and AlaGln + GlyGln showed the highest rate ( P <  0.05) of MII, monospermic fertilization, and the lowest rate ( P <  0.05) of ammonia accumulation in the media.
Conclusion:  AlaGln and GlyGln in IVM and IVF media were more stable and effective than the individual amino acids in reducing the accumulation of ammonia, and increased the rate of porcine oocyte MII and monospermic fertilization in vitro . (Reprod Med Biol 2007; 6 : 165–170)     

Analysis of limited fertility in intracytoplasmic sperm injection of sperm obtained by electroejactulation

Background and Aims :  We correlated findings in semen from patients with ejaculatory dysfunction with results of in vitro fertilization using their electroejaculated sperm.
Methods and Results :  Electroejaculation was carried out in six patients with the above-mentioned criteria for a total of eight times. Sperm was obtained in six attempts. Intracytoplasmic injection of these sperm was performed in 156 eggs. Sixty-seven eggs were fertilized; most of these were injected with motile sperm. Two women became pregnant, both after injection with motile sperm. As previously reported, electroejaculated sperm showed low motility and a low fertilization rate, but even motile sperm had a low fertilization rate.
Conclusion :  The results of the present study suggest the importance in fertilization of undetermined factors in addition to sperm motility. (Reprod Med Biol 2004; 3 : 9–12)     

Effect of fatty acids on boar sperm motility viability and acrosome reaction

Aim  The present study was undertaken to determine which fatty acids improve motility viability and increase acrosome reaction (AR) in boar spermatozoa. Methods  Boar spermatozoa were washed, swum-up and incubated at 37°C for 4 h in TALP medium supplemented with myristic, palmitic, stearic, lignoceric, oleic, linoleic, arachidonic, docosahexaenoic and palmitoleic acid. Sperm motility, viability and AR were evaluated during 4 h of incubation. Results  Results show that oleic and linoleic acid significantly improved (P < 0.05) the motility and viability of boar spermatozoa. The AR was significantly improved (P < 0.05) by oleic and arachidonic acid in almost all incubation periods. When combinations of oleic, linoleic and arachidonic acid were studied for motility, viability and AR, it was found that oleic plus linoleic acid significantly increased (P < 0.05) motility, whereas arachidonic plus oleic acid significantly increased (P < 0.05) AR. Conclusion  Unsaturated fatty acids, especially arachidonic acid, can improve boar sperm motility and AR. A combination of arachidonic and oleic acid is important for inducing boar sperm AR.     

Effect of sperm-immobilizing antibodies on the acrosome reaction of human spermatozoa.

OBJECTIVE: To study by a triple stain technique the effect of sperm-immobilizing antibodies on the acrosome reaction of human spermatozoa. DESIGN: The spermatozoa were allowed to swim up and culture in a medium containing 7.5% (vol/vol) serum with sperm-immobilizing antibodies or control serum up to 6 hours. Sperm mobility was analyzed, and the percentage of live acrosome reacted spermatozoa was determined. SETTING: Samples were collected from patients referred to university hospital infertility clinics. MATERIALS: Serum samples were drawn from seven patients with sperm-immobilizing antibodies. All the sera were heat activated and stored at -40 degrees C until use. Semen samples were taken from two healthy donors. RESULTS: During culture for 6 hours, the percentage of live sperm showing the acrosome reaction increased significantly (P less than 0.01) in the control group but not in the sperm-immobilizing antibodies group. However, the inhibitory effect of sperm-immobilizing antibodies on the acrosome reaction was reversed when sperm was reincubated in medium with control serum (P less than 0.01). CONCLUSIONS: Sperm-immobilizing antibodies block fertilization at least in part by inhibiting the acrosome reaction of human spermatozoa.     

蛋白C抑制剂对人精子顶体反应和顶体素活性的影响

本研究应用提纯的尿PCI 进行对精子顶体反应和顶体酶的抑制试验。当洗涤后的精子与PCI 一起在体外孵育,其活动率和顶体反应未见降低;但当精子提取物或猪纯顶体素与PCI一起孵育时,其酰胺酶活性明显受到抑制且与PCI 剂量反应时间相关。这种抑制效应可能是通过形成PCI-顶体素复合物而起作用的,尽管这种复合物对SDS不稳定。精液中PCI 的高浓度(220±41.0mg/l)可能通过抑制由于精于变性或受损时释放出的顶体素或其它蛋白酶活性,从而保护生殖道上皮或精子本身不受酶的降解。     

Seminal plasma fructose and glucose in normal and pathological conditions

A total number of ninety four semen samples were analysed which included normozoospermia (39), oligozoospermia (6), oligoasthenozoospermia (10), asthenozoospermia (15) and azoospermia (24). A positive correlation (r = 0.344) was existing between sperm count and motility in normozoospermia. Seminal plasma was estimated for fructose (94) and glucose (73). It was found that there was a statistical difference between the values of oligozoospermia and all other groups studied for fructose. A strong positive correlation (r = 0.394) was existing between percentage of motility of spermatozoa and fructose in normozoospermia showing that fructose is very essential for increased number of motile spermatozoa. Likewise a relationship (r = 0.451) was seen between sperm count and glucose in normal. This gives an impression that both these are regulated by one source.     

Effects of platelet activating factor on mouse sperm function

Platelet activating factor (PAF) has been implicated in a variety of reproductive processes. This study was designed to investigate the effect of PAF and the specific PAF receptor antagonist, CV-3988, on capacitation and the acrosome reaction in mouse spermatozoa using an in vitro fertilization (IVF) system. When spermatozoa were preincubated for 30 min in medium containing PAF (10^–7 to 10^–11 M), a significant increase in the fertilization rate with both cumulus-free and zona-free oocytes was observed. In contrast, treatment of the spermatozoa with 10^–5 M CV-3988 caused a significant decrease in both sperm motility and fertilization rates with zona-intact and zona-free oocytes. This suppression was reversed by the addition of PAF. Furthermore, the acrosome reaction was enhanced by PAF treatment of spermatozoa in a dose-dependent manner. This stimulation of the acrosome reaction by PAF required the presence of calcium ions in the medium. While 10^–5 M CV-3988 inhibited the acrosome reaction, the inhibition was also reversed by the addition of PAF. These results suggest that PAF can stimulate not only the capacitation process but also the acrosome reaction, both of which are dependent on extracellular calcium.     

Identification and characterization of human acrosomal antigen defined by a monoclonal antibody with blocking effect on in vitro fertilization.

A monoclonal anti-human sperm antibody (Mab 1A1) has been produced by fusion of myeloma cells with splenocytes from a BALB/c mouse immunized with in vitro capacitated human spermatozoa. Immunofluorescence studies with Mab 1A1 show that it recognizes an antigen(s) (Ag 1A1) which is located in the acrosome of human spermatozoa. As shown by Western blotting experiments, 1A1 antigen represents a family of proteins with Mr ranging from 20 kDa to 34 kDa. Immunofluorescence observations on epitope exposure and location suggest that during in vitro capacitation of human spermatozoa, Mab 1A1 epitope-bearing molecules are concentrated in regularly arranged granules in the acrosome. After long-term incubation the epitope is exposed on the apical acrosome surface exhibiting a spot-like arrangement. The 1A1 epitope is widely distributed among mammalian species: boar, ram, mouse and rat acrosome is intensively stained by Mab 1A1. The antibody inhibits in vitro fertilization mainly by blocking sperm attachment to and penetration through the zona pellucida when included in the medium for the in vitro fertilization of mouse, porcine and human oocytes.     

Effects of human follicular fluid on spermatozoa that have been cocultured with human oviductal cells

Objective: To investigate the sequential effects of human oviductal cells and human follicular fluid (hFF) on various sperm functions.Design: Laboratory experimental study.Setting: University gynecology unit.Patient(s): Fallopian tubes were from patients undergoing tubal ligation or hysterectomy. Semen was from men attending the subfertility clinics.Intervention(s): Spermatozoa were treated with [1] 6 hours in Earle’s balanced salt solution (EBSS-BSA; control); [2] 5 hours in EBSS-BSA and 1 hour with hFF (hFF); [3] 5 hours with oviductal cells and 1 hour in EBSS-BSA (coculture); and [4] 5 hours with oviductal cells and 1 hour with hFF (sequential).Main Outcome Measure(s): Motility, acrosome reaction, zona binding, and oocyte fusion.Result(s): Groups II and III spermatozoa had similar motility and were better than that of group I. Group IV displayed higher motility parameters than the other groups. Human follicular fluid induced acrosome reaction. The incidence of acrosome reaction in group IV was significantly lower than that in group II. Group III did not affect the acrosome reaction. Spermatozoa in groups II–IV had lower zona binding capacity than those in group I. Human follicular fluid stimulated oocyte penetration, whereas oviductal cells suppressed this effect of hFF.Conclusion(s): Oviductal cells maintained the fertilizing capacity of spermatozoa, whereas hFF facilitated the fertilization process of oviductal spermatozoa.     

Identification of 66 kDa phosphoprotein associated with motility initiation of hamster spermatozoa

Background and Aims:  Sperm motility is regulated by protein phosphorylation. The 66 kDa protein obtained from hamster sperm flagella was phosphorylated at serine residues associated with the motility initiation. In order to understand the regulatory mechanism of sperm motility, the 66 kDa protein was identified in the present study.
Methods:  The 66 kDa protein was purified by 2-D gel electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry, liquid chromatography-tandem mass spectrometry and peptide sequencer.
Results:  The 66 kDa protein was tubulin β chain.
Conclusion:  The 66 kDa protein is one of the tubulin β chain isoforms and phosphorylated in relation to the motility initiation. (Reprod Med Biol 2004; 3 : 133–139)     

Clinical outcomes of two different endometrial preparation methods for cryopreserved-thawed embryo transfer in patients with a normal menstrual cycle

Aim:  To compare the clinical outcomes of cryopreserved-thawed embryo transfer among patients with a normal menstrual cycle who had natural or hormone-replacement cycles.
Methods:  From January 2004 to June 2006, cryopreserved embryos following conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) were thawed and transferred in a total of 720 natural cycles and 136 hormone-replacement cycles.
Results:  Cryopreserved-thawed embryo transfer in patients who had a natural or hormone-replacement cycle resulted in clinical pregnancy in 43.1% and 40.4%, respectively; a rate of miscarriage of 14.5% and 23.6%, respectively; and a rate of ongoing pregnancy and delivery of 36.5% and 30.9%, respectively. None of these differences were statistically significant.
Conclusions:   Patients with a normal menstrual cycle who have natural or hormone-replacement cycles can be expected to have comparable clinical outcomes with cryopreserved-thawed embryo transfer. (Reprod Med Biol 2007; 6 : 53–57)     

The effects of calmodulin and it's antagonist, W-7, on the acrosome reaction and fertilization of human spermatozoa

The acrosome reaction of mammalian spermatozoa has been shown to be dependent upon an influx of Ca2+ following capacitation. Recently it was shown that calmodulin which activates various enzymatic activities in a calcium-dependent manner is contained in the acrosomal portion of sperm obtained from diverse species including human. Therefore calmodulin appears to be the primary target for Ca2+-dependent regulatory process involved in the acrosome reaction. This report examines the effects of calmodulin and it's antagonist, W-7, on the acrosome reaction in human spermatozoa and the fertilization with zona free hamster eggs. The results are as follows: 1) In the experiment on fertilization in mBWW medium with 30nM of calmodulin, there was no significant difference between the calmodulin and the control. (2) In fertilization in mBWW medium with 2.5 to 25 microM of W-7, there were no significant changes in the fertilization rate, but there was a significant decrease in the fertilization rate when the concentration of W-7 was elevated up to 50 microM. (3) When fertilization in mBWW was performed using spermatozoa pretreated with W-7, the fertilization rates were more markedly and promptly elevated with insemination times than the control. (4) When the eggs and spermatozoa were pretreated with W-7 before insemination and then placed in mBWW medium, the fertilization rate was markedly decreased. (5) Triple stain method and transmission electron microscopy showed that a large proportion of spermatozoa had undergone the acrosome reaction in mBWW medium containing W-7 with normal manner. From the results given above, it appears that calmodulin plays an important role in the acrosome reaction and fertilization in human spermatozoa.     

The improvement in fertilizing ability of cryopreserved mouse spermatozoa using laser-microdissected oocytes

Aim:  The C57BL/6 mouse strain is now commonly used for producing transgenic/knockout strains. However, the fertilizing ability of these spermatozoa decreases as a result of cryopreservaion. Although the micromanipulation technique has been established to increase their fertilizing ability, it requires a considerable degree of technical skill. In the present report, we investigate the simple microdissection of zona pellucida by laser to increase the fertilizing ability of cryopreserved spermatozoa.
Methods:  C57BL/6J spermatozoa were cryopreserved using a solution consisting of 18% raffinose/3% skim milk. Oocytes of the same strain were placed in PB1 medium containing 0, 0.25, 0.50 or 0.75 mol sucrose. The zona pellucida of oocytes was microdissected by laser with different pulse lengths selected from 0.45 to 0.65 ms. Microdissected oocytes were then fertilized with cryopreserved spermatozoa, and the subsequent development of embryos was assessed.
Results:  When oocytes were microdissected in PB1 medium without sucrose, 81.5% of the oocytes were fertilized. The fertilization rates increased significantly as the pulse length was lengthened when compared with oocytes with intact zona pellucida. Furthermore, normal offspring were obtained in all experiments.
Conclusion:  The fertilizing ability of cryopreserved spermatozoa is improved when oocytes with their zona pellucida microdissected by laser were used. (Reprod Med Biol 2006; 5: 249–253)     

The sirtuin 1 activator YK 3-237 stimulates capacitation-related events in human spermatozoa

Research questionDoes sirtuin-1 (SIRT1) have a role in the human spermatozoa capacitation process?DesignHuman spermatozoa were incubated for 6 h in a capacitating medium in presence or absence of the specific SIRT1 activator, YK 3-237. Several sperm parameters were determined by flow cytometry: viability, acrosome reaction and mitochondria membrane status. Sperm motility was determined objectively by computer-assisted semen analysis. Sperm capacitation status was evaluated by the extent of protein tyrosine phosphorylation and by the percentage of spermatozoa with the acrosome reacted by a calcium ionophore challenge.ResultsSIRT1 was detected in the connecting piece of human spermatozoa where a lysine acetylation pattern was mainly found along the sperm tail. SIRT1 activation accelerates the occurrence of a phenotype associated with human sperm capacitation, with no differences seen in the lysine acetylation pattern. After 1 h of co-incubation of YK 3-237 with human spermatozoa, tyrosine phosphorylation levels were comparable to control levels after 6 h of incubation in capacitating conditions. In addition, the activator improved sperm responsiveness to a Ca²⁺ ionophore (A23187) challenge determined by an increase in acrosome-reacted spermatozoa (P = 0.025). Importantly, sperm viability and mitochondrial activity-related parameters assessed by flow cytometry were not affected by YK 3-237.ConclusionYK 3-237 induces capacitation-related events in human spermatozoa such an increase of tyrosine phosphorylation levels and acrosome-reacted spermatozoa after the ionophore challenge. Together, these results show that YK 3-237 affects human spermatozoa capacitation-related events by a mechanism independent of protein lysine acetylation but dependent on bicarbonate and calcium.     

The kinetics of the acrosome reaction of human spermatozoa and its correlation with in vitro fertilization.

OBJECTIVE: To study the reaction pattern of acrosome reaction in human semen and correlate it to the results of in vitro fertilization (IVF). DESIGN: The percentage of acrosome-reacted spermatozoa of 41 IVF semen samples was determined after 0, 2, 4, and 24 hours of incubation in human tubal fluid medium supplemented with 10% human pool serum. SETTING: St. Radboud Hospital, Catholic University of Nijmegen, The Netherlands. PATIENTS: Forty-one IVF couples. INTERVENTIONS: None. MAIN OUTCOME MEASURE: Acrosome reaction was determined using fluorescein isothiocyanate conjugated concanavalin A lectin. To avoid false-positive signals from dead spermatozoa, the sperm viability was determined. RESULTS: Three kinetic patterns of acrosome reaction could be distinguished: (1) normal reacting pattern (percentage of acrosome-reacted spermatozoa less than 10% at 2 hours and greater than 5% at 4 hours; 75% fertilization in IVF); (2) a quickly reacting pattern (percentage of acrosome-reacted spermatozoa greater than 10% at 2 hours; 22% fertilization in IVF); and (3) a nonreacting pattern (percentage of acrosome-reacted spermatozoa less than 5% at all time intervals studied; 15% fertilization in IVF). CONCLUSIONS: The timing of acrosome reaction and the percentage of acrosome-reacted spermatozoa are very important parameters in IVF.