Easily decapitated spermatozoa defect: a possible cause of unexplained infertility.

We report the first 16 cases of a new sperm abnormality which we call 'easily decapitated spermatozoa defect'. This was discovered during intracytoplasmic sperm injection (ICSI) in couples with unexplained infertility. Semen analysis was normal, but minimal micromanipulation for ICSI resulted in decapitation of the spermatozoon during immobilization. For some oocytes the head and tail were injected separately, in others the intact sperm was injected after minimal immobilization. A fertilization rate of 47.5% was obtained using ICSI. Conventional in-vitro fertilization (IVF) on sibling oocytes (three cases) or in a previous cycle (three cases) resulted in total failure of fertilization. All patients reached the embryo transfer stage and three pregnancies resulted. Findings on electron microscopy in four cases included spermatozoa with degeneration or absence of the basal plate, abnormalities of the proximal centriole and degeneration of the midpiece with a large cytoplasmic droplet. We conclude that an occult sperm abnormality presenting as easily decapitated spermatozoa during ICSI could be a cause of unexplained infertility, as it resulted in total failure of fertilization in conventional IVF. Further research is necessary to investigate this sperm abnormality.     

A simple method for assessment of the human acrosome reaction of spermatozoa bound to the zona spellucida: lack of relationship with ionophore A23187-induced acrosome reaction

Acrosome reactions induced by the calcium ionophore A23187 [GenBank] andzona pellucid a (ZP) were studied. Sperm samples were obtainedfrom fertile men or men with normal semen analysis and normalsperm-ZP binding. Oocytes were obtained, with the consent ofthe patients, after the failure of fertilization in vitro. Motilespermatozoa selected by a swim-up technique were incubated with10 µM A23187 [GenBank] for 1 h, four oocytes for 2 h or solubilizedZP (4 ZP/µl) for 2 h. Spermatozoa bound to the ZP weredislodged and collected in a small volume of phosphate-bufferedsaline by aspirating the oocytes with a glass pipette with aninner diameter (120 µm) slightly smaller than the diameterof the oocyte. The acrosome status of the spermatozoa was determinedusing fluorescein-labelled Pisum sativum agglutinin. The proportionof spermatozoa undergoing the acrosome reaction on the ZP at2 h varied over a wide range (5–99%), but the agreementbetween results for the same semen sample exposed to differentgroups of oocytes was good: the standard deviations of the differencesbeing 9%. Pre-incubation of spermatozoa for 2 h did not increasethe ZP-induced acrosome reaction. Re-incubation of ZP with thesame sperm suspension for 2 h after removing ZP-bound spermatozoafrom the first 2 h incubation produced a significantly lowerZP-induced acrosome reaction in the second incubation (22 ±16%) than in the first incubation (30 ± 14%; P < 0.001,n = 20). There was no significant difference in the ZP-inducedacrosome reaction with oocytes with ZP which had or had notbeen penetrated by spermatozoa during the in-vitro fertilizationinsemination. Pre-incubation of spermatozoa with solubilizedZP blocked sperm-ZP binding. However, the acrosome reactioninduced by solubilized ZP (4 ZP/µl) was significantlylower than the acrosome reaction induced by intact ZP (10 ±5 and 30 ± 13% respectively, n = 11, P < 0.001), butthere was a high correlation (Spearman r = 0.822, P < 0.01)between the results. On the other hand, although the averageof the acrosome reaction was similar for A23187 [GenBank] (42%) and forZP (43%), there was no significant correlation between the resultsfor the two stimuli (n = 60). In conclusion, a useful methodfor assessing the ZP-induced acrosome reaction has been developedusing oocytes which failed to fertilize in vitro. The lack ofa relationship between the results of the chemical (A23187 [GenBank] )and physiological (ZP) stimali for the acrosome reaction inthe same subjects questions the biological basis of using A23187 [GenBank] for tests of sperm function. Solubilized human ZP in a concentrationthat blocks sperm-ZP binding is a less efficient inducer ofthe acrosome reaction than is intact ZP. It is possible thatthe three-dimensional structure of the ZP is important for inductionof the acrosome reaction or that spermatozoa which bind to theZP are more likely to acrosome react Assessment of the physiologicalacrosome reaction for diagnosis of sperm defects which interferewith the fertilization process should be concentrated on thespermatozoa which are capable of binding to the ZP.     

Andrology: Successful fertilization and establishment of pregnancies after intracytoplasmic sperm injection in patients with globozoospermia

Globozoospermia or round-headed spermatozoa is a rare type ofteratozoospermia where the acrosome is absent resulting in maleinfertility with no known therapy. A few studies have shownthat round-headed spermatozoa cannot bind to or penetrate thezona pellucida, and no normal fertilization has been observedin in-vitro fertilization (IVF) after insemination of humanoocytes with round-headed spermatozoa. In this study, the fertilizationcapacity of round-headed spermatozoa after intracytoplasmicsperm injection (ICSI) into human oocytes has been examined.In pre-clinical experiments, 45 oocytes were injected; 41 oocyteswere intact after injection, 15 oocytes were fertilized normally,and 13 of these 15 oocytes developed further in vitro. ICSIwas carried out in 11 treatment cycles of seven infertile coupleswith globozoospermia. Normal fertilization and embryo transferoccurred in four cycles (three patients). Positive serum humanchorionic gonadotrophin was observed in three cycles (two patients);one patient had a pre-clinical abortion and the other patientbecame pregnant twice: the first pregnancy was ectopic and thesecond pregnancy is a twin pregnancy which is currently at 16weeks of gestation.     

ERp57 is a potential biomarker for human fertilization capability

Human infertility is a growing concern and while many assisted reproductive technologies exist, their success rates are low. Thus, developing tests, possibly by assessing proteins involved in fertilization, that could predict the outcome of these technologies is of great significance. To identify candidate proteins, we used two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF techniques and detected the ERp57 protein from human testis protein profile. Immunohistochemistry showed that ERp57 was mostly located in spermatogenic cell cytoplasm from spermatocytes to the spermatozoa phases and in Leydig cells of human testes; it was also present at low levels in Sertoli cells. ERp57 was evident in human spermatozoa, primarily in the acrosome and tail; moreover, it appeared to translocate to the equatorial segment after the acrosome reaction. During sperm capacitation, the ERp57 protein underwent post-translational modification. Blocking ERp57 with antibodies significantly inhibited human sperm from penetrating zona-free hamster oocytes in a dose-dependent manner. Finally, expression levels of ERp57 were associated with fertility; they were decreased dramatically in IVF patients with low fertilization rates compared with those with high rates or to fertile sperm donors. Taken together, these results show that ERp57 is a component of human sperm acrosome proteins, which play a critical role in gamete fusion. Furthermore, ERp57 could be a novel phenotype marker for male infertility and has the potential to be used to assess sperm selection for IVF.     

Sperm treatment with extracellular ATP increases fertilization rates in in-vitro fertilization for male factor infertility

Previous work from our laboratory has revealed that extracellular ATP is a rapid and potent activator of human sperm acrosome reaction and fertilizing ability. In the present study, we assessed the effects of in-vitro sperm incubation with ATP on fertilization and embryo development in couples undergoing in-vitro fertilization (IVF) for male factor infertility. Oocytes from 22 women undergoing ovulation induction were divided in two groups and inseminated in vitro either with selected spermatozoa from the corresponding partner suffering from male factor infertility pre-incubated with ATP (2.5 mM) for 1 h, or with spermatozoa incubated with 0.9% NaCl solution (control group). After insemination, fertilization was assessed by the presence of pronuclei and then by embryo cleavage. The fertilization rate in the group of oocytes inseminated with ATP-treated spermatozoa improved significantly with respect to the control group (65.7 versus 42.5%, P < 0.01). No significant differences were observed in embryo cleavage and embryo quality. Embryos from both treated and control groups were transferred together in 20 transfer procedures, and in two couples fertilization was not obtained. Nine pregnancies occurred: one biochemical, one miscarriage, and seven patients delivered 9 healthy babies. Two pregnancies were twin with an overall pregnancy rate of 40.9% per cycle and of 45% per transfer. In conclusion, the results of the present study demonstrate that, in humans, extracellular ATP induces a significant increase of sperm fertilizing potential, as these findings are a rationale for the use of ATP for in-vitro treatment of human spermatozoa during IVF.     

Cryopreservation of single human spermatozoa

A procedure is described that allows cryopreservation and efficient post-thaw recovery of either a single or a small group of human spermatozoa. This is achieved by injecting them into cell-free human, mouse or hamster zonae pellucidae before the addition of cryoprotectant. The method involves a combination of physical micromanipulation procedures and glycerol-mediated cryoprotection. Zonae were tracked by positioning them in straws between two small air bubbles prior to freezing. Spermatozoa from poor specimens were cryopreserved and their fertilizing ability after thawing was compared with that of fresh spermatozoa from fertile men. Human eggs used for fertilization testing were either 1 day old or in-vitro matured. Only 2% of the frozen zonae were lost and >75% of spermatozoa cryopreserved in this manner were recovered and prepared for intracytoplasmic sperm injection. The feasibility of cryopreserving a single spermatozoon was assessed. Fifteen motile spermatozoa were frozen in 15 zonae, of which 14 were recovered after thawing. Ten were injected into spare eggs, of which eight became fertilized. Spermatozoa recovered mechanically from human zonae fertilized the same proportion of oocytes as fresh fertile control spermatozoa. The recovery and fertilization rates with spermatozoa frozen in animal zonae were 87 and 78% respectively. The fertilization rate was marginally higher (P < 0.05) than that for spermatozoa frozen in human zonae, perhaps because the latter may have acrosome reacted more frequently. The zona pellucida appears to be an ideally suited sterile vehicle for storage of single spermatozoa.      

Establishment of a capillary-cumulus model to study the selection of sperm for fertilization by the cumulus oophorus

BACKGROUND: Spermatozoa have to traverse the cumulus oophorus before fertilization in vivo. Evidence suggests that the cumulus oophorus plays an important role in the fertilization process. We describe the establishment of a capillary-cumulus oophorus model with which to study the action of cumulus mass on the function of human spermatozoa. METHODS: Human cumulus oophorus was aspirated into a glass capillary. Spermatozoa were allowed to pass through the cumulus mass in the capillary from one end of the capillary. The spermatozoa that had traversed the mass were collected at the other end of the capillary and underwent sperm function analyses. RESULTS: Compared with those spermatozoa cultured in medium alone, spermatozoa exposed to the cumulus mass were more likely to have normal morphology, be capacitated and acrosome reacted, with a distinct motility pattern and better zona-binding capacity. CONCLUSION: A novel in vitro model for spermatozoa penetration through the cumulus oophorus was established. The model can be applied to investigate the effect of the cumulus oophorus on sperm functions.     

Induction of acrosome reaction in human spermatozoa used for subzonal insemination.

Human spermatozoa were injected into the perivitelline space of oocytes from 43 couples (44 cycles) in whom fertilization had failed in conventional in-vitro fertilization (IVF). The spermatozoa were treated to enhance the percentage of acrosome-free spermatozoa either by incubation for 24 h in T6 medium with 50% follicular fluid (v/v) or by incubation for 24 h in T6 medium followed by electroporation and incubation for a few hours in T6 medium with 3.5 mM pentoxifylline. After these two procedures, the mean percentage of acrosome-free spermatozoa increased to 35.5 and 53.9% respectively. Up to three spermatozoa were injected into the perivitelline space of metaphase II oocytes; few oocytes were damaged during the injection procedure. The overall fertilization rate was 30.9% of the 433 oocytes that were intact after subzonal insemination. Only 3% of the injected oocytes had more than two pronuclei. The cleavage rate of the fertilized oocytes was 80%. There was no difference in the fertilization and cleavage rates between the two sperm treatment procedures. One, two or three embryos were replaced in 34 cycles and seven patients became pregnant. In three of the four ongoing pregnancies, prenatal diagnosis by amniocentesis indicated a normal karyotype.     

Recombinant human albumin supports hamster in-vitro fertilization

BACKGROUND: Serum albumin is normally required to support sperm capacitation and IVF, but its mechanism of action is not well understood. Commercial serum albumin preparations are contaminated with a variety of other proteins and compounds, and their biological activity is variable. Recombinant human albumin (rHA) might replace serum albumin for IVF. METHODS: rHA was examined for its ability to capacitate hamster spermatozoa and to support fertilization in vitro. A standardized hamster IVF system was used to compare the capacitation-supporting activities of rHA and two commercial preparations of bovine serum albumin (BSA) in a chemically defined culture medium. Epididymal spermatozoa were incubated for 4 h at 37 degrees C under 5% CO(2) in air in either the basic medium containing rHA, one of the two BSA preparations or no protein, and then cultured in the same medium with ovulated oocytes for another 4 h. The experiment was replicated five times. RESULTS: Spermatozoa incubated in protein-free medium fertilized only one oocyte (2% of total), significantly less than any of the other three treatment conditions (P < 0.01); spermatozoa incubated in medium containing rHA or BSA fertilized 86-93% of oocytes. There were no differences between the three albumin-containing treatment groups. CONCLUSION: rHA is equivalent to commercial serum albumin preparations in its ability to support sperm capacitation and fertilization in this test system. This finding has considerable practical implications for human IVF and may also help efforts to elucidate the mechanism of sperm capacitation.     

Andrology: High fertilization prediction by flow cytometric analysis of the CD46 antigen on the inner acrosomal membrane of spermatozoa

The study was set up to determine the relationship between thehuman sperm acrosome reaction and fertilization in couples undergoingroutine in-vitro fertilization (IVF) treatment. Prospectivedata analysis was carried out on all IVF patients during a 6month period. Exceptions were those patients having insufficientsperm concentration to allow both acrosome reaction determinationand insemination. The main outcome measures were the predictionof fertiliza tion in IVF patients using flow cytometric analysisof the spontaneous and ionophore-induced acrosome reaction [givingthe acrosomal response to ionophore challenge (ARIC) score]in the male partner's spermatozoa versus standard analyticalmethods of sperm motion parameters and morphology. Stepwiselogistic regression indicated only two independent factors predictiveof fertilization: ARIC score (x² = 109.6, P < 0.0001) andpost-Percoll % motility (x² 8.8, P < 0.003). Of patientswith an ARIC score of >10, 92% had >30% of oocytes fertilized;100% of patients with an ARIC score of <10 had <30% fertilizationof oocytes. The sensitivity and specificity of the assay systemwere 1.00 and 0.82 respectively. The results would indicatethat the ARIC test as measured by flow cytometric analysis ofCD46 binding is a sensitive and specific assay for use in theprediction of fertilization in IVF patients, thus enabling directchannelling of those patients with ARIC scores of <10 intothe more invasive micro-assisted fertilization schemes.     

Unexplained in-vitro fertilization failure: implication of acrosomes with a small reacting region, as revealed by a monoclonal antibody.

To determine the acrosomal characteristics related to in-vitro fertilization (IVF) outcome, spermatozoa from 50 men whose wives had resorted to IVF have been studied by indirect immunofluorescence microscopy with anti-human pro-acrosin monoclonal antibody 4D4 (mAb 4D4), prior to and after incubation in a capacitating medium. The antibody labelled only the acrosomal principal region (APR), revealing its shape (i.e. normal, small or amorphous) and its status (i.e. unreacted, partially or totally reacted). The IVF outcome distinguished: (i) spermatozoa which were able to fertilize at least one oocyte in vitro (group I; n = 25) and (ii) spermatozoa which failed to fertilize any oocyte in vitro (group II; n = 25). The semen characteristics of the two sperm groups, including the acrosome morphology, were similar according to conventional analysis. The mAb 4D4 detected in both the whole and the swim-up sperm cell fractions a lower percentage of normal APR in group II (< 50% for 10 patients in group II versus one patient from group I), which was related to a higher percentage of small APR. Moreover, after 21 h incubation, group II had a lower acrosomal loss index. The spermatozoa of five patients of this infertile group II did not undergo acrosomal modification whereas spermatozoa of all group I patients underwent the acrosomal reaction. The data showed that the relationship between acrosomal anomalies and IVF failure is mainly due to an increased incidence of acrosomes with a reduced size of the region involved in the acrosome reaction. Immunodiagnosis of this acrosomal region by means of mAb 4D4 is informative for IVF outcome.     

The spontaneous acrosome reaction of human spermatozoa incubated in vitro

Motile human sperm populations were prepared from liquefiedsemen (10 donors x 3 replicates) using Percoll density gradientsat 30–60 min post-ejaculation. Sperm suspensions wereincubated in a complex ’synthetic tubal fluid‘ culturemedium (STF) at 37°C under 5% CO₂ in air for up to 36 h.Parallel aliquots were incubated with 50 µM A23187 toinduce maximum acrosome loss (AR_max). Acrosome reactions wereassessed using both the triple-stain (TS) technique and fluorescentpeanut agglutinin (PNA) lectin-labelling. During incubation,the proportion of TS acrosome reacted spermatozoa increasedfrom 9.1 to 54.3% with AR_max being 68.3%. Spermatozoa showingintact acrosomes by PNA labelling decreased from 68.4 to 26.1%over 36 h of incubation (AR_max = 13.8%). Simultaneously, spermatozoashowing complete acrosomal loss (no PNA labelling) increasedfrom 8.1 to 27.0% (AR_max= 46.3%). Therefore, while only 23.5%of cells were actually undergoing acrosomal changes at the startof incubation, this had increased to 46.9% after 36 h (AR_max=40.7%). These experiments clearly show that even in selectedpopulations, not all human spermatozoa are capable of undergoingan acrosome reaction. However, the incidence of acrosomal changesafter 36 h of incubation did approach the AR_max. These levelsof spontaneous occurrence of the human sperm acrosome reactionwere higher than those reported in many other in-vitro incubationstudies: an improvement that may be attributable to the morephysiological nature of the STF culture medium     

Fate of the acrosome in ooplasm in pigs after IVF and ICSI

BACKGROUND: ICSI bypasses the sperm-oolemma interactions that, in normal fertilization, depend on completion of the acrosome reaction. Morphological changes in the acrosomes of sperm in the ooplasm were therefore examined following IVF and ICSI using pig gametes. METHODS: In-vitro-matured porcine oocytes were used for ICSI or IVF. Oocytes were then stained with fluorescein isothiocyanate-conjugated peanut agglutinin lectin (FITC-PNA), which specifically labels the outer acrosomal membrane of boar sperm and the cortical granules (CG) in porcine oocytes. This was followed by observation under a confocal laser scanning microscope. RESULTS: In ICSI, PNA showed the presence of disintegrated acrosomes that classified into four categories. Heterogeneous chromatin decondensation was observed in the sperm with intact/disintegrated acrosome, whereas acrosomes were barely detected in oocytes which had formed a male pronucleus. Both in ICSI and IVF, PNA-positive tails were concomitantly observed with one type of disintegrated acrosome, which was considered to be acrosome-reacted. The disappearance of CG in activated oocytes after ICSI was similar to that after IVF. CONCLUSIONS: The PNA-binding properties of sperm head components introduced into the ooplasm during ICSI are different from those after IVF. The delay of sperm chromatin decondensation is associated with that of acrosomal disassembly. Acrosomes appear to disintegrate in the ooplasm whether or not the acrosome reaction has taken place. Oocytes undergoing ICSI appear normally activated in terms of meiotic resumption and CG exocytosis.     

Should ICSI be the treatment of choice for all cases of in-vitro conception?

The objective of this study was to examine different clinical scenarios of in-vitro conception, viz. fertilization with conventional IVF, IVF with high insemination concentration (HIC) and intracytoplasmic sperm injection (ICSI), and assess on a sibling oocyte comparison the hypothesis that ICSI should be performed in all cases requiring in-vitro conception. ICSI with husband's spermatozoa had a higher incidence of fertilization as compared with IVF or IVF with HIC with donor spermatozoa (if previous failure of fertilization had occurred) for unexplained infertility. Similarly, ICSI with husband's spermatozoa had as high an incidence of fertilization as IVF with donor spermatozoa for patients with severe oligozoospermia, asthenozoospermia and/or teratozoospermia, even when the spermatozoa were not selected for their morphology. Two studies were performed to assess ICSI in potential oocyte-related failure of IVF, viz. when fertilization occurred in >50% of oocytes for one group of patients, and in <50% of oocytes in a second group. In both of these studies a significant proportion of the oocytes that failed to fertilize with conventional IVF eventually fertilized after ICSI. The overall conclusion was that ICSI as a first option offers a higher incidence of fertilization, maximizes the number of embryos and minimizes the risk of complete failure of fertilization for all cases requiring in-vitro conception. However, among other concerns, current knowledge of ICSI as an outcome procedure does not provide the confidence to use this process in all cases of IVF for the time being.     

Andrology: Successful treatment of severe male factor infertility in 100 consecutive cycles using intracytoplasmic sperm injection

In this report, we present the results of our first 100 consecutivecycles of intracytoplasmic sperm injection (ICSI). Overall,fertilization occurred in 98% of cycles and embryos were transferredin 94% (2.6 embryos per cycle). About 50% of patients had embryosfrozen. The overall fertilization rate was 71%, of which 4%were abnormally fertilized (three pronuclei). A total of 30clinical pregnancies were established (32% per transfer), resultingin 18 singleton, six twin and one triplet ongoing pregnancies.The implantation rate per embryo was 15%. There were no significantdifferences in the fertilization or pregnancy rates betweenpatients Who had only occasional motile spermatozoa in the ejaculate,semen that was too poor for routine in-vitro fertilization (IVF),or who had failed routine IVF and/or subzonal sperm injection(SUZI). A group of 18 patients were treated with both ICSI androutine IVF on their first cycle because of the high likelihoodof failed fertilization due to poor sperm morphology (<20%normal). In this group, ICSI oocytes had a fertilization rateof 76% compared to only 15% for the routine IVF (control) oocytes,and six patients conceived after transfer of ICSI embryos (33%),indicating that ICSI can be used successfully on 50% of theoocytes if fertilization failure is expected. Similarly, patientswho had failed to become pregnant with SUZI achieved excellentresults after ICSI. There were no significant differences betweenICSI and routine IVF in the proportions of grade 1, 2 or 3 embryoson day 3 post-oocyte recovery. In conclusion, we have achievedresults comparable to those reported from Belgium and we havefound that ICSI is universally applicable to all forms of severemale factor infertility. ICSI produces fertilization, pregnancyand freezing rates comparable to routine IVF with normozoospermicsamples and has none of the drawbacks of other assisted fertilizationtechniques.     

A new test for the assessment of sperm- zona pellucida penetration: relationship with results of other sperm tests and fertilization in vitro

The spermatozoa of some patients attending for in-vitro fertilization(IVF) fail to penetrate the zona pellucida in vitro. A testhas been devised to identify these cases. It is based on thenumber of spermatozoa penetrating into the zona pellucida, whichwere counted after removing spermatozoa bound to the zona surfaceby vigorous aspiration of each oocyte through a narrow gauge(120 µm) glass pipette. The oocytes were collected from197 patients undergoing IVF treatment with their own gametes;79 with no oocytes fertilized and 118 with some oocytes fertilized.Sperm motility, morphology and DNA normality (acridine orangestain) were also measured. The relationships between sperm testresults and IVF rate were examined by logistic regression. Theproportions of penetrated zonae, normal sperm morphology andnormal DNA were the most significant factors related to IVFrate in the whole group. Also, in patients with 30 spermatozoabound per zona pellucida or with normal sperm morphology 30%,the proportion of penetrated zonae and normal DNA were mostsignificant. Oocytes from 42 patients who had zero fertilizationand low sperm-zona binding (average, 2.2 spermatozoa/zona pellucida)were re-incubated with normal donor spermatozoa: large numbersof spermatozoa bound (average, 88 spermatozoa/zona pellucida)and each zona was penetrated by at least one spermatozoon. Inconclusion, the percentage of zonae penetrated was the variablemost significantly correlated with IVF rate. Penetration ofthe zona was also strongly related to fertilization rates inpatients without defects of sperm morphology and sperm-zonabinding. In patients where all zonae were penetrated, poor fertilizationmay be due to sperm morphology and DNA abnormalities. Failureof sperm-zona binding and penetration in vitro in patients withfailure of fertilization was mainly due to sperm defects andnot oocyte defects     

Intracellular calcium increase and acrosome reaction in response to progesterone in human spermatozoa are correlated with in-vitro fertilization

In this study we have investigated responsiveness to progesteronein spermatozoa from a group of unselected male partners of couplesundergoing in-vitro fertilization (IVF). We evaluated progesterone-stimulatedintracellular Ca²⁺ ([Ca²⁺]_i) and percentage increase in acrosomereaction in the same sperm sample used for oocyte inseminations.[Ca²⁺]_i was measured with a fluorimetric method, while the acrosomereaction was assessed using a fluorescent probe (fluoresceinisothiocyanate-labelled peanut lectin). The average percentage[Ca²⁺]_i as well as the rate of increase in the frequency ofacrosome reaction following progesterone challenge were significantlylower (P < 0.005) in the group of patients with a fertilizationrate <50%. In addition, significant correlations betweenthe fertilization rate and the progesterone-stimulated [Ca²⁺]_iand acrosome reaction increases (r = 0.78 and r = 0.79 respectively)were observed. Furthermore, in cases of fertilization failure,no increase of [Ca²⁺]_i or acrosome reaction was observed inresponse to progesterone with the exception of one case. Ourresults indicate that [Ca²⁺]_i and acrosome reaction increasesin response to progesterone can be of value in the predictionof sperm fertilizing ability. As the two parameters were significantlycorrelated to each other (r = 0.86), the two assays have similarIVF predictive value and might be used interchangeably as adiagnostic tool in the assignment of male patients to the differentkinds of assisted fertilization techniques.     

Microsurgical fertilization and teratozoospermia

Semen parameters were correlated with the outcome of partial zona dissection (PZD) in 42 couples with male factor infertility. Although fertilization rates were reduced, 12% of the embryos implanted following replacement. Spermatozoa from teratozoospermic sperm populations were able to fuse with oocytes following zona penetration through the artificial gaps. PZD followed by insemination with less than 5% normal spermatozoa led to 20 embryos which, upon replacement, did not implant. Motility and sperm count were not clearly correlated with the outcome of PZD and are therefore less useful indicators for patient selection. Teratozoospermic patients who previously failed to fertilize were compared to a group of similar patients who had not attempted IVF before. Although fertilization was significantly improved in first-time patients, 41% of the patients whose spermatozoa were initially unable to fertilize had at least one embryo when PZD was performed. Several pregnancies were established in this group. Subzonal sperm insertion (SZI) and PZD were compared in 19 patients using sibling oocytes. A significant fraction of spermatozoa from infertile men were able to fuse with the oolemma when directly inserted into the perivitelline area. Using a sucrose solution to shrink the ooplasm, only 1% of the oocytes were damaged during SZI. Monospermic fertilization rates following PZD and SZI were 15 and 16%, respectively. Both micromanipulation methods were successful in most patients. However, in two small groups of patients, only one technique resulted in fertilization.     

Frequency of disordered zona pellucida (ZP)-induced acrosome reaction in infertile men with normal semen analysis and normal spermatozoa-ZP binding

Results of zona pellucida (ZP)-induced acrosome reaction (AR) are reported for 186 normospermic men with unexplained infertility and compared with 34 normal fertile men and 54 patients with disordered ZP-induced AR (DZPIAR) diagnosed after failure of standard IVF. For each ZP-induced AR test, four oocytes that failed to fertilize in IVF were incubated for 2 h with 2x10(6)/ml motile spermatozoa. Spermatozoa tightly bound to the ZP were recovered by aspirating the oocytes with a pipette and the AR assessed using pisum sativum agglutinin labelled with fluorescein. The standard deviation of the difference was 5.2% for repeated tests for ZP-induced AR on different ejaculates from 54 men. The ranges for the ZP-induced AR were 3-98% for normospermic infertile men, 24-95% for fertile men and 0-16% for DZPIAR patients. In the normospermic group, there was a significant correlation between ZP-induced AR and sperm concentration (Spearman r = 0.238, P < 0.001). Using ZP-induced AR < or =16% as the threshold for diagnosis of DZPIAR, the frequency of this condition in normospermic infertile men would be 25%. Thus DZPIAR is common with normospermic idiopathic infertility and this condition should be diagnosed before assisted reproductive technology since it requires intracytoplasmic sperm injection.     

Effects of human hydrosalpinx fluid on in-vitro murine fertilization

Patients with hydrosalpinges show a decrease of both fertility and clinical outcome of IVF and embryo transfer treatment. Several reports have demonstrated the negative effects of hydrosalpinx fluid (HSF) on embryo development and implantation. The aim of this study was to determine whether human HSF, collected from infertile patients, might exhibit a deleterious effect on gametes and fertilization using a murine IVF system. Murine gametes were co-incubated during IVF until first cleavage with human HSF diluted to 50% from four patients (HSF1-4). It was demonstrated that HSF affected fertilization, as determined by the count of the 2-cell embryos. Pre-incubation of spermatozoa with HSF during capacitation significantly lowered the percentage of 2-cell embryos (P < 0.05). While HSF1-3 had no significant effect on motility and viability of spermatozoa, HSF4 almost completely affected their survival. In contrast, pre-incubation of ovulated oocytes surrounded by their cumulus cells with HSF before IVF did not impede first cleavage. Taken together, these results suggest that HSF has a cytotoxic effect on spermatozoa and/or impairs the fertilization process, probably by altering capacitation/acrosome reaction and/or ligand(s)-receptor(s) interactions. Hydrosalpinges may be partly associated with sterility through HSF inhibitory effects on fertilization.