Correlation between testicular histology and outcome after intracytoplasmic sperm injection using testicular spermatozoa

A comprehensive study is presented of a series of 124 infertilemen undergoing testicular sperm retrieval for intracytoplasmicsperm injection (ICSI). In this study we correlated the histologicalchanges observed in the testicular tissue with the results ofthe wet preparation and the outcome after ICSI using testicularspermatozoa. In all patients with normal spermatogenesis andhypospermatogenesis spermatozoa were recovered from the wetpreparation. The sperm recovery rate was 84% in patients withincomplete germ-cell aplasia and maturation arrest, while inpatients with complete germ-cell aplasia or maturation arrestthis figure was 76%. In these patients more specimens were sampledand fewer spermatozoa were recovered. Since no spermatozoa wererecovered in only 10 patients, ICSI with testicular sperm wasperformed in the remaining 114 couples (91.9%). The normal fertilizationrate was 57.8%. The fertilization rate was significantly lowerin couples among whom the husband showed germ-cell aplasia andmaturation arrest. Overall, 55.2% of normally fertilized oocytesdeveloped into embryos showing 50% of anucleate fragments. Therewere no major differences between the different histologicalcategories in terms of embryonic development in vitro. The overallpregnancy rates per testicular sperm extraction (TESE) procedure,per ICSI procedure and per transfer were respectively 36.3,39.5 and 43.7%. The overall implantation rate per embryo (sacs/embryosreplaced) was 20.3%. A lower implantation rate was observedin couples among whom the husband had maturation arrest (notstatistically significant). The above data show that testicularbiopsies may have an important therapeutic role in the managementof infertility in azoospermic patients.     

Pregnancies after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa

In 25 patients (14 suffering from obstructive azoospermia, sixfrom non-obstructive azoospermia, three from astheno-azoospermiaand two from absence of ejaculation) spermatozoa were extractedfrom testicular biopsies. Intracytoplasmic sperm injection (ICSI)with fresh testicular spermatozoa was performed in 18 cases;spermatozoa in excess were cryopreserved in pills. No pregnancieswere achieved. In the remaining seven patients, testicular spermatozoawere retrieved and cryopreserved during a diagnostic testicularbiopsy. After thawing, sperm motility was assessed in 17 cases(68%), and 18 ICSI with cryopreserved testicular spermatozoawere performed. The mean two-pronuclear (2PN) fertilizationrate was 59%, the mean cleavage rate was 92%, and six clinicalpregnancies were achieved, all of them still ongoing (pregnancyrate 33%). A comparison of the results of ICSI carried out withfresh or cryopreserved testicular spermatozoa showed that themean 2PN fertilization rates per cycle (53 compared with 55%),mean cleavage rates per cycle (99 compared with 96%) and embryoquality were not significantly different In conclusion, cryopreservationof testicular spermatozoa is feasible, even in patients withnon-obstructive azoospermia, and the results of ICSI with frozen-thawedtesticular spermatozoa are similar to those obtained using freshtesticular spermatozoa. Cryopreservation of testicular spermatozoamay avoid repetition of testicular biopsies to retrieve spermatozoafor successive ICSI cycles in patients in whom the only sourceof motile spermatozoa is the testicle.     

Fertility with testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermic men

In non-obstructive azoospermia spermatozoa can usually onlybe isolated from the testicles, and thus the most promisingtreatment model is testicular sperm extraction (TESE). Hormoneconcentrations, testicular volume determinations and testicularbiopsy results are not uniform enough to select potential candidatesfor successful TESE and intracytoplasmic sperm injection (ICSI)approaches in advance. The aim of this study was to assess theefficacy of using ICSI with testicular spermatozoa in casesof non-obstructive azoospermia and to compare the inclusioncriteria and sperm existence in the testicles in sperm obtainableand non-obtainable groups. All men showed either complete orincomplete (n = 14) maturation arrest in spermatogenesis, severehypospermatogenesis (n = 10) or Sertoli cell-only syndrome (n= 5) in their testicular biopsies. Only 14 out of a total of29 men provided enough spermatozoa for the ICSI procedure, whileno spermatozoa were found in the testicular samples of the remaining15 men. Out of 123 oocytes obtained from 14 females, 101 wereinjected with the husbands' testicular sperm cells. Total fertilizationfailure was observed in three cases. Of 39 oocytes fertilized,38 cleaved. The fertilization and cleavage rates were 38.6 and97.4% respectively. The pregnancy rate was 20.7% per initiatedcycle. In the group from whom spermatozoa were obtainable, thepregnancy rate was 42.9% per initiated cycle and 54.5% per embryotransfer. A total of six pregnancies were achieved, of whichtwo Were twins and four were singletons. One singleton pregnancyresulted in abortion in the first trimester. There was no statisticaldifference concerning the serum follicle stimulating hormoneconcentration, testicular volume and biopsy results in groupsin which spermatozoa were obtainable or not. In conclusion,although the association of TESE with ICSI obtained pregnanciesfor some patients with non-obstructive azoospermia, furtherstudies are needed to determine the inclusion criteria for successfulTESE.     

Outcome of intracytoplasmic sperm injection with testicular spermatozoa in obstructive and non-obstructive azoospermia

From 1 August 1993 until 30 September 1994, 69 couples sufferingfrom azoospermia underwent testicular sperm extraction and intracytoplasmicsperm injection. In 50 couples with obstructive azoospermiaa total of 631 meta-phase-II oocytes were injected after testicularsperm extraction yielding a 2-PN fertilization rate of 57%.In female patients <40 years of age an ongoing pregnancyrate per transfer of 42% (14/33) was obtained. So far, eighthealthy babies have been born, including two singletons andthree twin gestations. In 19 couples with non-obstructive azoospermiaa total of 264 metaphase-II oocytes were injected after testicularsperm extraction, yielding a 2-PN fertilization rate of 58%.An ongoing pregnancy rate per transfer of 31% (5/16) was established.So far, six healthy babies have been born including one singleton,one twin and one triplet gestation.     

Fertilization and early embryology: Ongoing pregnancies and birth after intracytoplasmic sperm injection with frozen--thawed epididymal spermatozoa

In seven patients who did not become pregnant following microsurgicalepididymal sperm aspiration (MESA) and intracytoplasmic sperminjection (ICSI), a subsequent ICSI was performed using previouslycryopreserved supernumerary epididymal spermatozoa without re-operatingon the husband. During the original MESA procedure a mean spermconcentration of 12.3x10⁶/ml was achieved. The supernumeraryspermatozoa were cryopreserved for later use. After thawingfrozen epididymal spermatozoa a mean concentration of 1.9x10⁶spermatozoa/ml was obtained in straws containing a total volumeof sperm suspension of 250 µl. From 68 intact oocytesinjected with frozen—thawed epididymal spermatozoa, atwo pronuclear fertilization rate of 45% and a cleavage rateof 82% were obtained. A total of 17 embryos were replaced inthe seven patients, resulting in two ongoing singleton pregnanciesand one twin delivery. Six embryos were cryopreserved. In conclusion,it would appear mandatory to cryopreserve supernumerary spermatozoaduring a MESA in order to avoid subsequent further scrotal surgery.     

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.     

Pregnancy and birth after intracytoplasmic sperm injection with spermatozoa from a patient with tail stump syndrome

Tail stump syndrome, which may be associated with primary ciliarydyskinesia, is also associated with morphological defects ofthe flagellum resulting in severe asthenozoospermia. Until recently,these morphological anomalies caused definite male infertility.Today, however, new methods such as micromanipulation techniquesprovide a rational therapy for this patient group. A pregnancyfollowed by living offspring was achieved following the intracytoplasmicinjection of immotile spermatozoa from a patient with tail stumpsyndrome.     

High implantation and pregnancy rates with testicular sperm extraction and intracytoplasmic sperm injection in obstructive and non-obstructive azoospermia

Thirty-two infertile couples with obstructive and non-obstructiveazoospermia were included in this study. Testicular sperm extraction(TESE) was performed in 16 obstructive azoospermic cases wheremicrosurgical sperm aspiration (MESA) or percutaneous spermaspiration (PESA) were impossible because of totally destroyedepididymis and 16 non-obstructive azoospermia cases with severespermatogenetic defect where the testicles were the only sourceof sperm cells. A total of 288 oocytes was obtained from 32females and 84% were injected. The fertilization rates (FR)with 2 pronuclei (PN) and cleavage rate were 50.8 and 68.2%respectively. A total of 15 pregnancies was achieved (53% perembryo transfer), nine from the obstructive and six from thenon-obstructive group. Four pregnancies resulted in clinicalabortion (26.6%). The ongoing pregnancy rate was 39.2% per embryotransfer (ET) and 343% per started cycle. A high implantationrate was also achieved (26.6% in non-obstructive and 30% inobstructive azoospermia group). Using testicular spermatozoain combination with ICSI in both obstructive and non-obstructiveazoospermic groups, high implantation and pregnancy rates canbe achieved.     

High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy

In cases requiring microsurgical epididymal sperm aspiration(MESA) for congenital absence of the vas deferens (CAVD) orirreparable obstructive azoospermia, often no spermatozoa canbe retrieved from the epididymis, or there may even be no epididymispresent. We wished to see whether testicular biopsy with testicularsperm extraction (TESE) in such cases could yield spermatozoathat would result in successful fertilization and pregnancy(despite the absence of epididymal spermatozoa) using intracytoplasmicsperm injection (ICSI). In the same setting during the same2-week period, 28 patients with CAVD or irreparable obstructionwere treated; 16 consecutive fresh MESA—ICSI cycles and12 cycles which required testicular biopsy with testicular spermextraction (TESE—ICSI) were performed. Normal two-pronuclearfertilization rates were similar in both groups: 45% for epididymalspermatozoa and 46% for testicular biopsy-extracted spermatozoa.Cleavage rates were also similar (68% for epididymal and 65%for testicular spermatozoa). The ongoing pregnancy rates inthis series were 50 and 43% respectively. We conclude that epididymalspermatozoa and testicular spermatozoa yield similar fertilization,cleavage and ongoing pregnancy rates using ICSI. When epididymalspermatozoa cannot be retrieved, a testicular biopsy can beperformed and the few barely motile spermatozoa thus obtainedcan be used for ICSI. It appears that all cases of obstructiveazoospermia can now be successfully treated.     

Pregnancies achieved with testicular and ejaculated spermatozoa in combination with intracytoplasmic sperm injection in men with totally or initially immotile spermatozoa in the ejaculate

The efficacy of intracytoplasmic sperm injection (ICSI) employingtesticular and ejaculated spermatozoa was assessed in 24 coupleswith totally or initially immotile spermatozoa. No criteriawere employed in selecting which patients would be treated withtesticular or ejaculated spermatozoa. The men were chosen atrandom. Testicular spermatozoa obtained by testicular spermextraction were used in 14 and ejaculated spermatozoa were usedin 10 of these couples. In all cases, asthenozoospermia wastotal in their basal semen sample. In 12 male partners, spermatozoawere totally immotile before and after Percoll gradient fractionation(totally immotile). In the remaining 12 men, spermatozoa initiallyshowed a total absence of motility; however, some of the spermatozoahad showed very poor motility (0.1%) after Percoll gradientfractionation and a 13–2.0 h incubation period (initiallyimmotile). Of these 24 total asthenozoospermic males, 14 alsohad total terato-zoospermia. The fertilization and cleavagerates in the testicular and ejaculated sperm groups were 533and 963 and 543 and 94.4% respectively. One cycle resulted incomplete fertilization failure, and in 23 embryo transfer cyclesa total of 10 pregnancies were obtained (41.6%). Eight pregnancieswere achieved in the testicular sperm group, while only twopregnancies were obtained in the ejaculated sperm group. Fourpregnancies, two from the ejaculated sperm group and two fromthe testicular sperm group, resulted in clinical abortions inthe first trimester. Of the remaining six pregnancies, two havealready resulted in healthy births and four pregnancies arenow in the second or third trimester in the testicular spermgroup. Using testicular spermatozoa in combination with ICSIcan be an alternative mode of treatment in cases with totallyor initially immotile spermatozoa in the ejaculate. Very lowpregnancy rates have been obtained and no ongoing pregnancyhas been achieved using ejaculated spermatozoa in these cases.     

Timing of sperm and oocyte nuclear progression after intracytoplasmic sperm injection

We investigated the time course of human oocyte activation afterintracytoplasmic sperm injection (ICSI) by observing the oocytechromosome configuration at different times after injection.One day old human oocytes were injected with spermatozoa andsubjected to cytogenetic analysis at 2, 3, 4 and 5 h after injection.We found that anaphase is initiated in the vast majority ofthe oocytes between 2 and 3 h after injection, and that by 4–5h after injection most of the oocytes have reached the chromatinmass stage. Two distinguishable stages of sperm nucleus transformationwere observed. The first phase — swelling — wasreached within 2 h after the injection and was independent ofoocyte activation. The second phase — the ‘brush’-likestage or decondensed chromatin stage — was found onlyin activated oocytes. Moreover, this stage was not reached beforethe chromatin mass stage (late telophase) of the oocyte. Thesame proportion of metaphase II oocyte chromosome configurationsand unchanged sperm nuclei was found at any given time afterinjection. We conclude that: (i) ICSI allows users to obtainan almost synchronized population of activated oocytes; (ii)anaphase II is initiated in the majority of oocytes not laterthan 2–3 h after injection and telophase II is reached5 h after injection; and (iii) there are two distinguishablephases of sperm nucleus transformation after ICSI: oocyte activationindependentswelling of the sperm head and oocyte activation-dependent chromatindecondensation which is coupled to the beginning of oocyte chromosomedecondensation.     

Andrology: Pregnancy after intracytoplasmic sperm injection of metaphase II oocytes with spermatozoa from a man with complete retrograde ejaculation

Retrograde ejaculation is an uncommon cause of infertility,which has been treated successfully with different kinds ofartificial reproduction technique, e.g. cervical cap artificialinsemination by husband, intra-uterine and intraperitoneal insemination,standard in-vitro fertilization, pronuclear stage transfer andgamete intra-Fallopian transfer. All these techniques requirea minimal number and motility of spermatozoa obtained afterpost-masturbation voiding. In some cases, only very few spermatozoawith very poor or no motility are found in the urine voidedimmediately after masturbation. In such a case, where no morethan 14 spermatozoa were recovered over a 3 h search, intracytoplasmicsperm injection of metaphase II oocytes led to the developmentand replacement of three fair embryos, resulting in an ongoingtwin pregnancy. This technique opens up perspectives for thetreatment of men with complete retrograde ejaculation and quasi-azoospermicpost-voiding specimens.     

Obstetric outcome of 424 pregnancies after intracytoplasmic sperm injection

An evaluation of the outcome of pregnancies resulting from intracytoplasmicsperm injection for severe male factor infertility was conductedby analysing the data obtained from the patients and/or theirobstetrician/gynaecologist on standardized questionnaires. Thedata from 424 pregnancies between April 1991 and September 1994were analysed. Early pregnancy loss before 16 weeks occurredin 99 cases (23.3%), including 48 clinical abortions (11.3%),47subclinical pregnancies (11.1%) and four ectopic pregnancies(0.9%). Vanishing twins and triplets, which could be regardedas early embryonic wastage, were found in 36 cases (8.5%). Onepregnancy was interrupted at week 15 of gestation because ofanhydramnios, and four pregnancies (0.9%) ended in spontaneouslate abortions before 26 weeks. A total of 320 pregnancies (75.5%)resulted in the birth of at least one child; 222 of these (69.3%)were singletons, 93 were twins (29.1%) and five were triplets(1.6%). The problems of prematurity and low birthweight wereespecially related to the multiplicity of pregnancies. Furthermore,from among the total of 423 babies born, we have observed threecases of stillbirth and five cases of neonatal mortality. Theperinatal mortality rate was therefore 18.9 per 1000 births.The results of this study show that the obstetric outcome ofthese pregnancies was similar to that obtained after conventionalin-vitro fertilization and other assisted reproduction techniques.     

Blastocyst transfer following intracytoplasmic injection of ejaculated, epididymal or testicular spermatozoa

Recent studies indicate a strong paternal influence on embryo development and progression of the embryo to the blastocyst stage. The aim of this study was to compare, during extended culture, the in-vitro development of embryos resulting from intracytoplasmic sperm injection (ICSI) of ejaculated spermatozoa (group 1, n = 347), epididymal (group 2, n = 22) or testicular (group 3, n = 18) spermatozoa from obstructive azoospermic and testicular spermatozoa from non-obstructive azoospermic (group 4, n = 31) subjects. Fertilization and blastocyst formation rates were significantly lower in group 4 (P < 0.05). The incidence of expanded and hatching blastocysts was significantly lower in group 4 (P < 0.05). Overall in 93.2% ejaculate ICSI cycles, blastocysts were transferred on day 5. This was significantly higher than the 62% day 5 transfers in the non-obstructive azoospermic group (P < 0.05). Implantation rate per embryo was significantly higher in the ejaculate ICSI group compared with the other groups (P < 0.05). Clinical pregnancy per transfer was similar between groups; however, significantly fewer multiple pregnancies were encountered in the non-obstructive azoospermic group (P < 0.01). In conclusion, the source of the spermatozoa, most likely to be indicative of the severity of spermatogenic disorder, affects the rate of blastocyst formation and blastocyst implantation. Spermatozoa from non-obstructive azoospermic subjects, when utilized for ICSI, result in embryos that progress to the blastocyst stage at a lower and slower rate and implant less efficiently.     

Fertilization and early embryolgoy: Human oocyte activation following intracytoplasmic injection: the role of the sperm cell

The aim of this study was to investigate whether the human spermatozoonparticipates in the activation of human oocytes following intracytoplasmicsperm injection (ICSI) and if so, by what mechanism. In thefirst series of experiments, we randomized human oocytes whichhad remained unfertilized after in-vitro fertilization (IVF)or ICSI, for intracytoplasmic injection with live spermatozoa,spermatozoa presumed to be dead and no spermatozoa. Secondly,unfertilized human oocytes and freshly ovulated mouse oocyteswere randomized for intracytoplasmic and sub-zonal injectionwith human sperm cytosolic fraction (CF) before and after heattreatment. We found that oocyte injection with initially motilespermatozoa induces human oocyte activation at a significantlyhigher rate than injection with dead spermatozoa (61 versus0%; P < 0.001) or injection without a spermatozoon (61 versus14%; P < 0.001). Intracytoplasmic injection of CF activatedboth human and mouse oocytes at the same rate as sperm injectionof human oocytes (activation rates of 70 and 65% respectively).This effect was greatly reduced by heat treatment of the CF.From these experiments we conclude firstly that the human spermatozooninjected intracytoplasmically contributes to human oocyte activationand secondly that the spermatozoon releases into the oocytea heat-sensitive, intracellularly active factor, which is notspecies-specific.     

Testicular needle biopsy, open biopsy, epididymal aspiration and intracytoplasmic sperm injection in obstructive azoospermia

Testicular or epididymal spermatozoa were obtained for in-vitrofertilization and intracytoplasmic sperm injection ICSI) in27 cycles out of 33 (in six men the azoospermia proved to havetesticular causes). Testicular needle biopsy carried out inaddition to surgical open biopsy proved to be an effective methodto obtain spermatozoa for ICSI from patients with obstructiveazoospermia. Thus it might be possible to replace scrotal operationsby simple needle biopsies. Embryos resulting from ICSI withtesticular spermatozoa were used in 19 transfers that resultedin six pregnancies. One pregnancy resulted from six embryo transfersfrom ICSI after microsurgical-epididymal sperm aspiration (MESA).The normal fertilization rates with testicular (37.3%) and MESAspermatozoa (53.7%) did not differ significantly from each other,but with testicular spermatozoa the rate was significantly lowerthan that obtained with ejaculated spermatozoa and ICSI (59.7%)in the matched couples. The abnormal fertilization of oocyteswith one pronucleus was significantly higher with testicularspermatozoa than with ejaculated spermatozoa in the controlcouples.     

Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men

The evident ability of the intracytoplasmic sperm injection (ICSI) procedure to achieve high fertilization and pregnancy rates regardless of semen characteristics has induced its application with spermatozoa surgically retrieved from azoospermic men. Here, ICSI outcome was analysed in 308 cases according to the cause of azoospermia; four additional cycles were with cases of necrozoospermia. All couples were genetically counselled and appropriately screened. Spermatozoa were retrieved by microsurgical epididymal aspiration or from testicular biopsies. Epididymal obstructions were considered congenital (n = 138) or acquired (n = 103), based on the aetiology. Testicular sperm cases were assessed according to the presence (n = 14) or absence (n = 53) of reproductive tract obstruction. The fertilization rate using fresh or cryopreserved epididymal spermatozoa was 72.4% of 911 eggs for acquired obstructions, and 73.1% of 1524 eggs for congenital cases; with clinical pregnancy rates of 48.5% (50/103) and 61.6% (85/138) respectively. Spermatozoa from testicular biopsies fertilized 57.0% of 533 eggs in non-obstructive cases compared to 80.5% of 118 eggs (P = 0.0001) in obstructive azoospermia. The clinical pregnancy rate was 49.1% (26/53) for non-obstructive cases and 57.1% (8/14) for testicular spermatozoa obtained in obstructive azoospermia, including three established with frozen-thawed testicular spermatozoa. In cases of obstructive azoospermia, fertilization and pregnancy rates with epididymal spermatozoa were higher than those achieved using spermatozoa obtained from the testes of men with non-obstructive azoospermia.     

Testicular biopty gun needle biopsy in collecting spermatozoa for intracytoplasmic injection, cryopreservation and histology.

Using testicular spermatozoa from either open biopsy (29 cycles) or biopty gun needle biopsy (49 cycles), a total of 81 intracytoplasmic sperm injection (ICSI) cycles among 57 couples were carried out from January, 1994 to September, 1997. In six cycles, no spermatozoa were obtained, and in three cycles spermatozoa from both needle and open biopsies were used. The fertilization (37% after open and 41% after needle biopsy) and pregnancy rates (29% per embryo transfer compared with 16% per embryo transfer) were similar after both open and needle biopsies. Five pregnancies were achieved among the 14 couples with non-obstructive azoospermia of the male partner, four of these after needle biopsy. It was possible to use cryopreserved testicular spermatozoa after both needle and open biopsies, and one pregnancy started after using cryopreserved testicular spermatozoa in both groups. Histological needle biopsy was carried out in 62 cases, and they were all diagnostic, giving 15-20 cross-sections of seminiferous tubuli per biopsy. Testicular needle biopsy using a 14 gauge biopsy needle gave a sufficient amount of tissue and spermatozoa for ICSI, cryopreservation and histology, even in non-obstructive azoospermia. This technique is simpler and cheaper than open biopsy and, hence, it can be regarded as the optimal method for the retrieval of testicular spermatozoa.     

Oocyte morphology does not correlate with fertilization rate and embryo quality after intracytoplasmic sperm injection

The fertilization rates and further development of 528 humanmetaphase IT oocytes directly injected by a single spermatozoonwere analysed with respect to their morphological features atthe light microscopy level at the time of retrieval. The deviationsof oocyte morphology which were most frequently observed, afterremoval of cumulus cells, were dark incorporations, dark zonapellucida, large peri-vitelline space, spots, vacuoles, refractilebodies and irregular shape. These deviations correlated neitherwith the fertilization rate nor with the embryo quality score,as compared to ‘ideal’ oocytes. Since the majorityof oocytes displayed deviations from the ‘ideal’morphotype but were still fertilized and developed in cultureat a normal rate, they were probably as normal as ‘ideal’oocytes. Since some of these morphotypes, such as refractilebodies, have been shown to be associated with failure of fertilization,it seems that intracytoplasmic sperm injection may be an appropriatemethod of treatment for couples in whom repeated failure ofin-vitro fertilization is associated with the retrieval of dysmorphicoocytes in the presence of normal semen characteristics.     

Aggressive sperm immobilization prior to intracytoplasmic sperm injection with immature spermatozoa improves fertilization and pregnancy rates

This study was conducted to determine whether the mode of spermimmobilization prior to intracytoplasmic sperm injection (ICSI)influences fertilization by immature spermatozoa. Of the 837ICSI cycles evaluated, 81 were performed with epididymal ortesticular spermatozoa; 35 cycles with epididymal spermatozoaimmobilized in the standard fashion resulted in fertilizationand pregnancy rates of 48.3 and 51.4% respectively. When a moreaggressive sperm immobilization technique (i.e. permanentlycrimping the sperm fiagellum between the midpiece and the restof the tail) was applied in 17 cycles, the resultant fertilizationand pregnancy rates were significantly (P < 0.05) higher:82.0 and 82.4% respectively. Similar increases in fertilizationand ensuing pregnancy rates were also observed in ICSI cycleswith the aggressive immobilization of frozen-thawed epididymalspermatozoa (eight cycles) versus standard immobilization (16cycles). However, the fertilization rates for ICSI using testicularspermatozoa (five cycles) were basically the same, regardlessof the immobilization technique. Furthermore, for ejaculatedspermatozoa (756 cycles), the fertilization rates followingaggressive sperm immobilization were also positively affected(73.4%), although no statistical differences in the clinicalpregnancy rates were found. Because aggressive immobilizationappears to affect sperm membrane pennea-bilization, the enhancedfertilization patterns observed in immature spermatozoa followingaggressive immobilization may suggest a different membrane constitutionin these spermatozoa. These findings indicate that immaturegametes may require additional manipulation to enhance the post-ICSIevents essential for adequate nuclear decon-densation.