A follow-up study of sperm preparation for IVF by swim-up in a solution of hyaluronate

Spermatozoa prepared for in-vitro fertilization (IVF) by swim-up in a balanced salt solution containing hyaluronate gave rates of fertilization, cleavage and pregnancy which were not significantly different from those obtained with sperm prepared by swim-up in standard IVF medium followed by centrifugation. However, the content of prostaglandin F2alpha in the final sperm suspension was higher using hyaluronate but this seemed to be of no consequence for IVF. Thus, preparation of normal sperm samples for IVF may be simplified by performing swim-up in a balanced salt solution containing hyaluronate.     

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.     

Human sperm--zona pellucida binding, sperm characteristics and in-vitro fertilization

The number of sperm bound to the zona pellucida (ZP) were countedon 660 oocytes that failed to fertilize in vitro from 162 patients.Oocytes from clutches in which some fertilized had significantlymore sperm bound to the ZP than did those from clutches in whichall oocytes failed to fertilize. Oocytes from patients in whomall were not fertilized and no sperm bound to the ZP were ableto bind normal fertile donor sperm after storage in ammoniumsulphate solution. The number of sperm bound to the ZP was significantlyrelated to the proportion of sperm with normal morphology andnormal intact acrosomes in semen, sperm concentration inseminated,sperm motility and viability. The number of sperm bound to theZP, sperm normal morphology, diagnosis of male infertility andsperm concentration in semen were significantly related to thefertilization rate by logistic regression analysis. Thus thenumber of sperm bound to the ZP is an indicator for IVF successand low binding appears to be more frequently associated withsperm defects than oocyte defects.     

Andrology: A new predictive test for in-vitro fertilization based on the induction of sperm acrosome reaction by N-acetylglucosamine-neoglycoprotein

Neoglycoproteins with N-acetylglucosamine residues (BSA-GIcNAc)induced specifically the acrosome reaction (AR) in human spermatozoa.Our objective was to investigate the relationship between thisphenomenon and the in-vitro fertilization (IVF) rate. Spermsuspensions from IVF protocols were incubated with BSA-GlcNAc(t), using calcium ionophore (i) or medium alone (c) as positiveor negative controls. When the normalized AR percentage ratio(STIM) (%ARt-%ARc): (%ARi-%ARc) was compared with fertilizationrate in 31 couples from our IVF programme, a positive correlationwas found (r = 0.46, P < 0.01). The fertilization rate inpatients with STIM 0.2 was higher than in non-responders (STIM< 0.2); 72 ± 7% compared with 5 ± 3%. The overallpredictive value of this test for adequate fertilization rate(>30%) was 87%, sensitivity 91% and specificity 78%. Falsepositives were 9% and false negatives 22%. For successful fertilizationrates (>60%), the results were: overall predictive value,84%; sensitivity 100%; specificity 64%. False positives were23% and no false negatives were found. The results indicatedthat the induction of AR in human spermatozoa by GlcNAc-neoglycoproteinscould be used to predict their fertilizing ability in vitro.     

Andrology: Nuclear decondensation of sperm head and failure at in-vitro fertilization: an ultrastructural study

The problem of unexplained male infertility was investigatedby electron microscopic study of spermatozoa from 51 males.The subjects were subdivided as follows: group A (n = 25) normalfertile males (controls), group B (n = 13) successful in-vitrofertilization (IVF) cases (fertilization rate >50%), groupC (n = 13) failed IVF cases. All subjects included in groupsB and C had a 6–12 year history of childlessness and IVFwas employed when other methods of assisted reproduction failed.The study of spermatozoa in fertile males (controls) was carriedout to establish baseline ultrastructural abnormalities. Inall 51 cases, an average of 330 (280–800) sperm headsand 660 (330–1190) sperm tails were studied. Decondensationof nuclear chromatin was observed in 70 ± 15% (mean ±SD) of spermatozoa in failed IVF cases, 16 ± 5% in successfulIVF cases and 7 ± 3% in controls. These results werefound to be statistically significant (P > 0.001). The meanvalue for motility of spermatozoa in all three groups was withinaccepted limits of normality. It is concluded that decondensationof nuclear chromatin seen by electron microscopy is one of themost important causes of male infertility. It is advocated thatelectron microscopic examination of semen should be carriedout in all cases of longstanding, unexplained male infertilitybefore embarking upon IVF programmes.     

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.     

Male factor as determinant of in-vitro fertilization outcome.

The effect of different semen parameters was evaluated in 200 consecutive couples in an in-vitro fertilization (IVF) programme. All semen analyses were performed on the native aliquot of semen which was subsequently prepared and used for in-vitro insemination. Morphology evaluation using strict criteria (kappa 0.46 and r = 0.565) was compared with progressive motile sperm density (kappa 0.37 and r = 0.333) and the conventional World Health Organisation (WHO) evaluation of morphology (kappa 0.31 and r = 0.378). Results show that morphology evaluation using strict criteria is the best predictor of IVF and density of progressively motile spermatozoa can be an optional method. The combined results of strict morphology and motile concentration progressively showed that if both parameters were below the cut-off points of 5% and 3 x 10(6)/ml respectively, the fertilization rate per oocyte was very low (18%). No pregnancies were achieved in this group. When both parameters were above the cut-off points, the fertilization rate per oocyte was high (72%) (P less than 0.005) and the pregnancy rate per embryo transfer was 27%. Predictive values indicate that morphology evaluation using strict criteria and the number of progressive motile spermatozoa can be used as patient selection criteria for infertility clinics.     

Andrology: Semen parameters as predictors of in-vitro fertilization: the importance of strict criteria sperm morphology

This study evaluated 120 couples undergoing in-vitro fertilizationtreatment to determine which semen parameter(s) predicted fertilizationand whether there was any consistent relationship between strictcriteria and standard assessment of sperm morphology. Strictcriteria morphology was the only significant predictor of fertilization(P = 0.0006, r² = 0.09), with a sensitivity of 94% and a specificityof 40%. A 12% cut-off point presented a negative predictivevalue of 98% and a positive predictive value of 22%. The probabilityof satisfactory fertilization is 40% with morphology <4%,which increases to 97% with normal morphology (12%). The receiveroperating characteristic curve deviated significantly from thediagonal with a 76% area wider the curve, making this a superiorpredictive test. This was augmented by likelihood ratios (LR)of 8.25 (LR +) for results with <4% normal morphology and0.15 (LR-) for results with 12% normal morphology by strictcriteria. While there was some correlation between strict criteriaand standard assessment of morphology (r²=0.35), the formerexplained only 12% (r²=0.12) of the variability in the latter.This study concludes that strict criteria morphology predictsfertilization, while other semen parameters do not. A 12% cut-offpoint makes strict criteria morphology an excellent predictorof satisfactory fertilization, while a value <4% is a goodpredictor of poor fertilization.     

Conventional in-vitro fertilization versus intracytoplasmic sperm injection in sibling oocytes from couples with tubal infertility and normozoospermic semen.

An auto-controlled study was conducted in couples with tubal infertility and normozoospermic semen. The fertilization rates and embryonic development in sibling oocytes treated, using the same semen sample, either by conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) at the same time were compared. Sibling oocyte-cumulus complexes (OCC) of 56 different couples with tubal infertility and normozoospermic semen were randomly divided in order of retrieval into two groups inseminated either by conventional IVF or by ICSI. Of the retrieved OCC in the same cohort, 53.0 +/- 31.2 and 62.0 +/- 26.6% showed two distinct pronuclei after conventional IVF and ICSI respectively (not significant). Complete fertilization failure occurred after conventional IVF in 12.5% (7/56 couples). After ICSI, the comparable figure was 3.6% (2/56). The number of cases was too small to apply a statistical test to this difference. Total cleavage rates were quite similar: 86.7 +/- 28.0 and 90.1 +/- 21% of the zygotes developed into transferable embryos after IVF and ICSI respectively (not significant). Similarly, no difference in embryo quality was observed. Although injection and insemination of the oocytes were performed at the same time in the two groups, at 42 h post-insemination more embryos were at the four-cell stage after ICSI (P < 0.001) than after conventional IVF, where more embryos were still at the two-cell stage (P < 0.02). Embryo transfer was possible in all 56 couples, resulting in 16 positive serum human chorionic gonadotrophin tests (28.6% per embryo transfer), from which a clinical pregnancy resulted in 15 couples. The best embryos were selected for transfer independently of the insemination procedure, but preferably from the same origin. There appeared to be no difference in implantation potency of the embryos obtained with either technique after the non-randomized transfers.     

Studies on psychosomatic implications of infertility-effects of emotional stress on fertilization and implantation in in-vitro fertilization

A case report shows the long and stony path of a couple whohad to go through eight attempts at IVF in 4 years until thedesired child was achieved. The psychosomatic implications ofthe various ‘failures’ of treatment become clearerwhen compared with the results of a psycho-endocrinologicalquestionnaire completed by 551 patients and 115 controls since1982. This questionnaire contains among others the Giessen personalitytest for the image of self, mother and father, a somatic complaintsscore to evaluate the severity of autoiiomic disorders, itemsfor the presence and severity of possible gynaeco-endocrinologicalsymptoms and items to evaluate the reference persons and theirchanges during childhood. The following results could be obtained.In a comparison of the means of 58 different somatic complaints,patients rated significantly higher than controls (P = 0.002).Women with menstrual disturbances, menstrual pains, acne orunclear skin or dry skin also suffered more from other complaints.These symptoms were also correlated with a higher score fordepressive mood. Those women who were not brought up by bothparents during their childhood also scored significantly higher(P < 0.0001) in the somatic complaints score. In the Giessenpersonality test patients appeared significantly more compulsive(P < 0.002) and depressive (P = 0.003) than the controlsand identified themselves more with their mothers. From 60 patientsin our IVF programme, 19 had no fertilization of oocytes andscored significantly higher (P = 0.008) on the social potencyscale than did the patients with fertilized oocytes. Women withunsuccessful treatments in terms of pregnancy scored significantlyhigher in the social resonance and social potency tests. Weinterpret this exaggerated positive attitude as an attempt toovercome inner fears, doubts and ambivalences. These resultssupport the importance of psychosomatic aspects in infertilityand reassure us of the necessity of including counselling inany infertility treatment, including IVF.     

Pilot study with 120 mg Andriol treatment for couples with a low fertilization rate during in-vitro fertilization.

Treatment with 120 mg/day of Andriol (testosterone undecanoate; Organon, The Netherlands) was given to 11 men whose semen had either failed to fertilize, or had resulted in a less than 33% fertilization rate in a first in-vitro fertilization (IVF) trial. Repeat IVF at the end of a 3 month treatment period resulted in a highly significant increase in the number of oocytes fertilized from 4/95 (4.2%) before to 23/87 (26.4%, P less than 0.001) after Andriol treatment. One couple attained spontaneous conception during the second month of Andriol intake and three pregnancies occurred among the remaining 10 cases undergoing repeat IVF, for a total ongoing pregnancy rate of 36.4%. Sperm concentration but not motility increased in the native semen after Andriol intake, but there were no significant changes in sperm characteristics after preparation. The improvement in pregnancy rate contrasts favourably with the results obtained by repeat IVF of untreated historical controls. The result obtained in the present pilot study should encourage the performance of a larger, placebo-controlled investigation protocol.     

In-vitro fertilization with donor sperm after failure of artificial insemination

This retrospective study compares results of artificial inseminationby donor (AID) and in-vitro fertilization (IVF) trials, andespecially of IVF treatment with fresh donor spermatozoa inpatients who were unsuccessfully treated on 18 occasions byAID using frozen semen. AID gives cumulative pregnancy rateof 54.6% after six trials. But after 18 trials, 22.4% of thepatients were still not pregnant. Treating them with IVF gavea probability of pregnancy as good as in the other IVF patients,53.9% after six trials. However, pregnancy loss rates differedresulting in cumulative normal pregnancy rates of 16.2% in AIDcycles ranking from 19 to 24 compared with 41.8% when goingto IVF. In ‘ex-ATD’ women, significantly more follicleswere punctured, but the number of oocytes obtained and fertilized,embryos replaced, and the incidence of pregnancy did not differfrom the other patients. Considering separately the variousinfertility factors encountered in the patients, the pregnancyrates still did not differ between ex-AID women and the others.Changing from long-term failed AID to IVF is certainly acceptableand even more so with the newly introduced technique of trans-vaginalpuncture. The female infertility factors involved in AID failureare corrected in IVF.     

Sperm proteome mapping of a patient who experienced failed fertilization at IVF reveals altered expression of at least 20 proteins compared with fertile donors: case report

The aim of this study was to compare the sperm protein expression profile (proteome map) from a patient who experienced failed fertilization at IVF with fertile controls. One patient and three fertile donor sperm samples were characterized using two-dimensional electrophoresis. Differences in protein expression were established using gel analysis software before attempted protein identification. Gel analysis of the fertile donor proteome maps revealed excellent reproducibility as well as very low intra-donor and inter-donor variability in the presence of protein spots. In the patient samples, we have noted 20 consistent differences in protein expression (six spots missing, three additional spots, four less abundant, seven more abundant) compared with the controls. Two proteins that were more intense in the patient have been conclusively identified as secretory actin-binding protein and outer dense fibre protein 2/2. In conclusion proteome variation between different fertile donors was very low. In contrast, the patient proteome exhibited 20 differences compared with controls, which we believe is an underestimate. These proteins merit further investigation to determine whether failed fertilization at IVF might be caused by abnormalities in their expression. This case report represents a proof of principle that proteomics may be useful to study defects in sperm function.     

Andrology: Acrosome reaction inducibility predicts fertilization success at in-vitro fertilization

We prospectively studied the ability of acrosome reaction (AR)inducibility to predict fertilization success in a group of232 infertile patients presenting sequentially for in-vitrofertilization (IVF). The median percentage of eggs fertilizedfor the overall patient population was 25% (interquartile range5–58%), with one to 29 oocytes available for insemination(median, five oocytes). The median percentage of eggs fertilizedat IVF increased as the percentage of spermatozoa able to undergoAR became greater: spermatozoa with a failed AR (5%) fertilizedonly 12% of eggs, while spermatozoa with AR values>9% fertilized50% of eggs. The assay had a specificity of 0.75, a sensitivityof 0.55 and an odds ratio of 2.9; thus, AR-positive patientsare 2.9 times more likely to achieve fertilization than patientswith a failed AR. Receiver operator characteristic (ROC) curveswere constructed for AR, sperm concentration and percentageof normal forms in semen. All three parameters proved to bepotentially useful in predicting the occurrence of fertilization,although AR and morphology appeared to be better than spermconcentration by ROC analysis. Patients were divided into fourclearly defined subgroups according to their traditional semencharacteristics, including morphology. The median percentageof eggs fertilized decreased as traditional semen characteristicsdeteriorated, from a median of 46% for patients with excellentsperm concentration, motility and morphology, to a median of29% for patients with suboptimal semen quality and a medianof 0% for patients with severely impaired semen. Within eachpatient subgroup, the median percentage of eggs fertilized was3-to 4-fold higher for individuals with a positive AR than forthose with a failed AR, indicating that AR has a greater effecton fertilization rate than traditional semen parameters includingmorphology. We now recognize that some men with good semen characteristicshave an unexpectedly poor AR and a markedly reduced fertilizationrate, while other men with poor traditional semen characteristicsunexpectedly retain AR and perform relatively well at IVF. Bycontrast to AR, morphology seemed to have little effect on fertilizationsuccess (two-way analysis of variance not significant). Thewife's age and oocyte quality were evenly distributed amongthe different patient subgroups, indicating that differencesin fertilization rate could not be attributed to either parameter.Our data indicate that AR has a much higher predictive valuefor IVF success than traditional semen parameters includingmorphology. We propose that AR assessment is a clinically usefuldiagnostic tool in determining a patient's likelihood of achievingfertilization at IVF.     

Costs in relation to effects of in-vitro fertilization.

A comparison has been made between in-vitro fertilization (IVF) and its most important alternative, tubal surgery. Medical results and financial costs have been taken into consideration. It is concluded that IVF must be regarded as a regular fertility treatment. The cost-effectiveness of IVF, in terms of the average treatment costs per full-term pregnancy, is approximately NLG 25,000, which is in line with that of tubal surgery. The deviation around the average is large, both for IVF and for tubal surgery.     

Cryopreservation of human oocytes and fertilization by two techniques: in-vitro fertilization and intracytoplasmic sperm injection

Human oocyte cryopreservation results in poor survival and subsequentfertilization rates. It has been suggested that freeze-thaw-inducedchanges in the zona pellucida may impair sperm penetration orattachment. The aim of this study was to compare fertilizationand cleavage rates in cryopreserved oocytes inseminated by conventionalin-vitro fertilization (IVF) or intracytoplasmic sperm injection(ICSI). A total of 220 oocytes, obtained from volunteers whohad undergone ovarian stimulation, were cryopreserved usinga slow freeze-rapid thaw protocol with 1.5 M propanediol asthe cryoprotectant. Surviving oocytes (n= 74, 34.4%) were randomlyallocated for fertilization by conventional IVF (group 1) orICSI (group 2) using cryopreserved spermatozoa from a singledonor of proven fertility. Fertilization was achieved in five(13.5%) of the oocytes in group 1 and 17 (45.9%) in group 2(P < 0.005), with only one oocyte in group 1 exhibiting normalfertilization as opposed to 16 (43.2%) in group 2 (P < 0.001).Similarly, one oocyte fertilized by IVF cleaved, while all fertilizedwith ICSI cleaved (P < 0.001). We conclude that althoughthe survival of oocytes is poor following cryopreservation,fertilization and cleavage rates can be enhanced significantlyusing ICSI. These data also suggest that the method of cryopreservationused in this study affected the zona pellucida, such that normalsperm attachment or penetration was impaired.     

A prospective controlled study of in-vitro fertilization, gamete intra-fallopian transfer and intrauterine insemination combined with superovulation.

The relative effectiveness of in-vitro fertilization (IVF), gamete intra-Fallopian transfer (GIFT) and intrauterine insemination (IUI) combined with superovulation in the treatment of infertility were compared in 151 couples undergoing a single cycle of treatment. Treatment was selected as appropriate (IVF for tubal disease, GIFT or IUI/superovulation for nontubal infertility) but possible bias due to non-randomization was overcome by all couples having had favourable fertilization in a previous cycle of IVF. Furthermore, in a preliminary study of initial IVF treatment in 265 couples from whom the study patients were drawn, implantation and pregnancy rates in the diagnostic groups were similar. In the definitive study comparing IVF, GIFT and IUI/superovulation, the pregnancy rate observed with GIFT was highest (40%) but this was not significantly higher than with IVF (28%) or IUI/superovulation (20%). However, the implantation rate per egg transferred by GIFT (21%) was significantly higher than the implantation rate per embryo transferred by IVF (11%). Although the pregnancy rates with GIFT were not statistically greater than with IVF, a significant advantage is likely to be observed in larger groups in view of the better implantation rate. The lower pregnancy rates with IUI superovulation are to be expected because of limited ovarian stimulation, they are nevertheless of comparative interest.     

Selective drop-out in successive in-vitro fertilization attempts: the pendulum danger.

Success rates from in-vitro fertilization (IVF) in the long term are dependent on selection procedures with regard to continuation into further IVF episodes. Publications on success rates in successive episodes will give incentives to adapt selection criteria, but if these publications do not deal explicitly with patient selection, the adaptations might change their direction every time: the pendulum danger.     

Andrology: An indiscriminate use of pentoxifylline does not improve in-vitro fertilization in poor fertilizers

In order to enhance fertilization in vitro, pentoxifylline (PTX)was used in couples showing low fertilization rates in previousin-vitro fertilization (IVF) attempts for the treatment of malefactorinfertility. Sibling oocytes were inseminated at random withspermatozoa prepared with or without PTX. After selection byPercoll gradient, sperm samples were divided into two equalaliquots. One aliquot was incubated in Earle's medium containing3.6 mM PTX (treatment group), the other aliquot was incubatedwith PTX-free Earle's medium (control group). After 30 min,both suspensions were washed twice. Sperm parameters after preparationdid not differ between treatment and control samples, and nordid the mean fertilization rates, which were 49.3 and 42.6%respectively. Cleavage characteristics and morphological qualityof the embryos were not significantly different between thetreatment and control groups. The results of this study demonstratethat indiscriminate use of PTX in an IVF programme increasesneither fertilization rate nor embryo transfer rate in poorfertilizers. More prospective research is needed to evaluatethe role of PTX in IVF in couples selected according to theeffect of the compound on sperm function, e.g. hyperactivationand acrosome reaction.     

Conversion of cycles involving ovarian hyperstimulation with intra-uterine insemination to in-vitro fertilization

Conversion to in-vitro fertilization (IVF) and embryo transferas an alternative to cancellation was offered in 27 consecutivecycles of controlled ovarian hyperstimulation and intra-uterineinsemination (IUI) cycles with excessive follicular developmentin patients with idiopathic infertility. IVF and embryo transferwas performed in 25 cycles, resulting in 13 pregnancies (52%),with 22% of couples having at least two embryos cryopreserved.The pregnancies have resulted in one singleton and two twinbirths, one spontaneous abortion, and nine ongoing pregnancies(including one triplet gestation). Four patients developed severeovarian hyperstimulation syndrome (OHSS) after IVF and embryotransfer, including two cases requiring paracentesis. Threeof four OHSS patients were pregnant, resulting in live birthsof healthy twins, one spontaneous abortion and one ongoing singletongestation. In two cycles a spontaneous luteinizing hormone (LH)surge occured, preventing oocyte retrieval. For these two women,drainage of all follicles except the five most likely to fertilize(18–22 mm diameter) was performed, followed by IUI, withno pregnancies or OHSS observed. Conversion of patients fromIUI cycles to IVF/embryo transfer cycles avoids cancellationof the very cycles with the best chance of achieving pregnancy.OHSS remains a problem, necessitating extensive pre-IVF counsellingand post-transfer vigilance.