Fertilization and early embryology: The factors affecting sperm binding to the zona pellucida in the hemizona binding assay

Sperm binding to the zona pellucida is a prerequisite for fertilization.The hemizona binding assay (HZA) is commonly used to evaluatethe zona-binding capacity of spermatozoa. The present studyreports three factors that affect HZA. They were the base mediumused, the protein source and the size of pipette used for removingloosely bound spermatozoa during HZA. The number of spermatozoabound on the hemizona was compared between (1) Earle's balancedsalt solution (EBSS) and Ham's F-10, and (2) between human serumand bovine serum albumin (BSA). Results indicated that EBSSand human serum both significantly increase the number of boundspermatozoa when compared to Ham's F-10 (P < 0.0001, pairedt-test) and BSA (P < 0.05, paired t-test) respectively. Pipettesof different diameters were used to study the effect of sizein removing loosely bound spermatozoa on hemizona. Data showedthat the diameter of the pipette should be 200 mm, in ordernot to remove bound spermatozoa excessively. These results emphasizethe importance of standardization of the protocol of the hemizonaassay worldwide to be able to compare results between differentlaboratories.     

Pre-freezing sperm preparation does not impair thawed spermatozoa binding to the zona pellucida.

The present study was conducted to assess the fertilizing potential of frozen-thawed spermatozoa, which were cryopreserved after separation on a Percoll gradient, or washed out of seminal plasma. For this purpose, binding to the zona pellucida and other characteristics of the treated sperm cells were compared with those of cryopreserved spermatozoa from the same original sample which were not manipulated before freezing. Semen specimens were obtained from 80 candidates for sperm donation. Percoll-treated sperm samples compared with the sibling, unprocessed controls had significantly higher values of sperm motility characteristics and per cent of cells with normal morphology after freezing and thawing. Sperm binding ability to the zona pellucida was not statistically different (109 +/- 8.1% and 94 +/- 6.7% in unprocessed and Percoll-treated samples respectively). Sperm specimens processed by washing had significantly higher values for motility characteristics than untreated sibling samples, but no differences were found between the treated and untreated samples for morphology and binding to the zona pellucida (hemizona index of 75 +/- 7.0% and 76 +/- 6.7% in unprocessed and washed samples respectively). These findings suggest that, judged by the binding assay, the aforementioned pre-freezing separation processes have no adverse effect upon the fertilizing potential of the thawed sperm cells. These procedures make it possible to optimize the progressive motile sperm cell concentration of the frozen specimen, which facilitates the storage of samples with good quality, even when the features of the original semen are sub-optimal.     

Andrology: Intracytoplasmic sperm injection does not require special treatment pf the spermatozoa

In order to assess whether specific treatment of spermatozoais required prior to intracytoplasmic sperm injection (ICSI),three methods of sperm preparation were compared in this study.These three methods were (A) incubation of spermatozoa withpentoxifylline (PTX) and 2-deoxyadenosine (DOA), (B) electroporationfollowed by incubation in medium with PTX, and (C) no furthertreatment with the Percoll gradient. Controlled comparisonswere carried out between method A and method B in 21 patients,and between method A and method C in 32 patients. There wasno difference in the rates of fertilization and embryo cleavagewhen ICSI was done with spermatozoa treated by procedures A,B or C. Furthermore, the sperm selection procedure prior toICSI was done in two different media: T6 medium containing 1.78mM CaCl₂·2H₂O and a final washing step after the Percollgradient in T6 medium containing 5.0 mM CaCl₂·2H₂O, andEarle's medium containing 1.78 mM CaCl₂·2H₂O. The resultsof ICSI on sibling oocytes from 12 patients revealed no differencein the fertilization and embryo cleavage rates between the twodifferent media used during the sperm selection procedures.In conclusion, it appears that high fertilization and pregnancyrates can be obtained in couples with severe male-factor infertilityby ICSI and that no special treatment of the spermatozoa priorto ICSI is required.     

Andrology: The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters

High success rates have been reported for the use of intracytoplasmicsperm injection (ICSI) in alleviating essentially andrologicalinfertility. However, neither the relationship between any ofthe sperm parameters and the result of ICSI nor the minimalsperm requirements for ICSI have been investigated so far. Inthis paper, our objective was therefore to study the relationshipbetween three basic sperm parameters (total sperm count, spermmotility and morphology) and the outcome of ICSI by retrospectiveanalyses of fertilization, embryo development and pregnancyrates in 966 micro-injection cycles, performed with ejaculatedsemen. The results showed that there was no important influencefrom either the type or the extent of sperm impairment on theoutcome of ICSI. Even in the most extreme cases of male-factorinfertility, where cryptozoospermia or total astheno- or totalteratozoospermia was diagnosed in the initial semen sample,high fertilization and pregnancy rates were obtained by ICSI.Only one condition had a strongly negative influence on theresult of ICSI: where an immotile (presumably dead) spermatozoonwas injected into the oocyte. Thus the only ultimate criterionfor successful ICSI is the presence of at least one living spermatozoonper oocyte in the pellet of the treated semen sample used formicro-injection.     

Andrology: CASE REPORT Pregnancy after intracytoplasmic sperm injection of spermatozoa extracted from frozen-thawed testicular biopsy

The case report illustrates the successful application of anew method of sperm extraction from a frozen-thawed testicularbiopsy specimen within an established programme of intracytoplasmicsperm injection.     

Andrology: Effects of platelet activating factor on human sperm function in vitro

The direct effects of platelet activating factor (PAF) and thespecific PAF receptor antagonist, CV-3988, on the fertilizingability of human spermatozoa were investigated. PAF (10^–7–10^–11M) increased the human sperm penetration rates in a sperm penetrationassay at all doses >10^–11 M. In contrast, treatmentof the spermatozoa with 10^–5 CV-3988 caused a significantdecrease in human sperm penetration of zona-free hamster oocytesand adversely affected sperm motility after 24 h of incubation.This suppression was reversed by the addition of PAF. The acrosomereaction was also enhanced by PAF treatment of spermatozoa butthis effect was not observed in calcium-free medium. While 10^–5M CV-3988 decreased the acrosome reaction, the inhibition wasalso reversed by the addition of PAF. These results suggestthat PAF may have a direct role in the fertilizing capacityof human spermatozoa. These findings also suggest that PAF mayhave a clinical application in an in-vitro fertilization programme.     

Andrology: Effectiveness of various sperm processing methods in removing seminal plasma from insemination media

The effectiveness of the wash and swim-up (1 and 2 wash cycle),two layer Percoll gradient, SpermPrep and underlay sperm preparationmethods in removing seminal plasma from insemination suspensionswas investigated. The number of wash cycles needed to rid spermsuspensions of seminal plasma (n = 15) was also determined.All sperm preparation methods were compared to the control washand swim-up method, performed using Earle's buffered salt solution(EBSS), without serum supplementation so as not to mask seminalplasma concentrations. Control processing comprised 1 ml semensubjected to two wash cycles in EBSS followed by a swim-up periodof 60 min under 5% CO₂ in air. The Percoll method comprised1 ml semen layered on a discontinuous 36 and 81% Percoll gradient(n = 14). In the SpermPrep method, 1 ml semen was run througha SpermPrep II column (n = 10), and underlay samples (n = 12)were processed by two wash cycles, after which sperm pelletswere resuspended in the remaining medium, layered under 1.2ml EBSS and allowed to swim up under 5% CO₂ in air for 1 h.Seminal plasma and supernatant fractions obtained after eachprocessing phase were stored at -20°C and protein concentrationswere determined by spectrophotometry. The wash and swim-up methodwas the most effective in removing seminal plasma from spermsuspensions, followed by the two-layer Percoll gradient, underlayand finally the SpermPrep II processing methods.     

Andrology: Assessment of human sperm functional changes after in-vitro coincubation with cells retrieved from the human female reproductive tract

Human spermatozoa must undergo functional changes prior to fertilization;however, the site of this physiological event is still unclear.To evaluate the influence of the female reproductive tract onsperm fertilizing capacity, fertile sperm samples were coincubatedwith endometrial, oviductal, granulosa and cumulus cells, follicularfluid and maternal serum. Sperm penetration into the zona-freehamster ova and motion parameters were measured daily for 72h. Compared to control samples, endometrial and oviductal cellcultures did not alter sperm fertilizing capacity or their movementcharacteristics. Sperm coincubated with follicular fluid, granulosaor cumulus cells exhibited a significantly higher ability topenetrate zona-free hamster ova for up to 48 h. Sperm motilityincreased at 4 h in the presence of follicular fluid and serum.At 24 h sperm velocity and amplitude of lateral head displacementsignificantly declined in sperm samples exposed to serum, andvelocity also declined in follicular fluid and with coincubationusing ovarian follicle cells. Sperm motility and velocity decreasedat 48 h in the presence of serum, follicular fluid, cumulusor granulosa cells. Our findings may suggest that specific secretoryfactors produced in the human pre-ovulatory ovarian follicleenhance human sperm fertilizing capacity.     

Andrology: Mitochondrial disease and reduced sperm motility

Mitochondrial dysfunction reduces aerobic energy productionand results in symptoms from various tissues, depending on metabolicdemands. Mitochondrial adenosine triphosphate (ATP) is essentialfor sperm motility. Sperm motility was investigated in a patientwith a mitochondrial disease caused by reduced activity of themitochondrial enzyme complexes I and IV, and in two controlsubjects. Spermatozoa were cultured in media containing variousenergy substrates. Motility was judged by light microscopy,and ultrastructure by transmission electron microscopy. In thepatient with mitochondrial disease, 12% of the spermatozoa weremotile in the medium containing only glucose. There was a threefoldincrease in motile spermatozoa when pyruvate. and succinatewere present together with glucose. In contrast, the spermatozoaof both control subjects had best motility in the presence ofsubstrates for complex I, and no further increase was observedwhen succinate was added. Glucose and pyruvate enter the respiratorychain at complex I, and succinate at complex II. Electron microscopyof spermatozoa from the patient with mitochondrial disease revealedmitochondria with increased matrix, thickening of membranes,parallelization of cristae and lipid inclusions, which are characteristicfindings in mitochondrial disorders. Abnormal mitochondria werealso found in a spermatid, suggesting that the ultrastructuralchanges of mitochondria are primary rather than secondary todegeneration of the spermatozoa. The results indicate that mitochondrialdysfunction causes reduced sperm motility in some men.     

Diversity of the inhibitory effects on fertilization by anti-sperm antibodies bound to the surface of ejaculated human sperm

BACKGROUND: The presence of anti-sperm antibodies (ASA) in males can reduce fecundity. However, it has been shown that there is a diversity of ASA bound to the sperm surface. This study was performed to investigate the inhibitory effects on fertilization by ASA in males. METHODS: ASA were detected using the direct-immunobead test (D-IBT) in 509 semen samples. In some cases, the direct-sperm immobilization test (D-SIT) was carried out. The fertilizing ability of infertile males with ASA was determined as follows; (i) an IVF fertilization rate of >/=50%, (ii) a hemizona index (HZI) of >/=50%, and (iii) pregnancy established without the use of ART. RESULTS: In total, 18 (3.54%) infertile males had ASA on the sperm surface. Except for one male with an absolute indication for ICSI because of severe asthenozoospermia and two males who dropped out of this study, fertilizing ability in 15 males could be determined. Four (26.7%) men did not satisfy the criteria. The existence of sperm immobilizing antibodies on the surface of ejaculated sperm had no impact on fertilization. In four (57.1%) of seven patients who had IB-bound sperm of >/=80%, fertilizing ability was inhibited, while none of the eight patients who had <80% IB-bound sperm had an inhibitory effect on fertilization. There was a significant difference between the two groups (P = 0.01). CONCLUSIONS: Some sperm-bound antibodies are related to the inhibitory effects on fertilization, indicating that a diversity of sperm-bound antibodies exists in males. This result might be one of the reasons for the controversy of the relationship between ASA and male immunological infertility. Based on the present study, a sperm-zona pellucida binding assay should be performed for appropriate decision making in infertile males with ASA.     

Andrology: Factors affecting pentoxifylline stimulation of sperm kinematics in suspensions

Sperm suspensions were prepared either by the ‘swim-up’technique or by a Percoll gradient method before pentoxifyllinewas added to improve sperm motion characteristics. They weresubsequently washed and then incubated with oocytes in in-vitrofertilization (IVF) programmes. The curvilinear velocity andlateral head displacement were 20% higher in Percoll gradient-separatedsamples compared with samples prepared by the swim-up technique(P > 0.001), sperm motion characteristics being assessedby computer-assisted semen analysis. The dose-response studyin which samples were separated by Percoll gradient/swim-upmethod showed that pentoxifylline gave maximum enhancement ofsperm motion characteristics at a concentration of 2.8 mM/l,when the curvilinear velocity and lateral head displacementwere significantly increased (P > 0.001). However, when pentoxifyllinewas removed by washing, the enhanced motion characteristicswere reduced or lost in the process. Surprisingly, washing hada significant effect on the control samples, the motion characteristicsbeing Increased significantly (P > 0.001). This study castsdoubt on the usefulness of pentoxifylline in IVF programmes.     

Andrology: Percutaneous epididymal sperm aspiration for obstructive azoospermia

We report the use of percutaneous epididymal sperm aspirationas a simpler and more acceptable alternative to microscopicepididymal sperm aspiration for patients with obstructive azoospermiain whom bypass surgery is not feasible or has not been successful.Some contamination of the aspirate with blood is inevitable,but with careful sperm preparation techniques this can be reducedsubstantially in the final aliquot used for assisted conception.Spermatozoa with active forward progression may be used forgamete intra-Fallopian transfer treatment, but when this capacityis absent intracytoplasmic sperm injection is recommended. Threepregnancies were obtained in seven couples and a set of twinshas been delivered.     

Immunology: Diversity of the blocking effects of antisperm antibodies on fertilization in human and mouse

The blocking effects of complement-dependent sperm immobilizingantibodies in the sera of infertile women and monoclonal antispermantibodies against humans and mice on fertilization were investigated.The hemizona assay (HZA) and sperm penetration assay (SPA) wereused to study the inhibitory effects of sera from 22 infertilepatients positive for sperm immobilizing antibodies. Use ofthese tests allowed us to differentiate whether the antibodyblocked sperm—zona pellucida tight binding and/or spermpenetration into the ooplasm. The zona pellucida penetrationassay (ZPA) was also used to study the effects of four monoclonalantibodies (mAbs) on human sperm penetration into the zona pellucida.Seven mAbs against murine spermatozoa were tested for theirinhibitory effects on in-vitro fertilization (IVF) and HZA inmice. Of 22 patient sera with sperm immobilizing antibodies,21 (95.5%) inhibited HZA attachment and penetration, whereasthis did not occur in any of 13 patient sera without these antibodies.However, 19 of 22 (86.4%) patient sera with sperm immobilizingantibodies and eight of 13 (61.5%) patient sera without theseantibodies inhibited the SPA. Two (2C6, 1G12) of four mAbs againsthuman spermatozoa showed strong inhibitory effects in all theassays (HZA, ZPA and SPA). One mAb (3B10) did not inhibit HZAbut blocked ZPA and SPA. Another mAb (H6-3C4) seemed to haveno inhibitory effects on fertilization. Two (Vx 5 and Vx 8)of seven mAbs against murine spermatozoa inhibited IVF in micebut did not block mouse HZA. These findings suggest that antispermantibodies block fertilization at specific stages. Some of themmay inhibit sperm capacitation and thus prevent all processesof fertilization that follow. Some other antibodies may notaffect capacitation and sperm binding to zona pellucida butinhibit the acrosome reaction, followed by the blocking of spermpenetration through zona pellucida and ooplasm.     

Comparison of the ability of two sperm preparation techniques to remove microbes

The ability of a new sperm preparation method--self migration in sodium hyaluronate (SH)--to remove microbes from samples was compared to that of the traditional method of centrifugation/swim up (CS). Two 1-ml aliquots were taken from 20 semen samples used for inseminations, and prepared by each of the methods. Samples for culture were taken immediately before wash and at 2 and 24 h after. Microbes were found in all raw semen samples with a mean concentration of 47.8 x 10(3) colony forming units per ml (c.f.u./ml). After preparation and 2 h incubation with and without penicillin, both CS and SH were found greatly to reduce the number of isolates. There were no differences between the ability of the two methods to reduce microbe concentration, although SH recovered more progressive motile spermatozoa than CS (P less than 0.05). When penicillin preparation was omitted, both the CS and SH methods resulted in significantly more isolates after 24 h of incubation. This effect of penicillin preparation was also evident after only 2 h of incubation with CS, but not with SH. No clinical signs of infection were observed among women receiving treatment. We conclude that sperm preparation by self migration in sodium hyaluronate is a simple and safe method to remove microbes and to recover motile spermatozoa.     

Intrauterine insemination and comparison of two methods of sperm preparation

In a study of intrauterine inseminations (IUI) after clomiphene stimulation, a randomized comparison was made between a new method of sperm preparation, self migration in sodium hyaluronate (SH), and a traditional method, centrifugation and swim up (CS). After two IUI cycles with either SH or CS, the sperm preparation method was swapped and the patients received another two IUI cycles. Interjacent cycles of natural intercourse after clomiphene treatment served as the control. The SH method resulted in a significantly higher percentage recovery of progressive motile spermatozoa than the CS method, 17.7% versus 8.6% (P less than 0.01). The sperm samples were prepared by SH in 68 cycles and by CS in 57 cycles, resulting in six and five pregnancies, respectively. Pregnancies were obtained in 11 of 125 IUI cycles (8.8%) and in 3 of 124 control cycles (2.4%) (P less than 0.05). The pregnancy rate following IUI was highest in the patients with cervical factor (35%) and asthenozoospermia (23%), while none became pregnant in the group with oligozoospermia. In the unexplained infertility group, no difference between the pregnancy rates in IUI cycles and control cycles was seen. SH is a simple and rapid method of sperm preparation and it appears to give a high recovery of motile spermatozoa and a number of pregnancies which is comparable to that of CS. Treatment with IUI in cycles with a simple stimulation protocol seems to be valuable in cases involving either a cervical factor or asthenozoospermia.     

Andrology: Conventional in-vitro fertilization versus intracytoplasmic sperm injection for patients requiring microsurgical sperm aspiration

Intracytoplasmic sperm injection (ICSI) has been successfulin cases of extreme oligoasthenozoospermia in achieving pregnanciesvia in-vitro fertilization (IVF) with the lowest imaginablesperm counts. In azoospermia caused by congenital bilateralabsence of the vas deferens (CBAVD), it has been shown thatepididymal spermatozoa can be retrieved in large numbers, butfertilization rates using conventional IVF are low. Furthermore,no fertilization has ever been possible using testicular spermatozoawith conventional IVF. In the most extreme case of absence ofthe epididymis, spermatozoa can only be retrieved from maceratedtesticular biopsy specimens. In such cases, all that can beseen are free-floating Sertoli cells with many spermatids attached,and only occasional spermatozoa per high power field which haveonly the barest, occasional, slightly twitching motion. Theobjective of the present study was to determine whether ICSIcould achieve better results than conventional IVF with microsurgicalaspiration of spermatozoa (MESA). ICSI (using epididymal ortesticular spermatozoa) from men with CBAVD or irreparable obstructiveazoospermia, achieved good fertilization and normal embryosin 82% of cases, compared to 19% with conventional IVF. Therewas an overall fertilization rate of 45%, with 85% progressingto normally cleaving embryos using ICSI, compared to 6.9% usingconventional IVF. The pregnancy rate with ICSI/MESA was 47%per stimulated cycle (normal delivery rate was 30%), comparedto 4.5% with conventional IVF. These results were achieved inpatients who had consistently failed to fertilize in previouscycles with MESA and conventional IVF. We conclude that althoughcomplex mechanisms (facilitated by epididymal passage) may berequired by spermatozoa for conventional fertilization of humanoocytes (whether in vivo or in vitro), no such mechanisms arerequired for fertilization after direct microinjection. Becauseof the consistently good results using epididymal spermatozoawith ICSI in comparison to conventional IVF, and also the goodresults in extreme cases requiring testicular tissue spermatozoa,ICSI may be man dated for all future MESA patients with CBAVD,or with irreparable obstructive azoospermia.     

Andrology: Evaluation of a computer-aided semen analysis system with sperm tail detection

The aim of this study was to evaluate the Stroemberg-Mika cellmotion analyser (SM-CMA) which uses tail detection in orderto discriminate between immotile spermatozoa and other particles.Analysis of the spermatozoa by the SM-CMA can easily be checkedon a video monitor. The semen samples were from donors and patientsvisiting the fertility unit of the University Hospital, Hanzeplein,The Netherlands. Both fresh semen samples and purified spermsuspensions were used to estimate sperm counts and motilitycharacteristics. We tested the use of the x10 objective insteadof the x20 and we assessed the ways in which motility characteristcswere influenced by temperature. We found a considerable discrepancybetween sperm concentrations measured manually and by computerizedanalysis, both in semen samples and in purified sperm suspensions.The SM-CMA correctly recognizes motile spermatozoa, but underestimatesimmotile ones. Although temperature affects motility characteristics,in routine measurements the influence of short cooling periods,which are unavoidable, was nil. We found that using the x10objective can be useful, especially at low sperm concentrations.In our opinion, the SM-CMA system is, despite some shortcomingsin its user-interface, a useful and versatile instrument forexamination of human semen samples, with desirable features.     

Andrology: Variability of human sperm response to immediate and prolonged exposure to pentoxifylline

Pentoxifylline improves some motility characteristics of humanspermatozoa, but the variability of response to this drug hasnot been clearly defined. We used computer-assisted sperm motionanalysis to examine the in-vitro response of spermatozoa topentoxifylline. Individuals (n = 31) with normal sperm countswere randomly selected and their spermatozoa exposed to differentconcentrations of pentoxifylline. Further tests on a subgroupof individuals examined the longevity of spermatozoa in responseto this agent. Straight line velocity (VSL) was only improvedat 0.1 mM and the major effect of the drug was on curvilinearvelocity (VCL) and lateral head displacement (ALH). Prolongedexposure to pentoxifylline enhanced sperm motion only at 0.1mM. Higher concentrations produced dose-dependent detrimentaleffects on all the motion characteristics. There was considerableinter-individual variability in both VCL and ALH response rangingfrom little or no detectable response to a 40% increase abovecontrol value. The maximum response was most commonly seen ata concentration of 2 mM pentoxifylline.     

Andrology: The ability of the hemizona assay to predict human fertilization in different and consecutive in-vitro fertilization cycles

The objective of this prospective study was to examine the abilityof the hemizona assay (HZA) to predict fertilization outcomeof mature, pre-ovulatory oocytes under in-vitro fertilization(IVF) conditions. Since a large number of patients were evaluatedover a long period, the power of the HZA to prognosticate fertilizationresults in the same and subsequent (consecutive) IVF cyclesof those same patients was assessed. For IVF, only metaphaseII oocytes were used. For the HZA, both fresh oocytes donatedby patients at the time of IVF and oocytes recovered from surgicallyremoved ovarian tissue (and salt-stored) were used, and bisectedby micromanipulation techniques. Matching hemizonae were co-incubatedeither with spermatozoa from the patient (test) or from a fertileman (control) for 4 h. The number of spermatozoa tightly boundto the zona was counted. Patients (n = 112) were divided intotwo groups based on HZA results (expressed as HZA index or HZI):HZI 30% (n = 72) and <30% (n = 40). The patients with HZI<30% had significantly lower fertilization rates in boththe HZA—IVF cycle and in subsequent cycles compared topatients with HZI 30% (P < 0.03). Linear discriminant analysisindicated the HZA to have a sensitivity of 84%, and positiveand negative predictive values of 85 and 70% respectively, forprediction of fertilization outcome in a total of 233 cycles.It was concluded that the HZA is a good predictor of fertilizationrate in vitro, and can be used in the IVF setting to supplyadditional clinical information in malefactor patients.     

Optimizing sensitivity of the human sperm motility assay for embryo toxicity testing

The human sperm motility assay was used as a measure of quality control in the IVF laboratory. The effects of albumin supplementation and incubation time on the sensitivity of the human sperm motility assay were investigated. The assay was also compared with mouse embryo development. The human sperm motility assay and mouse embryo development assays were performed on 25 items commonly used in IVF laboratories. Sperm motility assay was conducted after 2, 4, 6, 8, 24 and 48 h incubation intervals under standard embryology conditions. A calculated sperm motility index value <0.75 was used to indicate sperm toxicity. It was found that optimum sensitivity (P < 0.01) of the human sperm motility assay was attained in the absence of albumin after 4, 8 and 48 h incubation periods. Items identified to be sperm toxic within 8 h by the human sperm motility assay were considered to be of clinical significance due to the close concordance of these results with mouse embryo development.