Fertilization and early embryology: Premature chromosome condensation of the sperm nucleus after intracytoplasmic sperm injection

A cytogenetic-cytological study was performed on unfertilizedhuman oocytes (first polar body visible) after intracytoplasmicsperm injection (ICSI) with respect to the rate of prematurelycondensed sperm chromosomes (G₁-PCC). Out of 163 prepared oocytesderived from 41 ICSI cycles, 133 (-82%) could be analysed successfully.A total of 60 oocytes (45.1%) showed metaphase II chromosomesin the haploid range along with an intact sperm head and 27oocytes (20.3%) were missing the sperm head, but two of themshowed an approximately diploid set of chromosomes; 38 oocytes(28.6%) exhibited the maternal metaphase II chromosomes as wellas G₁-PCC of the sperm nucleus showing a remarkable variationin the degree of condensation. Ten ICSI cycles (each followedby an embryo transfer) were characterized each by 2–3oocytes demonstrating G₁-PCC. It is concluded that the maincause of failed fertilization after ICSI is the failure of oocyteactivation. When the sperm nucleus is able to act with the chromosomecondensing factors and the oocyte does not become activated,this will lead to the induction of PCC. Absence of the spermhead might be due to injection or ejection of the spermatozoonin the perivitelline space except for two cases in which fertilizationmight have occurred. Finally, the observation of both a singlechromatin region (n = 6) or two chromatin regions (n = 2) indicatedoocyte activation which, however, was followed by developmentalarrest.     

Fertiliza1tion and early embryology: Ultrastructural analysis of fertilization failure after intracytoplasmic sperm injection

Human oocytes that failed to display signs of fertilizationby 44 h after intracytoplasmic sperm injection (ICSI) were processedfor electron microscopic analysis. All oocytes were arrestedat metaphase II. The first polar body contained intact corticalgranules and chromosome clumps, which were not surrounded bya nuclear envelope but still associated with microtubules. Whena second globular body was present, it always showed the sameultrastructure, indicating that it had originated from fragmentationof the first polar body and not from the resumption of the secondmeiotic division. The most prominent organelles of the oocytecytoplasm were the smooth endoplasmic reticulum and mitochondria.In the oocyte cortex, cortical granules were intact, with nosigns of incipient or incomplete cortical reaction. Oocyte chromosomeswere found in the oocyte periphery near the locality of thefirst polar body extrusion. They consisted of dense aggregatesof chromatin associated with microtubules. The chromatin ofthe injected spermatozoon was demembranated and partially decondensed.In some cases, vesicular and tubular structures, apparentlyof oocyte origin, were associated with the periphery of thesperm chromatin mass but they never formed a continuous layer.These data suggest that fertilization failure after ICSI isbasically a failure of oocyte activation.     

High fertilization and implantation rates after intracytoplasmic sperm injection

Previously reported better fertilization rate after intra-cytoplasmicsingle sperm injection (ICSI) than after subzonal inseminationof several spermatozoa was confirmed in a controlled comparisonof the two procedures in 11 patients. Intracytoplasmic sperminjection was carried out in 150 consecutive treatment cyclesof 150 infertile couples, who had failed to have fertilizedoocytes after standard in-vitro fertilization (IVF) proceduresor who were not accepted for IVF because not enough motile spermatozoawere present in the ejaculate. A single spermatozoon was injectedinto the ooplasm of 1409 metaphase II oocytes. Only 117 oocytes(8.3%) were damaged by the procedure and 830 oocytes (64.2%of the successfully injected oocytes) had two distinct pronucleithe morning after the injection procedure. The fertilizationrate was not influenced by semen characteristics. After 24 hof further in-vitro culture, 71.2% of these oocytes developedinto embryos, which were transferred or cryopreserved. Only15 patients did not have embryos replaced. Three-quarters ofthe transfers were triple-embryo transfers. High pregnancy rateswere noticed since 67 pregnancies were achieved, of which 53were clinical, i.e. a total and clinical pregnancy rate of 44.7%and 35.3% per started cycle and 49.6% and 39.2% per embryo transfer.A total of 237 supernumerary embryos were cryopreserved in 71treatment cycles.     

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.     

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.     

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.     

Comparison of clinical outcome after cryopreservation of embryos obtained from intracytoplasmic sperm injection and in-vitro fertilization

The impact of intracytoplasmic sperm injection (ICSI) on cryopreservedzygotes and embryos was evaluated by comparing embryo survivaland implantation between embryos derived from ICSI and thosederived from standard insemination procedures. The study includedpatients whose excess zygotes and embryos were cryopreservedbetween September 1993 and December 1994 and who subsequentlyunderwent a frozen embryo transfer. Embryo survival, clinicalpregnancy rates per transfer and pregnancy outcome were compared.Three hundred and thirty eight cryopreservation cycles, duringwhich 1471 embryos were cryopreserved, were included in thisstudy. Of those, 961 were derived from oocytes fertilized byinsemination in vitro and 510 were derived from oocytes fertilizedby ICSI. A total of 690 of the embryos (451 in the inseminationgroup and 239 in the ICSI group) have since undergone a thawcycle. The embryo survival rates were similar between the twogroups (70.5 and 73.2%, insemination and ICSI respectively)and were not significantly affected by the stage at cryopreservation.There was no significant difference in pregnancy rates per transfer(31.8 and 32.3%), the preclinical pregnancy loss rate (16.7and 23.8%), or the clinical miscarriage rate (16.7 and 23.8%)between the insemination and the ICSI groups respectively. Itis concluded that ICSI does not have an adverse impact on thesurvival and successful implantation of cryopreserved and thawedembryos.     

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

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

In-vitro matured metaphase-I oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-II oocytes after intracytoplasmic sperm injection.

About 4% of all the oocytes denuded prior to intracytoplasmic sperm injection (ICSI) are in metaphase-I (MI). Frequently, these oocytes achieve meiosis after a few hours of in-vitro culture and are available for ICSI on the day of oocyte retrieval. In this retrospective study, the aim was to evaluate the fertilization rate and the developmental capacity of these in-vitro matured MI oocytes. After controlled ovarian stimulation using human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) in 896 ICSI cycles, 1210 MI-to-MII-matured oocytes were injected approximately 4 h after in-vitro culture and 8803 MII oocytes were injected immediately, or later, after denudation. The fertilization rate of in-vitro matured oocytes was significantly lower than that of mature MII oocytes (52.7 and 70.8% respectively, P < 0.00l). Embryo quality was only slightly different as regards the numbers of good quality embryos: 47.4% good quality embryos were obtained in the in-vitro matured oocyte group, whereas 53.2% good quality embryos were obtained in the MII oocyte group (P < 0.05). The same proportions of excellent (5.7 and 7.0%, NS) and fair quality (17.6 and 15.3%, NS) embryos were obtained for in-vitro matured and mature oocytes respectively. Embryos derived from in-vitro matured oocytes were transferred only if they were of better quality or if there were not enough mature oocyte derived embryos available. Fifteen transfers involved only embryos derived from in-vitro matured oocytes: 11 single embryo transfers and four transfers of two embryos, resulting in one singleton pregnancy and the birth of a healthy baby. It may be concluded that in cycles with few MII oocytes it might be worthwhile to inject in-vitro matured MI oocytes in order to increase the number of embryos available for transfer.     

Comparison between intracytoplasmic sperm injection and in-vitro fertilization (IVF) with high insemination concentration after total fertilization failure in a previous IVF attempt.

The aim of this prospective study was to evaluate whether couples with total fertilization failure in a previous in-vitro fertilization (IVF) attempt should be offered an additional IVF treatment with elevated insemination concentration or should be treated with intracytoplasmic sperm injection (ICSI). In 23 cycles 228 sibling metaphase II (MII) oocytes were randomly divided: 143 and 85 oocytes were utilized for ICSI and IVF respectively. Of the 143 injected (ICSI) oocytes, 90 (62.9%) were normally fertilized (two pronuclei), whereas 21 (14.7%) oocytes were damaged by the ICSI procedure. Of the fertilized oocytes 72 (80%) developed into transferable embryos. No fertilization at all was observed in the 85 sibling MII oocytes which were inseminated (P < 0.001). In all 23 cycles at least one embryo, obtained by ICSI, could be replaced. Eight pregnancies were achieved of which six resulted in the delivery of nine healthy children. In conclusion, for couples with no or almost no fertilization of oocytes in previous IVF attempts, ICSI appeared to be far superior to an additional IVF attempt with further elevated insemination concentrations.     

Analysis of 76 total fertilization failure cycles out of 2732 intracytoplasmic sperm injection cycles

From October 1992 to December 1994, 2732 cycles of treatmentby intracytoplasmic sperm injection (ICSI) were carried outin couples mainly with severe male-factor infertility. The overallfertilization rate in these 2732 cycles was 71% of intact oocytes.However, in 76 (72 couples) of these cycles, none of the injectedoocytes became fertilized,so the total fertilization failurerate was 3% (76/2732 cycles). Details of these 76 cycles wereanalysed. The results show that total fertilization failureafter ICSI may be explained by different factors related to(i) semen characteristics (only immotile or round-headed spermatozoafor ICSI) or (ii) the oocytes (number, abnormal morphology,damage after ICSI). Of 26 couples, 22 achieved fertilizationin their subsequent ICSI cycles. In conclusion, total fertilizationfailure after ICSI for the treatment of severe malefactor infertilitywas mainly caused by the poor viability of the spermatozoa usedfor injection; it was also associated with a low number andpoor quality of oocytes. Repeated ICSI treatment may be usefulor necessary in couples with total fertilization failure.     

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.     

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.     

Fertilization and early embryology: Factors affecting activation and fertilization of human oocytes following intracytoplasmic injection

Intracytoplasmic sperm injection (ICSI) has dramatically alteredthe treatment of severe male factor infertility, resulting inimproved fertilization and pregnancy rates. The purpose of thisstudy was to investigate oocyte activation and fertilizationin aged human oocytes following ICSI. Non-viable spermatozoawere injected into 24 h old human oocytes in the presence andabsence of calcium and were assessed for evidence of activationand fertilization 16–19 h after the injection procedure.Sham injections were also carried out to assess the effect ofthe injection procedure itself and the presence of calcium inthe injection medium on oocyte activation. Non-viable spermatozoainjected in the presence of 1.78 mM calcium were capable ofnormally fertilizing aged human oocytes and the resulting zygotesunderwent cleavage. None of the oocytes injected with non-viablespermatozoa in the absence of calcium were fertilized normally,although the rates of activation following all treatments weresimilar.     

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.     

The usefulness of a piezo-micromanipulator in intracytoplasmic sperm injection in humans

Intracytoplasmic sperm injection (ICSI) has wide clinical application. In order to achieve good results with this method, it is important to restrict the possibility of oocyte injury as much as possible, and securely inject spermatozoa into the ooplasm. For this purpose, we clinically applied piezo-ICSI, which employs a micromanipulator with piezoelectric elements, to humans, and compared the results with those obtained by conventional ICSI. Conventional ICSI and piezo-ICSI were used in 279 cycles and 335 cycles respectively. Piezo-ICSI showed significantly more favourable results, with a survival rate of 88.1% (conventional ICSI: 81.4, P < 0.001), a fertilization rate of 79.4% (conventional ICSI: 66.4%, P < 0.001), and a pregnancy rate of 23.1% (conventional ICSI: 14.9%, P < 0.05). In piezo-ICSI, the needle used is not sharpened and has a flat tip. However, deformation of the oocyte during insertion of the needle is restrained by vibration of the piezo, and the oolemma is punctured readily and securely by the piezo pulse, at the site where the spermatozoon is injected. Piezo-ICSI is a promising new technique for human ICSI that should improve the survival, fertilization and pregnancy rates after ICSI.     

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.     

Treatment of severe male immunological infertility by intracytoplasmic sperm injection

A total of 29 infertile couples (group A) with male antispermantibodies detected by the mixed antiglobulin reaction (MAR)and partly by flow cytometry (n = 21) were treated using anintracytoplasmic sperm injection (ICSI) technique to assistfertilization. In all, 22 of them had shown a poor fertilizationrate (6%) in previous in-vitro fertilization (IVF) treatments.The fertilization and cleavage rates in ICSI, 79 and 89% respectively,were similar to those in a MAR-negative group (group B; n =20) injected because of male infertility (68 and 93% respectively).A third group (group C; n = 37) with male immune infertilitywas treated by conventional IVF. All these couples had at leastone oocyte fertilized, but the overall fertilization rate (44%)in group C was significantly poorer (P < 0.001) than thatin the two ICSI groups. However, the embryo quality was lowerin group A compared with that in the other groups. A total of13 pregnancies resulted in group A (46%), of which five endedin miscarriage. None of the six pregnancies (30%) in group Baborted during the first trimester. These results reveal, forthe first time, that ICSI offers a good chance of fertilizationfor couples with male immunological infertility. However, post-fertilizationevents may compromise these results because of factors not yetclearly understood.     

Obstetric outcome of pregnancies after the transfer of cryopreserved and fresh embryos obtained by conventional in-vitro fertilization and intracytoplasmic sperm injection.

This study reports the obstetric outcome of pregnancies obtained after the transfer of cryopreserved or fresh embryos where the initial procedure was standard in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Pregnancies obtained after frozen IVF (n = 245) or frozen ICSI (n = 177) were compared with a control group of pregnancies after fresh embryo transfer in standard IVF (n = 245) and ICSI (n = 177) cycles were selected as controls. The controls were matched according to maternal age, parity and date of embryo transfer. In the standard IVF group, the biochemical pregnancy rates in the cryopreserved and fresh groups were 18.8 and 9.8% respectively (P < 0.01). In the ICSI group, the biochemical pregnancy rates in the cryopreserved and fresh groups were 16.4 and 6.8% respectively (P < 0.01). The miscarriage rates were comparable between the cryopreserved and fresh groups. However, in the frozen ICSI group the miscarriage rate (26.0%) was significantly higher than in the frozen conventional IVF group (13.1%) (P = 0.001). The frequencies of preterm deliveries, infants with very low birthweight and intrauterine deaths were similar in the groups. The low birthweight rates in the frozen IVF (16.1%) and ICSI (12.1%) groups were significantly lower than those in the fresh IVF (32.2%) and ICSI (32.7%) groups (P < 0.001). The major malformation rates in the frozen IVF (2.4%) and ICSI (2.9%) groups were not different from the major malformation rates in the fresh IVF (4.5%) and ICSI (2.4%) groups. In conclusion, the cryopreservation process had no negative impact on the outcome of pregnancies over 20 weeks of gestation. Long-term follow-up studies are needed in order to prove the safety of the freezing-thawing process.     

Oolemma characteristics in relation to survival and fertilization patterns of oocytes treated by intracytoplasmic sperm injection

The aim of this study was to analyse the various reactions displayedby the oolemma to the penetrating pipette during intracytoplasmicsperm injection (ICSI) and correlate them with clinical factors,oocyte survival and fertilization patterns. Three types of oolemmaresponses were observed: normal breakage, when the injectionneedle created an invagination that ruptured at the approximatecentre of the egg; sudden breakage, when the membrane brokewithout creating a funnel; and difficult breakage, when themembrane did not break or broke after several penetration attempts.A total of 2928 oocytes were analysed with the following observations:73.9% (n = 2164) experienced normal breakage, 11.8% (n =345)sudden breakage, and 14.3% (n = 419) difficult breakage. Thesurvival rate and number of normally fertilized oocytes weresignificantly lower and the incidence of digynic oocytes wassignificantly higher in the sudden breakage group; furthermore,in this group a significantly shorter length of stimulationwas observed along with lower serum oestradiol concentrationswhen compared to oocytes experiencing normal and difficult breakagepatterns. These recorded patterns were predictive of the survivaland fertilization ability of the injected oocytes, as well asthe incidence of digyny. The link between membrane behaviourand various clinical parameters appears to indicate a correlationbetween the modality of stimulation and oolemma characteristics.