Prospective follow-up study of 423 children born after intracytoplasmic sperm injection

In order to evaluate the safety of the intracytoplasmic sperminjection (ICSI) procedure, a prospective follow-up study of423 children born after ICSI was carried out. The aim of thisstudy was to compile data on karyotypes, congenital malformations,growth parameters and developmental milestones. Before startingthe infertility treatment, couples were asked to participatein a follow-up study including genetic counselling and prenataldiagnosis. The follow-up study of the child was based on a visitto the paediatrician-geneticist at birth or at 2 months of age,at 1 year and at 2 years of age when a physical examinationfor major and minor malformations and a psychomotoric evaluationwere done. Between April 1991 and September 1994, 320 pregnanciesobtained after ICSI led to the birth of 423 children (222 singletons,186 twins and 15 triplets). Prenatal diagnosis determined atotal of 293 karyotypes, one of which was abnormal (0.3%), andfour were benign familial structural aberrations, all inheritedfrom the paternal side. A total of 14 (3.3%) major malformationswere observed, defined as those causing functional impairmentor requiring surgical correction. Neurological or developmentalproblems at the age of 2 months were found in 14 children, fourof whom were multiples. Compared to most registers of childrenborn after assisted reproduction and to registers of malformationsin the general population, the figure of 3.3% major malformationsis within the expected range. Before drawing any firm conclusion,further careful evaluations of the available data are necessary.     

Comparative follow-up study of 130 children born after intracytoplasmic sperm injection and 130 children born after in-vitro fertilization

The safety of intracytoplasmic sperm injection (ICSI) as a novelprocedure of assisted fertilization may be assessed by the healthof the children born. In a prospective followup study of childrenborn after assisted procreation, 130 children born consecutivelyafter ICSI were compared with 130 control children born afterin-vitro fertilization (IVF). In both groups, mothers were matchedfor age and had the same standard treatment protocol. Therewere 74 singleton, 50 twin and six triplet children in eachgroup. Prenatal karyotyping and ultrasound screening, physicalexamination at birth and developmental milestones, with a follow-upat 2 months and 1 year, were recorded. Prenatal karyotypes wereobtained in 100 of the 130 children in the ICSI group comparedwith 22 of the 130 children in the matched IVF group. All karyotypeswere normal except for one prenatally detected mosaicism, whichwas not confirmed at birth. Four major malformations were detectedin the ICSI group (holoprosecencephaly, femur fibula ulna syndromeand palatoschisis in two children), compared with six in thematched IVF group (coarctation of the aorta, palatoschisis,hypospadias, unilateral cryptorchidism, soft tissue syndactilyand ll--hydroxylase deficiency). In the ICSI and IVF groups,mean ± SD birth weights were 2.94 ± 0.67 and 2.80± 0.73 kg, lengths were 48.46 ± 3.56) and 47.47± 5.78 cm, and head circumferences were 33.79 ±2.20 and 31.19 ± 8.88 cm respectively. Among the ICSIsingletons, the mean ± SD birth weight was 3.28 ±0.58 kg and among the twins it was 2.60 ± 0.43 kg; forthe IVF singletons and matched twins the mean ± SD birthweights were 3.19 ± 0.56 and 2.36 ± 0.61 kg respectively.In conclusion, there was no difference in the paediatric follow-upof 130 children born after ICSI and 130 children born afterconventional IVF in age-matched control patients.     

Subzonal insemination, partial zona dissection or intracytoplasmic sperm injection? An easy decision?

This review aims to analyse and compare the results to dateof subzonal insemination (SUZI), partial zona dissection (PZD)and intracytoplasmic sperm injection (ICSI) to evaluate criticallywhether it is now possible to replace SUZI and PZD by ICSI.It appears that ICSI is a much more efficient assisted reproductiontechnique than SUZI and PZD for resolving cases of severe maleinfertility and/or repeated failure of conventional in-vitrofertilization (IVF). For ICSI compared with SUZI and PZD, fertilization(49.4, 17.7 and 16.8% respectively), percentage of patientsreaching embryo transfer (91.0, 55.1 and 23.3% respectively),percentage of transfers performed with two or three embryos(83.3% ICSI and 39.3% SUZI), pregnancy rate per embryo replacement(28.2, 18.7 and 16.5% respectively) and pregnancy rate per oocyteretrieval (24.8, 10.3 and 3.8% respectively) are all improved.In addition, cases of severely impaired semen characteristics,which were condemned to infertility for life with conventionalIVF, SUZI or PZD, can now be treated and resolved efficientlywith ICSI.     

Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. Report of a second series of 300 consecutive treatment cycles

Subzonal insemination (SUZI) and intracytoplasmic sperm injection(ICSI) were carried out in 300 treatment cycles in couples unableto be helped by coventional in-vitro fertilization treatment.More oocytes were damaged by ICSI (13.5%) than by SUZI (7.1%).The normal fertilization rate was substantially higher afterICSI (51.0%) than after SUZI (14.3%) and was related to thesemen characteristics. The cleavage rate was similar for bothprocedures (77%). After 217 embryo transfers (72.3% of the treatmentcycles) 66 pregnancies were established, i.e. pregnancy ratesof 22.0% per started cycle and 30.4% per embryo transfer. Sofar, pregnancy loss has occurred in 27.3% of the pregnancies,nine healthy children have been born after eight deliveriesand 41 clinical pregnancies are progressing uneventfully. Chorionicvillus sampling or amniocentesis have been performed in 35 pregnanciesand 39 normal fetal karyotypes have been obtained after cytogeneticanalysis.     

A Danish national cohort of 730 infants born after intracytoplasmic sperm injection (ICSI) 1994-1997.

This national cohort study included all clinical pregnancies obtained after intracytoplasmic sperm injection (ICSI) registered in Denmark between January 1994 and July 1997 at five public and eight private fertility clinics. Laboratory and clinical data were obtained from the fertility clinics. The couples answered a questionnaire regarding the pregnancy and the health of the child (response rate 94%). Data validation was carried out through discharge charts. The mean age of the women was 32.1 years. In 84.2% of couples, male factor was the main reason for performing ICSI, and in 4.8% epididymal spermatozoa were used. The mean number of embryos replaced was 2.3 (range 1-3) and in 95% of cases fresh embryos were transferred. Only 183 women (28.5%) underwent prenatal diagnosis, resulting in 209 karyotypes with seven (3.3%) chromosome aberrations. Six major chromosomal abnormalities (2.9%) and one inherited structural chromosome aberration (0.5%) were found, but no sex chromosome aberrations. The frequency of multiple birth, Caesarean section rate, gestational age, preterm birth, and birth weight were comparable with previous studies. The perinatal mortality rate was 13.7 per 1000 children born with a gestational age of 24 weeks or more. In 2.2% (n = 16) of the liveborn infants, and in 2.7% (n = 20) of all infants, major birth defects were reported by the parents. Minor birth defects were found in nine liveborn infants (1.2%). In conclusion, the results of this study on outcome of ICSI pregnancies are in line with earlier reports, except that no sex chromosome abnormalities were found.     

Micro-assisted fertilization in patients who have failed subzonal insemination

During the past 3 years we have used direct injection of spermatozoainto the cytoplasm of the oocyte (DISCO) in an attempt to procureconception in vitro where subzonal insemination (SUZI) has failed,Acknowledging the paucity of information about this invasiveprocedure in humans, it was clearly the only alternative forsome patients. A total of 58 patients (cycles) who had had previouslyfailed SUZI elected for this approach. Sibling oocytes, wherepossible, were used as a comparison between DISCO, SUZI, partialzona dissection (PZD) and microdrop in–vitro fertilization(IVF). Fertilization and embryo transfer was achieved by 66%(n = 38) of patients. Patients with embryos derived solely fromDISCO (n = 19) produced four clinical pregnancies (21%), fourpatients had embryo transfer from SUZI embryos only with nopregnancies, and 15 had embryo transfer from a mixture of SUZIand DISCO embryos, with one dizygotic twin pregnancy from acombined SUZI and DISCO embryo transfer (7%). Twins, one maleand one female, have been delivered, there has been one miscarriageand two pregnancies are ongoing. Of the 627 oocytes, 251 wereused for DISCO and 71 (28%) were fertilized, 296 were used forSUZI and 29 (10%) were fertilized; 70 were used for PZD, two(3%) were fertilized. and none of the 10 undergoing microdropIVF were fertilized. Of the 58 patients (cycles) 31% (n = 19)had fertilization with DISCO only, 8% (n = 4) with SUZI only,28% (n = 15) with both SUZI and DISCO, while only one (2%) hadfertilization with PZD. The incidence of fertilization in sampleswith <5% normal forms was higher (37%) after DISCO than SUZI(15%). Of four cases with globozoospermia one achieved fertilizationwith DISCO only, and of cight cases of 100% immotile spermatozoa,two achieved fertilization with DISCO only and one with SUZIand DISCO. We conclude from this series of patients that DISCOmay not only be an advantage when SUZI fails but may becomethe first micro–assisted fertilization approach     

Andrology: Preferences for intracytoplasmic sperm injection versus donor insemination in severe male factor infertility: a preliminary report

With the advent of intracytoplasmic sperm injection (ICSI),our programme noted a drop in the number of couples using donorinsemination (DI) for severe male factor infertility. Over thefirst 8 months in which our infertility programme offered bothtreatments, 27 consecutive couples scheduled for ICSI and 15consecutive couples scheduled for DI were evaluated Since allpatients in our infertility programme beginning in-vitro fertilization(TVF) with planned ICSI or starting DI undergo a semi-structuredpsychological interview, the psychologist's clinical notes aswell as the medical chart were reviewed and coded retrospectivelyto determine factors related to a couple's treatment choice.Couples who chose IVF-ICSI over DI had a higher occupationalstatus and included husbands with higher educational levels.Their most common motivation was to have the husband's biologicalchild (93% of couples in the ICSI group). The most common motivationfor choosing DI (60% of DI couples) was that IVF was not financiallyaffordable. Choice of treatment was not related to psychologicaladjustment, the husband having prior biological children, orhis risk of passing on a genetic defect to offspring. Thesepreliminary data raise the concern that, with the success ofICSI, DI may change in the USA from being an option dictatedby semen quality to a second choice treatment utilized for economicreasons.     

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.     

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.     

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.     

Pregnancies, growth and development of children conceived by subzonal injection of spermatozoa.

Subzonal injection of spermatozoa (SUZI) was one of the first micromanipulation techniques efficient in treating male factor infertility and unexplained in-vitro fertilization failures. The aim of this retrospective study was to evaluate the in-vitro development of embryos conceived by SUZI, the obstetric outcome, the rate of congenital malformations and subsequent follow-up in children. Fifty-five pregnancies were obtained between 1991 and 1994 (54 after fresh embryos were transferred and one after cryopreserved embryos were transferred). Among the 50 clinical pregnancies, there were seven miscarriages (14%) and two ectopic pregnancies (4%). Among the 41 resulting evolutive pregnancies, the discovery of one anencephaly led to a medical abortion. Forty deliveries including six twin pregnancies occurred, leading to the births of 45 live neonates and one stillbirth. The gender distribution of the offspring included 17 males and 29 females (ratio 0.59:1). Birth weight, length and head circumference were within the expected ranges. Two children presented a malformation: the first one had one thumb with congenital shelf and the second a polymalformative neurological syndrome. Growth curves were normal for all these children except one (weight above the 2 SD curve). Medical follow-up detected no pathological features in these children apart from a physical disability in one girl. In this small series a 4.2% rate of malformation was observed, particularly affecting the neural tube, in SUZI offspring. However, no firm conclusions can be drawn since the study was carried out on a small cohort. SUZI is no longer performed but these observations suggest that it is necessary to collect extensive data about children conceived by microfertilization.     

Simultaneous bilateral tubal pregnancy after intracytoplasmic sperm injection

Since the advent of assisted reproductive technology, the concernabout ectopic implantation of embryos has increased dramatically.Simultaneous bilateral tubal pregnancy is the least common typeof ectopic implantation of two embryos. In this report we presentthe first case of simultaneous bilateral tubal pregnancy afterintracytoplasmic sperm injection (ICSI) and embryo transfertreatment. The present case had no risk factor for ectopic pregnancy.Therefore, for early diagnosis and management of such cases,close clinical follow-up and routine ultrasonography followingICSI are necessary.     

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 and perinatal outcome of pregnancies following intracytoplasmic sperm mjection

The aim of this study was to describe the obstetric and perinataloutcome for births following intracytoplasmic sperm injection(ICSI). Of 210 infants born, 140 were singletons and 70 weretwins. There were no triplets or higher births. The multiplebirth frequency was 20%. Overall, 17% of deliveries were preterm,although for singleton pregnancies the incidence was reducedto 9%. The median birth weight of all live born infants was3168 g and of singletons 3470 g. Of all infants, 17% had a lowbirth weight (<2500 g) and 2% had a very low birth weight(<1500 g). Two major malformations occurred in two singletonchildren and four minor malformations occurred in four children.This was within the range of expected values in Sweden. Karyotypingwas performed in 58 pregnancies. All of them were normal. Theperinatal mortality was 0.5%. In conclusion, in this observationalstudy from Sweden of the first infants born after ICSI in ourprogramme, the incidence of multiple births, preterm births,low birth weight babies and congenital malformations was lowcompared with other series of in-vitro fertilization pregnanciesnot associated with ICSI.     

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.     

Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection

In this study we investigated whether morphology and chromatinanomalies in human spermatozoa can influence fertilization afterintracytoplasmic sperm injection (ICSI). We examined unfertilizedoocytes, using the fluorochrome Hoechst 33342, to determinewhether a relationship exists between failure of fertilizationand sperm chromatin quality. Sperm chromatin packaging qualitywas assessed using the chromomydn A3 (CMA3) fluorochrome, andthe presence of DNA damage in spermatozoa, using in-situ nicktranslation. Normal males present sperm parameters with a normalmorphology of >20%, CMA3 fluorescence of <30% and exhibitendogenous nicks in <10% of their spermatozoa. When patientswere separated according to these values no difference was observedin their fertilization rates after ICSL When the unfertilizedICSI oocytes were examined, we found that patients with CMA3fluorescence of <30% and nicks in <10% of their spermatozoahad only 17.5 and 21.6% respectively of their unfertilized oocytescontaining spermatozoa that remained condensed. In contrast,patients with higher CMA3 and nick values had a significantlyhigher number, 412 and 48.9%, of their unfertilized oocytescontaining condensed spermatozoa. Sperm morphology did not showany such pattern. The percentage of spermatozoa which had initiateddecondensation in unfertilized oocytes was not influenced bymorphology, CMA3 fluorescence or nicks. In light of these resultswe postulate that poor chromatin packaging and/or damaged DNAmay contribute to failure of sperm decondensation after ICSIand result in failure of fertilization.     

Follow-up of children born after ICSI

The comparison of outcome of assisted reproductive technology (ART) children and naturally conceived children may be hampered by the difference in characteristics of the infertile patients such as age and genetic risks. Follow-up studies are further hampered by the type of neonatal surveillance protocol, the number of individuals lost to follow-up, the size of the cohort study, and the lack of standardization, for example to define major anomalies. The limited available data on ICSI fetal karyotypes reveal that, in comparison with a general neonatal population, there is: (i) a slight but significant increase in de-novo sex chromosomal aneuploidy (0.6% instead of 0.2%) and structural autosomal abnormalities (0.4% instead of 0.07%); and (ii) an increased number of inherited (mostly from the infertile father) structural aberrations. Available data indicate that in 8319 liveborn ICSI children, the mean percentage who do not originate from singleton pregnancies was 40% (range 32.6-60.8% according to centre). Most multiples are twins, but there are also 4.4% triplets (in one survey 13.2%). This substantial increase in multiple pregnancies must be considered the most important complication of ART. The different percentages of major and minor congenital malformations cannot be compared, but overall the data in large and reliable surveys does not indicate a higher rate of malformations in ICSI children than in naturally conceived children. To date, only three studies have examined the medical and developmental outcome of ICSI children at 1 and 2 years. These do not reveal obvious problems, but in future further comparison of matched cohorts of children and case-control studies are needed before final conclusions can be drawn.     

Human oocyte activation after intracytoplasmic sperm injection

Oocyte activation is a series of events triggered by the fertilizingspermatozoon and necessary for the beginning of the embryonicdevelopment. Calcium plays a pivotal role in this process. Herewe used confocal laser scanning microscopy to examine the changesin the concentration of intra-cellular free calcium ([Ca²⁺])in human oocytes after intracytoplasmic sperm injection (ICSI).The first considerable but short (<2 min) increase in [Ca²⁺]1was detected immediately after the penetration of the micro-injectionneedle into the ooplasm. This rise by itself did not provokeoocyte activation and was also obtained after the injectionof medium without spermatozoa. After a lag period of 4–12h, oocytes that were subsequently activated initiated a secondperiod of [Ca2⁺]1 changes. These changes were sperm-dependentand followed one of two alternative patterns, a non-oscillatoryone and an oscillatory one. The non-oscillatory pattern resembledthe changes described previously during parthenogenetic activationof mammalian oocytes. The oscillatory pattern was similar tothe changes accompanying normal fertilization in different mammalianspecies. It is concluded that the initial [Ca²⁺]1 rise provokedby the ICSI procedure is not responsible for oocyte activation,and that a release of a sperm factor(s) is required to initiatethis process.     

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.     

Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection1

The aim of this study was to determine why oocytes remain unfertilizedor develop three pronuclei after intracytoplasmic sperm injection(ICSI). Unfertilized and abnormally fertilized oocytes werefixed in glutaraldehyde,stained with Hoechst 33342 and examinedby fluorescence microscopy to identify oocyte, sperm and polarbody DNA.One-pronuclear oocytes were considered to be unfertilized.Atotal of 285 unfertilized oocytes were examined (104 ICSI cycles).Overall, 83% of these oocytes were not activated (still at metaphaseII) while 17% had activated and formed a single (female) pronucleus.About 66% of the unfertilized, metaphase II oocytes containeda swollen sperm head, indicating that the oocyte was correctlyinjected but had failed to activate and complete the secondmeiotic division. Premature chromosome condensation of the spermDNA was evident in 6% of these metaphase II oocytes (4% of theunfertilized oocytes). The swollen sperm head was located amongthe oocyte chromosomes in 5%of the metaphase II oocytes. Othercauses of failed fertilization in the metaphase II oocytes werethe failure of sperm head decondensation (11%) and ejectionof the spermatozoon from the oocyte (23%). A similar patternwas observed in one-pronuclear oocytes (52%, swollen sperm head;28%, intact, undecondensed sperm head; 20%, ejection of thespermatozoon), which indicates that asynchronous pronucleardevelopment does not explain the presence of one-pronuclearoocytes. A total of 41 threepronuclear oocytes were examinedand all had a single polar body, which indicates that the retentionof the second polar body leads to the formation of the thirdpronucleus.In conclusion, this study demonstrates that: (i)the major cause of fertilization failure after ICSI is failureof oocyte activation; (ii) ejection of the spermatozoon intothe perivitelline space is not a major cause of fertilizationfailure;and (iii) sperm head decondensation and oocyte activationafter ICSI can occur independently.