The effects of the sperm motility activators 2-deoxyadenosine and pentoxifylline used for sperm micro-injection on mouse and human embryo development

The sperm motility stimulants 2-deoxyadenosine (DOA) and pentoxifylline(PTF), used to improve the success of insemination and spermmicro-injection for low motility sperm samples, were studiedfor their effects on the developmental capacity of mouse andhuman oocytes. When human oocytes were micro-injected with spermatozoain 3 mM DOA 80% of them became blocked at the 1-cell pronuclearstage. However, when spermatozoa in 3 mM PTF were used for micro-injectionor when spermatozoa were washed to remove DOA before micro-injectiononly a few oocytes (9–10%) were blocked. Pregnancies occurredin five of 14 patients into whom cleaving embryos from all threetreatments had been transferred, indicating that once cleavagewas initiated, development of embryos occurred at expected rates.Exposure of mouse oocytes to DOA for a short period during insemination(4–6 h) or a longer period during the pronuclear cellcycle (18 – 20 h) significantly reduced cleavage beyondthe 2-cell stage, resulting in a dramatic reduction in blastocystformation. PTF did not significantly reduce mouse embryo development.Similar results were obtained for oocytes inseminated in vitroor micro-injected with a spermatozoon into the perivitellinespace. Neither DOA nor PTF increased fertilization of mouseoocytes. PTF reduced fertilization, particularly in cumulus-enclosedoocytes and oocytes micro-injected with spermatozoa in PTF.We conclude that DOA is a potent inhibitor of embryo developmentand oocytes should not be exposed to DOA. Exposure of oocytesto PTF had little effect on their subsequent development buttreatment of cauda epididymal mouse spermatozoa can reduce theirfertilizing capacity.     

Fertilization and early embryology: Does zona pellucida thickness influence the fertilization rate?

In human in-vitro fertilization (IVF), the cumulus oophorusis routinely removed to assess fertilization and hence the thicknessof the zona pellucida is measurable. This study aimed to measurethe thickness of the zona pellucida and to assess its influenceon fertilization rate in IVF programmes. The zona pellucidathickness varies from 10 to 31 µm with a mean of 17.5µm. One-way analysis of variance revealed that in IVFtrials performed with normal semen, the zona pellucida of fertilizedoocytes (16.6 ± 3.2 µm) was significantly thinnerthan the zona pellucida of unfertilized oocytes (18.9 ±4.0 µm; P < 0.001). As measured on micro-injected oocytes,the zona pellucida thickness did not change between ovulationand 16–20 h after fertilization. Zona pellucida thicknesswas not related to ooplasm diameter. In conclusion, zona pellucidathickness appears to be an additional factor that should betaken into account when interpreting the fertilization rate.Zona pellucida thickness influences sperm penetration, evenwhen the spermatozoa are considered normal. From a clinicalpoint of view, a thick zona pellucida (22 µm) could bean indicator for the use of micro-injection procedures.     

Fertilization and early embryology: Intracytoplasmic single sperm injection of 1-day-old unfertilized human oocytes

Although the average fertilization rate in most in-vitro fertilization(IVF) centres is 60–70%, there are cases of complete orvirtually complete fertilization failures. The aim of our workwas to study the fertilization and the subsequent cleavage characteristicsof 1-day-old human oocytes treated by intracytoplasmic singlesperm injection (ICSI) after failing to fertilize during thestandard IVF procedure. A total of 115 metaphase II 1-day-oldunfertilized oocytes were collected from 23 patients. No additionaltreatment was applied to the oocytes or to the semen sample.A single spermatozoon from the patient's husband was injectedinto the cytoplasm of each of these oocytes 21–33 h afterovum retrieval. Injected oocytes were observed at 16–18h and again 42–44 h after the ICSI procedure. Of the injectedoocytes, 92% (n = 106) were intact after ICSI, 38% (n = 44)had two distinct pronuclei and there was no difference in thefertilization rate of oocytes when andrological and non-andrologicalpatients were compared. Similarly, there was no difference inthe fertilization rate after ICSI where patients with acceptableor good (> 15%) fertilization after standard IVF were comparedto patients who had poor (<15%) fertilization after IVF.There was no significant difference in the sperm concentrationor in the progressive forward motility (a + b motility) in thesegroups except where a + b motility of andrological and non-adrologicalpatients was compared. The majority (84%) of the normally fertilizedoocytes cleaved and most (77%) of these embryos showed <20%fragmentation 2 days after the ICSI procedure. From this studyit can be concluded that 1-day-old metaphase II oocytes whichhave failed to be fertilized after standard IVF procedure canbe fertilized and cleave when ICSI is performed on them theday after oocyte retrieval.     

Reducing the time of sperm-oocyte interaction in human in-vitro fertilization improves the implantation rate

Human oocyte development was evaluated after a reduced timeexposure to spermatozoa in vitro. A total of 119 patients wereassigned to two study groups in a randomized prospective studyin which each patient‘s oocytes were exposed to spermatozoafor either 1 h (group 1 – 58 patients) or the standard16 h incubation period (group 2 – 61 patients). The fertilizationrate obtained in group 1 was higher than in group 2 (285/393,73%, and 272/410, 66% respectively), suggesting that the spermatozoa-oocyteinteraction occurs within 1 h. This was confirmed in a studyin vitro using fluorescently labelled spermatozoa and normaloocyte-cumulus complexes. Spermatozoa enter the cumulus complexwithin 15 min, traverse the cumulus layer within 3 h, and firstappear in the oocyte cortex at 4 h post-insemination. The incidenceof polyspermy was higher in oocytes exposed to spermatozoa for16 h (3%) than for 1 h (1%). There was no difference in thecleavage rate or morphological characteristics of embryos fromboth study groups. However, when evaluating the timing of embryodevelopment, group 1 generated a significantly higher percentageof four to five cell embryos when compared to group 2 (55 versus39%; P < 0.001), documented at 40 h post-insemination. Theimplantation and pregnancy rates for group 1 were 11 and 28%,while the corresponding rates for group 2 were 8 and 15%. Thissuggests that a reduced exposure of oocyte to spermatozoa favoursembryo viability, possibly due to a decrease in potential damagefrom sperm metabolic waste products.     

Intracytoplasmic sperm injection in the mouse

Intracytoplasmic sperm injection (ICSI) into mouse oocytes involvesa very low survival rate. This study was designed to determinewhy ICSI frequently fails in mice. Metaphase II oocytes wereobtained from superovulated 4–6 week old F₁ hybrid mice.Spermatozoa were retrieved from the epididymis of 12–14week old F₁ hybrid mice. The spiked microinjection pipette usedto inject a spermatozoon into the ooplasm had outer and innerdiameters of 10 and 8 µm respectively. The oocytes usedin the first part of the study were not activated (group 1).Some oocytes were incubated with calcium ionophore for 5 min(group 2). The injected oocytes were evaluated 6, 20, 48 and72 h after injection. A total of 143 eggs in each group underwentICSI. In group 1, sperm heads escaped into the perivitellinespace. In all, 63 (47%) of the remaining oocytes were damagedduring the injection or had degenerated by the first evaluation.The survival rate was 53%, but fertilization did not occur.In group 2, 31 oocytes (22%) were damaged during microinjectionor soon degenerated. Two oocytes underwent accidental subzonalinsemination. Six oocytes were fertilized (4.2%) among the 78%of survivors. After injection, the sperm tail was found in thecytoplasm (27 and 31% in groups 1 and 2 respectively), the perivitellinespace (45% in both groups) or protruding through the zona pellucida(28 and 23% respectively). More oocytes degenerated when thetail remained in the cytoplasm, i.e. 78% in group 1 and 36%in group 2.     

Intracytoplasmic sperm injection in mice: increased fertilization and development to term after induction of the acrosome reaction

A technique has been developed for intracytoplasmic sperm injection(ICSI) in the mouse with a relatively low rate of lysis of oocytes(range 5–25% across experiments) and pronuclear formationin around one-third of the intact oocytes (range 30–38%across experiments) for untreated spermatozoa. The treatmentof spermatozoa with calcium ionophore, to induce the acrosomereaction (increases acrosome-free spermatozoa from 28 to 58%)before ICSI, increased pronuclear formation to {small tilde}60%(range 59–627percnt; across experiments) in intact oocytes.The pronuclear oocytes developed to blastocysts in vitro andto term when transferred to recipient foster mothers at ratesequivalent to zygotes formed after insemination in vitro. Therewas no benefit for fertilization rates of activating oocyteswith 8% ethanol before or after ICSI, nor was there any evidenceof parthenogenetic activation by the sperm solution used forICSI. This technique adds to other in-vitro fertilization techniqueswhich can be used to explore gamete interactions and to recoverbreeding in infertile strains and reproductively unfit mice.     

Cytoskeletal organization and zona sensitivity to digestion by chymotrypsin of frozen--thawed mouse oocytes

Mouse oocyte—cumulus masses were added to 1.5 dimethylsulphoxide (DMSO) + 20% fetal bovine serum (FBS) that had beenprecooled at +4°C, were frozen by slow cooling to an intermediatetemperature of –60°C before being plunged into liquidnitrogen at –196°C, subjected to a controlled thaw,expelled into 1.5 M DMSO + FBS at 4°C, and then washed inmedium + FBS at 37°C. Of 7733 oocytes treated, 78.4% wereviable (controls; no treatment: 94.2% of 2764 oocytes; cryoprotectantonly: 92.2% of 2991 oocytes). The oocyte losses were not dueto complete loss of all oocytes from some straws or mice, sinceanalysis of individual straws containing oocytes from a singlemouse revealed considerable inter-straw/mouse variation. Amongstsurviving oocytes, no significant differences between frozenand control oocytes in spindle, chromosomal or microfilamentorganization were recorded. Two significant differences wereobserved: (i) fewer frozen—thawed oocytes had zonae resistantto chymotrypsin digestion, and (ii) spindle organization incontrol oocytes, but not frozen—thawed oocytes, was improvedby 3 h incubation at 37°C. More of the abnormal than thenormal frozen—thawed and control oocytes were surroundedby zonae which were resistant to digestion by chymotrypsin.     

Significance of cumulus oophorus in in-vitro fertilization and oocyte viability and fertility

Fertilization and cleavage rates of human cumulus-intact oocytes incubated in vitro for 36-48 h with normal spermatozoa tended to be higher than those which were cumulus-denuded (73 versus 68%; 68 versus 56%, respectively); however, the difference was not significant. Nor were these differences significant when using sperm samples of various qualities (normozoospermic samples: 75 versus 70% fertilized oocytes; asthenozoospermic: 66 versus 64%; oligozoospermic: 64 versus 56%; oligoasthenozoospermic: 35 versus 33%). The beneficial effect of the human cumulus oophorus on the binding of human spermatozoa to denuded hamster oocytes and on head decondensation of human spermatozoa observed after 2 h of incubation (9.3 versus 7.0 bound spermatozoa per oocyte, P less than 0.05; 0.5 versus 0.3 decondensed sperm heads per oocyte, P less than 0.02) disappeared after 6 h. A protective effect of the cumulus oophorus on hamster oocytes preincubated in medium containing 50% human preovulatory follicular fluid was observed in the sperm penetration assay (fertilization rate of cumulus-intact: cumulus-denuded oocytes, 26 versus 13%, P less than 0.05) and confirmed using fluorescein diacetate stain (cumulus-intact oocytes: 86 versus 100% vitality, non-significant; cumulus-denuded oocytes: 64 versus 100%, P less than 0.01). These data suggest the accelerating effect of the human cumulus oophorus on fertilization in its early stages. Furthermore, the cumulus plays an important part in protecting the oocyte against adverse environmental influences.     

Micromorphometry and spermatozoa binding patterns of fertilized and unfertilized human oocytes after in-vitro fertilization

Human oocytes (n = 380) from 71 in-vitro fertilization patientswere measured 18 h after insemination to find out if certainparameters of oocyte morphology could be related to fertilization.In addition, the number and distribution patterns of spermatozoabound to or within the zona pellucida of 534 oocytes were analysed.The mean diameter of the human oocyte was 167.7 ± 9.5µm and its mean volume was 2.5 x 10⁶ µm³. Therewere no significant differences in diameter between 112 fertilizedand 168 unfertilized oocytes, although they displayed differencesin the size of the perivitelline space and the zona pellucida.The age of the patients had no significant effect on the morphometryof the oocytes. Sperm binding patterns did not correlate withfertilization. The number and distribution of the spermatozoaon the surface of the zona pellucida was extremely heterogeneousand was not related to the occurrence of fertilization. Allpossible binding and distribution patterns were in the samerange in both fertilized and unfertilized oocytes. In conclusion,the micromorphometry of human oocytes and their sperm bindingpatterns were not related to the occurrence of fertilization.     

Fertilization and early embrology: A study of the cumulus-corona cell complex in in-vitro fertilization and embryo transfer; a prognostic indicator of the failure of implantation

In-vitro morphological changes and proliferation of cells ofthe cumulus oophorus, in particular cells of the corona radiata,were seen to differ between individuals and between oocytesof the same cohort in an unselected series of 159 IVF cycles.Cell proliferation did not relate to indications for treatment,type and length of ovarian stimulation, age of patient, ovarianresponse and quality of oocyte, fertilization rate or embryomorphology. A direct relationship, however, was demonstratedbetween the proliferative capacity of these cells and the incidenceof clinical pregnancy. No pregnancies resulted when there wasan absence of morphological change or proliferation of thesecells. In cycles where the proliferative capacity of these cellsvaried between oocytes of a single cohort the pregnancy ratewas 40% per cycle, and in cycles where cells associated withall oocytes underwent extensive proliferation the pregnancyrate was 49% (P < 0.001). Thus, regardless of the apparentquality of gametes or embryo, no pregnancies arose if the cumulus–coronacomplex was incapable of replication. We have, therefore, identifiedtwo populations of oocytes based on the proliferative capacityof cumulus–corona cells in vitro, one being associatedwith a failure of implantation.     

In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium

In-vitro maturation (IVM) of oocytes is a promising technique to reduce the costs and avert the side-effects of gonadotrophin stimulation for in-vitro fertilization (IVF). The pregnancy rates from oocytes matured in vitro are much lower than those of in-vivo stimulation cycles indicating that optimization of IVM remains a challenge. Therefore, we investigated the effect of supplementation of the medium with gonadotrophins, oestradiol and epidermal growth factor (EGF) and the effect of retaining or removing the cumulus cells on nuclear and cytoplasmic maturation of immature oocytes. Human germinal vesicle (GV) oocytes obtained after gonadotrophin stimulation for intracytoplasmic sperm injection (ICSI) were cultured in a complex defined medium either supplemented with gonadotrophins, oestradiol and physiological concentrations of EGF (2 ng/ml) or gonadotrophins and oestradiol alone. The cumulus cells were either removed or kept intact. In GV stage oocytes cultured without cumulus (group I) significantly more oocytes reached the metaphase II (MII) stage at 30 h in media supplemented with EGF (64.3 versus 33.9%, P < 0.003). For oocytes cultured with intact cumulus (group II), more oocytes reached MII at 30 h than in group I, but there was no difference in medium with or without EGF supplementation (81.8 and 79.8% respectively). Cytoplasmic maturation of MII oocytes was judged from their capability to activate and fertilize after ICSI. In group I, the rates of activation and normal fertilization were similar. However, in group II, significantly more oocytes underwent normal fertilization in the EGF-supplemented than the unsupplemented group (71.7 versus 45.6%, P < 0.05). The cleavage rates of the fertilized oocytes were similar in the sibling oocyte subgroups cultured with or without EGF supplementation, but the overall cleavage rates were higher in cumulus-intact compared to cumulus-denuded oocytes (88.9 versus 47.8%, P < 0.001). Thus, supplementation of the maturation medium with EGF and maintenance of the cumulus during culture improve the nuclear and cytoplasmic maturation of human oocytes in vitro.      

Fertilization of human oocytes in capillary tubes with very small numbers of spermatozoa

Human oocytes can be fertilized with high rates of success underin-vitro conditions even if only low numbers of spermatozoaare used. A culture system has been developed in which fertilizationis performed in haematocrit capillary tubes (length 75 mm; i.d.0.8–0.9 mm). Oocytes were fertilized in 5–10µlof different sperm suspensions containing a total of 500, 1000,2000 and 4000 spermatozoa per oocyte (0.1–0.4 x 10⁶ spermatozoa/ml).Oocytes were obtained from 10 patients participating in an in-vitrofertilization programme; of these, 32 oocytes were fertilizedin capillary tubes and 32 oocytes were cultured using standardmethods (1 ml culture medium in tissue culture tubes; 0.1–0.2x 10⁶ spermatozoa/ml). The overall fertilization rate of oocytescultured in tissue culture tubes was 78% (25/32) and the fertilizationrates in capillary tubes using 4000, 2000, 1000 or 500 spermatozoaper oocyte were 71% (5/7), 86% (6/7), 60% (6/10)and 50% (4/8),respectively. The fertilization rate of mature oocytes was highercompared with immature oocytes when fertilization was performedin culture tubes (83 and 63%) or in capillary tubes (74 and44%). Fertilization in capillary tubes using a 10µl ofoocyte and spermatozoa suspension compared to 5µl seemedto provide better culture conditions, resulting in higher fertilizationand cleavage rates. These preliminary results indicate thatfertilization of human oocytes under in-vitrio conditions canbe achieved even with very low numbers of spermatozoa     

Successcul embryo transfer of cryopreserved and in-vitro fertilized rabbit oocytes

In-vitro fertilization experiments with frozen/thawed rabbitoocytes were performed to develop an effective technique tobe used for the in-vitro fertilization of cryopreserved humanoocytes. Ovulatory oocytes, colected from the oviduct of virgindoes 13 h after induction of ovulation by HCG injection, werecryopreserved slowly to –30°C and plunged directlyinto liquid nitrogen. A mixture of 1.5 M 1, 3-propanediol and0.1 M sucrose was used as a cryo-protectant. After thawing,the oocytes were incubated with in-vitro capacited sperm for5 h indefined Brackett's medium. Fertilized ova were culturedfor an additional 20 h until the 4-to-8-cell stage was reached.These embryos were transferred to pseudopregnant recipient rabbitswhich were ’asychronous‘ in the sense that theyhad been given an injection of HCG 30, 24 and 18 h before startingto do the embryo transfer. A 32% survival rate of frozen/thawedoocytes was achieved. The fertilization rate was 74% (181/264)in this study. A total of 53 embryos was transferred to theoviducts of six recipients of three different asynchronicityand four young were born. The highest implantation rate (includingresorptions) of 18% could be achived in this investigation byusing –6 h asynchronous recipients, While the overallimplantation rate was 9.4 %     

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.     

Fertilization and early embryology: Cytogenetic and fluorescent in-situ hybridization chromosomal studies on in-vitro fertilized and intracytoplasmic sperm injected 'failed-fertilized' human oocytes

This study was undertaken to establish baseline data on thechromosomal status of ‘failed-fertilized’ oocytesderived from in-vitro fertilization (IVF) or intracytoplasmicsperm injection (ICSI) procedures. A cytogenetic analysis wasundertaken on 162 IVF and 51 ICSI oocytes. In all, 82.1% (133/162)of the IVF and 78.4% (40/51) of the ICSI oocytes had metaphaseII (Mil) plates, of which 50.4% of the IVF and 47.5% of theICSI oocytes were analysed further. Chromosomes of the G-group(21–22) were identified with the majority of the anomalies.No overall significant difference in the aneuploidy rate wasfound for the IVF (37.3%) or ICSI (31.6%) oocytes, or with maternalage. However, chromosome anomalies, e.g. diploidy, fragmentedand broken chromatids, single sperm and oocyte chromatids, werefound in oocytes from IVF patients aged >36 years and inthe ICSI oocytes throughout the maternal age range (31–38years). The status of the polar body chromatin indicated thatthere was no overall significant difference in the maturationof the IVF and ICSI oocytes. Evidence of successful sperm deliverywas found in 72.5% (37/51) of the ICSI failed-fertilized oocytes.In this group there was a significant increase in the incidenceof premature chromosome condensation: 19.6% (10/51) containedsperm chromosomes, 7.8% (4/51) had swollen sperm heads, andthe remaining 45.0% had condensed sperm heads. The presenceof both sperm and Mil oocyte chromosomes was found in 19.6%(10/51) of the ICSI and 8.6% (14/162) of the IVF failed-fertilizedoocytes. Specific fluorescent in-situ hybridization DNA probeswere used to re-analyse the chromosomes of karyotyped ‘failed-fertilized’IVF oocytes and, for the first time, applied to the karyotypedchromosomes of failed-fertilized ICSI oocytes. The hybridizationefficiency was 86–95% for the centromere probe and 100%for probes 21 and 18.     

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.     

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.     

Effect of timing of oocyte denudation and micro-injection on survival, fertilization and embryo quality after intracytoplasmic sperm injection

In human in-vitro fertilization (IVF), the oocytes are surrounded by cumulus and corona cells at the time of insemination so that their maturity cannot easily be evaluated. The best IVF results are obtained if the oocytes are inseminated 2-6 h after retrieval. In the intracytoplasmic sperm injection (ICSI) procedure, the oocytes are denuded by enzymatic and mechanical treatment in order to be able to perform the injection. As a consequence, the nuclear maturity of the oocytes can be evaluated and only those that have extruded the first polar body are injected. However, metaphase-II oocytes that have not yet reached cytoplasmic maturity cannot be recognized. The purpose of this study was to investigate the effect of different timing of cumulus- corona cell removal and injection on the outcome of ICSI. For this we allowed the oocytes to complete in-vitro cytoplasmic maturation in two different culture conditions: (i) surrounded by their cumulus and corona cells or (ii) totally denuded. We performed three different studies on sibling oocytes obtained after a standardized buserelin/human menopausal gonadotrophin (HMG) protocol. We investigated the effect of early (1-2 h after retrieval) and late (5-6 h after retrieval) oocyte denudation and injection on the survival and fertilization of the injected oocytes and on embryo cleavage after fertilization. We found no statistically significant differences between early and late injection, indicating that after a standardized buserelin/HMG protocol the metaphase-II oocytes do not need time for further cytoplasmic maturation. Furthermore, a different timing of cumulus-corona cell removal has no effect on the outcome of ICSI, suggesting that the surrounding cells are not necessary for survival, fertilization and cleavage after ICSI.      

Fertilization and early embryology: Intracytoplasmic sperm injection of mouse oocytes with 5 mM Ca2+ at different intervals

The objective of this investigation was to determine whetherintracytoplasmic sperm injection (ICSI) can be performed inthe mouse. Metaphase II oocytes were obtained from F1 hybridmice (C57BLx CBA) by i.p. injections of 10 IU pregnant mare'sserum gonadotrophin (PMSG) and human chorionic gonadotrophin(HCG) administered 48 h apart. Oocytes with cumulus oophoruswere retrieved 13–14 h post HCG. Cumulus was dispersedwith 0.1% hyaluronidase. Mouse spermatozoa were obtained fromthe cauda epididymides of males of the same strain. The spermatozoawere processed by the standard swim-up procedure. The harvestedspermatozoa were then incubated for 1.5 h to allow capacitation.Healthy oocytes were injected with 3–4 pi 5 mM Ca²⁺, followedby one live morphologically normal spermatozoon into the cytoplasmat intervals of 0, 0.5, 1, 2 and 3 h. The proportion of 2-cellembryos that developed from oocytes injected with Ca²⁺ and spermatozoaranged between 29.5 and 36.5% in all groups, with no statisticaldifference between treatments. Chromosomal analysis showed thattwo-thirds of the ICSI-derived 2-cell embryos were diploid.The proportion of parthenogenetically activated embryos in theICSI groups was similar to that in the control group (8–10%)which was injected with Ca²⁺ and polyvinyl pyrrolidone only.The proportion of blastocysts that developed in culture fromthe ICSI-derived 2-cell embryos was of the order of 36–42%.Some blastocysts were used for cell number counts. There wasa significant increase in total and inner cell mass counts ofblastocysts in which the spermatozoon was injected at 2 and3 h following Ca²⁺. The remaining blastocysts were transferredto day 3 pseudopregnant mice, of which 33% subsequently becamepregnant. Of the blastocysts transferred, 16–25% developedto term in vivo. No deformities were observed in the pups. Webelieve this is the first report of live-birth following mouseICSI.     

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

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