Modification of human sperm function by human follicular fluid: a review

This review assesses whether human follicular fluid (hFF) is able to modify human sperm function in vitro. Addition of hFF has been found to stimulate the motility of washed human spermatozoa, to increase the percentage of hyper-activated spermatozoa, to induce the acrosome reaction, to attract spermatozoa to the site of fertilization and to facilitate penetration of the oocyte by spermatozoa. It is possible that hFF could provide a favourable environment around the oocyte for fertilization by spermatozoa. Inclusion of hFF in gamete transfer medium may also improve the success rate of assisted reproduction technology. Purification of individual components in hFF which modify different aspects of sperm function awaits further investigation.     

Clinical importance of a micro-assay for the evaluation of sperm acrosome reaction using homologous zona pellucida

This study aimed to develop an acrosome reaction assay using microvolumes of solubilized human zonae pellucidae among 35 couples attending an in vitro fertilization programme. The sperm morphology of the men was classified as g-pattern (5-14% normal forms) and/or normal pattern (> 14% normal forms). All the couples had a history of repeated poor or failed in vitro fertilization rates from previous attempts. A zona-induced acrosome reaction test was performed using homologous 0.25 zona pellucida microl-1 incubated with spermatozoa to induce the acrosome reaction. Acrosome reactions were measured with FITC-PSA staining, and expressed as the difference between zona-induced and spontaneous acrosome reaction spermatozoa. The results indicated that microvolumes of solubilized human zona pellucida could successfully be used to determine the acrosome reaction status of spermatozoa. The results were compared with in vitro fertilization rates of metaphase II oocytes, and analysed with the receiver operating characteristics curve. Receiver operating characteristics analyses divided the patients into two groups: i.e. zona-induced acrosome reaction < 15% and > 15%. The sensitivity and specificity for zona-induced acrosome reaction results versus fertilization were 93% and 100%, respectively. The correlation coefficient between zona-induced acrosome reaction and in vitro fertilization was r = 0.94 (P < 0.0001). Zona-induced acrosome reaction data can be used as an indicator for fertilization failure, thus helping clinicians to refine the therapeutic approach for infertile couples prior to the onset of the treatment.     

Application of a 1.48-microm diode laser for bisecting oocytes into two identical hemizonae for the hemizona assay

Laser systems are very promising new technical tools in assisted reproduction. It was investigated if laser radiation can replace the mechanical cutting procedure via micromanipulator in the hemizona assay (HZA), a commonly used bioassay to determine the sperm-zona pellucida binding capacity. An oocyte was bisected precisely into two identical hemizonae with approximately 20 laser pulses (pulse length 30 msec) using a 1.48-microm diode laser. Compared with the conventional method using microscalpels for zona bisection, laser treated hemizonae showed equivalent sperm-binding and within the two groups there was no detectable difference between matching hemizonae in their capacity for tight sperm-binding. To evaluate whether laser radiation affects the outcome of the HZA when effects of certain substances are investigated, the spermatozoa were preincubated with human follicular fluid (hFF), which inhibits the binding of spermatozoa to zona pellucida in vitro. Supplementation with follicular fluid exerted an inhibitory effect in both groups. The hemizona index (HZI) showed no statistical differences between the two methods. Therefore, the 1.48-microm diode laser is a suitable new instrument for generating equally sized hemizonae. There is no use for holding pipettes and microscalpels, on the contrary, for performing the HZA the laser is a precise, very quick and easy to use new working tool.     

Extracellular signal-regulated kinase activation involved in human sperm-zona pellucida binding

In a previous study involving the inhibition the mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase (ERK), we found that the very specific MAPK kinase (MEK) inhibitor, PD098059, inhibited the zona pellucida (ZP) induced acrosome reaction. As an intact acrosome on the spermatozoa is a prerequisite in ensuring tight binding to the ZP, we investigated the zona binding potential of spermatozoa after PD098059 treatment of sperm, followed by exposure to solubilised human ZP and calcium ionophore (A23187). PD098059 treated spermatozoa, exposed to solubilised ZP, bound significantly more to the ZP, as compared to control spermatozoa also exposed to solubilised ZP (26.5 +/- 3.7 vs. 13.8 +/- 2.8, P < 0.05). No significant differences in binding to the ZP were observed between PD098059 treated and untreated sperm populations after A23187 exposure. These results can be interpreted to support the idea that the ZP-induced AR is the physiologically relevant exocytotic event, as it is the ZP-induced AR, and not the spontaneous (culture medium) or A23187 induced AR, that appears to be mediated through an ERK-mediated signal transduction process.     

Preincubation in peritoneal fluid decreases the follicular fluid-induced acrosomal reactivity of human spermatozoa

Summary. The aim of this study was to determine the effects of preincubation in peritoneal fluid on the follicular fluid-induced acrosomal reactivity of human spermatozoa in vitro. Thirty women participating in our IVF-EL program were given a GnRH-analogue, highly purified FSH and hCG in order to induce superovulation. Peritoneal and follicular fluids were aspirated during pick-up laparoscopy, centrifuged, filtered and frozen until use. An aliquot of swim-up suspension from nor-mospermic semen specimens ( n =30) was incubated with peritoneal fluid or HAM-F10 for 30–180 min, and follicular fluid (in volumetric proportion approximately 50/50 with peritoneal fluid) was subsequently added. The percentage of acrosomally-reacted spermatozoa was assessed using the FITC-conjugated Pisum sativum lectin before and after incubation in peritoneal fluid or control medium, as well as after follicular fluid addition. Peritoneal fluid was not able to stimulate acrosomal reactivity; further, preincubation in peritoneal fluid decreased, but not abolished, the follicular fluid-induced acrosomal reactivity. A longer pre-incubation in peritoneal fluid was associated with a lower percentage of reacted spermatozoa in response to the addition of follicular fluid. In conclusion, our data suggest that peritoneal fluid acts maintaining spermatozoa in an unreacted status in the upper female genital tract. After mixing with follicular fluid, a phenomenon that is likely to occur at ovulation, peritoneal fluid reduces, but does not abolish, the stimulating effect of follicular fluid on acrosomal reactivity.     

The hemizona assay (HZA) as an experimental model to evaluate the inhibition of sperm binding to the murine zona pellucida by isolated zona pellucida protein

Compelling evidence has demonstrated that zona binding represents gamete recognition by sperm binding with high affinity and specificity to complex glycoproteins of the zona pellucida. In the present study we evaluated the hemizona assay (HZA) in the investigation of the interaction of mouse spermatozoa with unfertilized murine oocytes and hemizonae after exposure to solubilized murine zonae pellucidae proteins. The zonae pellucidae were isolated from ovarian tissue following described mincing techniques. The sperm binding characteristics of murine spermatozoa were studied by using SDS-PAGE isolated ZP2 (+/- 120 Kd) and ZP3 (+/- 83 Kd) components of the zona pellucida. Sperm receptor activity was examined in a competitive gamete binding fashion using the HZA as an indicator of sperm/zona interaction. The results illustrated that isolated, solubilized ZP2 and ZP3 glycoprotein moieties of the zona pellucida inhibited sperm binding to hemizonae and oocytes when compared to controls, and that the HZA can be utilized as an internally controlled homologous bioassay to evaluate the effects of zona pellucida proteins on tight binding of spermatozoa to the zona pellucida.     

A new method to produce equally sized hemizonae pellucidae for the hemizona assay

The hemizona assay (HZA) is a valuable tool to study the binding potential and interaction of spermatozoa with the zona pellucida. Its accuracy strongly depends on the use of equally sized hemizonae. Usually, manipulator-guided microblades are used for cutting the zona pellucida. Recently, lasers were introduced for precise local thermolysis of the zona. The use of a 1.48 microm diode laser for the generation of hemizonae from human oocytes was investigated. This laser allowed drilling of equally sized hemizonae which were used for hemizona binding assays. It is concluded that the 1.48 microm diode laser system can be applied for the production of hemizonae. The method is easy to perform and offers a fast and efficient access to hemizonae of identical size.     

Ovarian stimulation protocol and the outcome of in vitro fertilization are not related to the ability of follicular fluid to induce sperm acrosome reaction

Summary. The ability to induce sperm acrosome reaction by follicular fluid is neither correlated with the stimulation regimen nor with the in vitro fertilization outcome.     

Sperm—oocyte interaction: studies on the kinetics of zona pellucida binding and acrosome reaction of human spermatozoa

Summary. Successful sperm-oocyte interaction depends, among other things, on sperm capacitation, which is defined by acrosomal and motility alterations. In the study described here the authors evaluated different aspects of this gamete interaction in humans. Specifically, the authors studied (1) the relationship between the number of spermatozoa bound to the zona pellucida and sperm concentration and incubation period, (2) the capacitation status and kinetics of acrosome reaction among the zona-bound spermatozoa, and (3) the effect of human follicular fluid on the zona-binding potential and acrosome status of spermatozoa from different men. The results indicated a concentration of 10⁷ cells ml⁻¹ after 15 min of coincubation to be the optimum for zona binding. The number of sperm bound after 0, 3 and 5 h of incubation was the same. In addition, spermatozoa incubated for 3 or 5 h underwent the acrosome reaction (range 9–43%) on the zona surface within 15 min of binding. The maximum percentage of acrosome-reacted spermatozoa was reached after 60 min of binding. Follicular fluid affected the sperm populations selectively, since it did not influence zona binding capacity in all cases. The data enhances the authors' understanding of critical events occurring before fertilization.     

Phosphatidylinositol 3-kinase inhibition enhances human sperm motility and sperm-zona pellucida binding

Various signalling pathways are involved in the regulation of sperm motility, capacitation, acrosome reaction and sperm-zona binding. Recent data pointed out an important role for phosphatidylinositol 3-kinase (PI3K) in human sperm motility. However, no study as of yet has been carried out to determine the effect of sperm treatment with the PI3K inhibitor LY294002 on other sperm parameters. In the present study, we investigated the role of PI3K on human sperm motility, acrosome reaction and sperm-oocyte binding by using this inhibitor. We demonstrate that in vitro incubation of washed unselected spermatozoa with LY294002 increased the percentage motility and progressive motility in asthenozoospermia patients as evaluated by computer-aided sperm analysis. The compound furthermore did not influence the acrosome reaction, whilst it (further) slightly enhanced sperm-oocyte binding. Our results therefore imply that PI3K negatively affects sperm motility and oocyte binding and might suggest a possible therapeutic role for PI3K inhibitors in the treatment regime for asthenozoospermia.     

Acrosome-reacted human sperm in insemination medium do not bind to the zona pellucida of human oocytes

In the literature there is still confusion whether acrosome-reacted sperm in medium can initiate primary binding to human zona pellucida (ZP). The ability of acrosome-reacted sperm to bind to ZP in vitro can be deduced by measuring the acrosome reaction (AR) of ZP-bound sperm compared with sperm in medium after incubation under different conditions inhibiting the ZP-induced AR. Motile sperm from fertile men, normospermic men and infertile men diagnosed with disordered ZP-induced AR (DZPIAR) were selected by swim-up (2 x 10(6) in 1 mL medium) and incubated for 1-2 h with four oocytes from failed in vitro fertilization (IVF). The acrosome status of sperm was assessed using pisum sativum agglutinin labelled with fluorescein. The ZP-induced AR was inhibited in experiments using sperm from DZPIAR patients, hyper-osmotic medium (400 mOsm/kg) and medium containing soybean trypsin inhibitor (SBTI; 4 mg/mL). Pre-treatment with calcium ionophore was used to create a sperm population with elevated AR. In all experiments with factors inhibiting the ZP-induced AR, the AR was significantly lower for ZP-bound sperm compared with sperm in medium: DZPIAR patients 4% vs. 15%, hyper-osmotic medium 3% vs. 12%, SBTI 2% vs. 12% and SBTI 3% vs. 23% after treatment with calcium ionophore. In conclusion, acrosome-reacted sperm in vitro have significantly reduced, in fact probably zero ability to bind to the ZP.     

Human follicular fluid stimulates motility and velocity of washed human sperm in vitro

Human follicular fluid collected by laparoscopic oocyte pick-up during IVF was studied with a computer-assisted semen analyser to evaluate the effect of hFF on human sperm motility and velocity. Freshly ejaculated human sperm were washed with phosphate buffered saline and mixed with either PBS or hFF. At various incubation periods of time, hFF increased both sperm motility and velocity as compared with control (P less than 0.01). After incubation of sperm with hFF at 37 degrees C and 5% CO2 in air for 0, 1, 3, 6, and 12 h, the amplitude increase of motility were 49%, 77%, 330%, 2020%, and 3340% when individual control motility was considered to be 100%. The amplitude increase of curvilinear velocity were 43%, 51%, 67%, 152%, 278%, respectively. Comparison of the motility and velocity of the sperm treated with hFF between 0 and 12 h, showed that hFF preserved both motility and velocity in vitro (P less than 0.01). The stimulatory effect of hFF was retained after boiling at 100 degrees C for 30 min, or after being filtered through Amicon membrane cones, but it disappeared if the hFF had been pre-treated with chymotrypsin. However, hFF did not stimulate the motility and velocity of unwashed sperm in freshly ejaculated human semen. A non-dialyzable and heat-stable factor(s) with a molecular weight below 50,000 in hFF may improve and maintain the motility and velocity of washed human sperm. Whether this factor could be used to improve pregnancy rate in assisted reproduction awaits further investigation.     

Acrosomal status of human spermatozoa after follicular fluid or calcium ionophore challenge in relation to semen parameters and fertilizing capacity in vitro

Summary.  The proportion of spermatozoa that undergo spontaneous acrosome reaction in vitro is relatively low. The proportion can be enhanced by incubation with either biological inducers such as follicular fluid or chemicals like calcium ionophore. It has been suggested that improper acro-somal reaction may be a cause of fertilization failure in vitro. The objectives of the present study were to assess the acrosomal status of human sperm following follicular fluid or calcium ionophore treatment and to analyse the relationship between spontaneous and induced acrosome reaction and fertilization rates in vitro by standard in vitro fertilization (IVF) technology. In all, 53 semen samples (22 normal and 31 subnormal) were studied. The effect of calcium ionophore A 23187 and follicular fluid was assessed using the fluorescence activated cell sorter. IVF results were evaluated in relation to the acrosome status of the sperm samples. Our results demonstrate that the effect of follicular fluid on the acrosomal status correlated positively with the effect obtained by the calcium ionophore (Pearson's correlation r = 0.45). A significantly higher percentage of maximal acrosome change ( P <0.02) was found in cases where fertilization occurred (19/27), than in sperm samples that did not achieve fertilization in vitro (8/27). The present finding that follicular fluid induced acrosome reaction can serve as a predictive tool which is as good as the ionophore treatment for assessing IVF outcome, supports the use of this method for clinical purposes.     

Development of a new, highly sensitive zona pellucida binding assay using a bioluminescence-enhanced detection system

To date, two different zona binding assays have been described in the literature. Both assays, however, require a large quantity of human zonae which vary immensely in quality. Furthermore, an inverted microscope with micromanipulation equipment is necessary, which makes both assays relatively complicated and time-consuming, and requires skilled staff. Therefore, we developed a new, highly sensitive zona binding assay using a bioluminescence-enhanced system which employs a pool of solubilized zona pellucida and is easier for routine use. In the detection system, light emission by the luciferin-luciferase system is measured. Because of the limited availability of human zonae pellucidae, this new assay was first developed in the porcine system. The new bioluminogenic substrate D-luciferin-O-beta-galactopyranoside (Lu-Gal) was synthesized, purified and characterized. Synthesis of Lu-Gal resulted in purity better than 99.998%. Analytical data and spectra were appropriate. In terms of the kinetic data, Lu-Gal is a highly sensitive and specific substrate for beta-galactosidase. Using the given chemical conditions, nonlabelled zonae bound competitively to boar spermatozoa, which resulted in a high sensitivity and specificity. By the addition of 10 nonlabelled zonae, the binding of labelled zonae was almost completely inhibited. Corresponding results were obtained when the bioluminescent system was compared with the hemizona assay. On the other hand, spermatozoa of other species (bull, hamster and man) showed only low binding to the porcine zonae or none at all. Competitive displacement was not observed, indicating the inter-species specificity of the assay.     

Comparison of two techniques to evaluate the acrosomal status of zona pellucida bound sperm in humans

In this work, we have compared two procedures that evaluate the acrosomal status of human sperm bound to the human zona pellucida. Motile sperm, selected by a Percoll gradient, were capacitated by incubation at 37 degrees C, 5% CO2, for 4.5 h, at 20 x 10(6) cells ml(-1). Then, the sperm were incubated with nonviable human oocytes for 10 min at 37 degrees C, 5% CO2. The oocytes with bound sperm were transferred to 500 microl phosphate-buffered saline (PBS) and washed to remove loosely bound sperm. The oocytes were then processed according to the procedures of Cross et al. (1986) or Liu & Baker (1996). In the Cross's procedure, the sperm were labelled while they were bound to the zona. In the Liu's procedure, the sperm were first dislodged from the zona into a droplet of PBS and labelled in there. Both procedures gave equivalent percentages of acrosome-reacted sperm. However, the total number of zona-bound sperm available for assessment with the procedure of Liu & Baker was greater than that of Cross et al. We suggest to use the former procedure to evaluate the acrosomal status of zona-bound sperm in humans. Moreover, this procedure also provided information about sperm ability to bind to the zona pellucida.     

Induction of acrosome reaction by low temperature is comparable to physiological induction by human follicular fluid

Summary. The acrosome reaction was induced by means of low temperature or human follicular fluid (FF) in spermatozoa from 18 patients. Live acrosome-reacted spermatozoa were determined with the triple-stain technique. Both inducers of acrosome reaction had a highly significant correlation with the percentage of acrosome-reacted spermatozoa after induction of the acrosome reaction: (r = 0.9190; P <0.0001) and the inducibility of AR (r = 0.8472; P <0.0001). This might indicate that both the non-physiological (low temperature) and the physiological (FF) inducers use the same signal transduction pathway, and demonstrates the usefulness of low temperature induction for diagnostic purposes.     

Assessment of sperm function in fertile and infertile men

Summary. The sperm function of fertile men (control), infertility patients (experimental), and men with varicocele were compared. The bioassays used were the follicular fluid-induced acrosome reaction, the binding to the zona pellucida, and the penetration of zona-free hamster oocytes. The percentage (mean ± SEM) of reacted spermatozoa was 35 ± 3 in the control, 22±1 in the experimental, and 22 ± 3 in the varicocele. The minimum value of acrosome reaction in control men was 20%. The mean number of zona-bound spermatozoa was 250 ± 30 in the control, 160 ± 28 in the experimental, and 196 ± 44 in the varicocele. The minimum number of zona bound spermatozoa in control men was 50. The mean number of hamster oocytes penetrated was 50 ± 8 in the control, 19 ± 3% in the experimental, and 10 ± 3 in the varicocele. The minimum number of oocytes penetrated in control men was 6%. In the experimental group, 22 men had a normal sperm function, 58 had 1 or 2 bioassays below the minimum (relative dysfunction), and 10 had all bioassay below the minimum (abnormal sperm function). The results of these bioassays could help to reclassify the infertile men in several subgroups.     

Analysis of the lipid content and the motility of human sperm after follicular fluid treatment*

Summary The effect of human follicular fluid (hFF) on the cholesterol and phospholipid content and the movement characteristics of human spermatozoa were studied. Semen was selected by a discontinuous Percoll gradient and incubated during in vitro capacitating conditions with B2 medium supplemented with hFF 20%. Percoll pelleted spermatozoa were incubated in either B2 (B2-Percoll) or B2 supplemented with hFF (hFF-Percoll). In hFF-Percoll, we observed a time-dependent (24 h) decrease in both the cholesterol and phospholipid contents (cholesterol: 10.1 vs. 8.7 nmol 10^-7 spermatozoa; phospholipids: 17.5 vs. 15.7 nmol 10^-7 spermatozoa, P < 0.05). This decrease in cholesterol and phospholipids in human spermatozoa was concomitant with a high straight line velocity, a high progressive motility percentage and an increased value of lateral head displacement without any significant alteration of the spermatozoal membrane. No modification of the cholesterol: phospholipid ratio after 2 and 24 h of incubation in either B2-Percoll (0.61, 0.54) in hFF-Percoll (0.59, 0.63) was observed when compared with original control semen. It is suggested that the decrease in cholesterol and phospholipids in hFF-Percoll may be taken into account for the changes of membrane modification as part of the capacitation process.     

The inhibition of the human sperm phosphatidylinosytol 3-kinase by LY294002 does not interfere with sperm/oocyte interaction

It has recently been reported that the selective inhibition of phosphatidylinosytol 3-kinase (PI3K) enhances human sperm motility. However, little information exists on a possible role of PI3K in other sperm functions involved in the fertilization process. In this study, we investigated whether LY294002 could affect human sperm ability to fuse with oocytes, by means of the hamster egg penetration test (HEPT). The effect on acrosome reactions (AR) and on sperm/zona pellucida (ZP) binding was also evaluated. The pre-incubation with scalar doses of LY294002 (0.1, 1 and 10 microm) did not interfere with sperm ability to fuse with oocytes either in the conventional version of the HEPT or in the version enhanced with progesterone (P). No interference with the stimulatory effect on AR exerted by P or mannose-bovine serum albumin (mannose-BSA) was revealed. Finally, LY294002 had no effect on sperm/ZP binding. These results indicate that the inhibition of PI3K by LY294002 does not interfere with sperm interaction with oocytes. This is noteworthy in the view of a possible clinical use of LY294002 as an in vitro stimulator of the sperm motility of asthenozoospermic patients for assisted reproduction techniques.     

精子功能指标在辅助生殖助孕方式选择中的作用

目的 评估精子功能参数对精子常规体外受精能力的预测价值,探寻有效选择受精方式的新指标.方法 回顾性分析2017年1月至2019年12月于我院首次行体外受精-胚胎移植(IVF-ET)短时受精的429个周期的临床资料,根据受精结局不同分为2组:受精失败或受精率＜30％的患者为早补救卵胞浆内单精子注射(R-ICSI)组(n=...