Analysis of the human sperm centrosomal function and the oocyte activation ability in a case of globozoospermia,by ICSI into bovine oocytes

BACKGROUND: Centrosomal function and oocyte activation ability of human sperm from a case of globozoospermia was assayed by heterologous ICSI into bovine oocytes. METHODS: Microtubules and chromatin configuration in bovine oocytes were examined by immunofluorescence after heterologous ICSI with human sperm from two fertile donors and from a globozoospermic man. RESULTS: The microtubule array from the sperm centrosome, the 'sperm aster' and the male pronucleus were observed in bovine oocytes, following ICSI with round-headed sperm from a globozoospermic man. The rate of sperm aster formation and the rate of male pronuclear formation in the bovine oocytes injected with fertile donor sperm were 57.9 and 92.5% respectively; the respective values for oocytes injected with round-headed sperm without artificial oocyte activation were 15.8 and 31.0%. Ethanol activation after ICSI improved male pronuclear formation (84.9%) but not sperm aster formation rate (32.3%) of the globozoospermic patient. CONCLUSIONS: These data indicated that sperm from this patient with globozoospermia have centrosomal dysfunction and low ability for oocyte activation compared with fertile donor sperm. The centrosomal dysfunction may be one of the reasons for infertility in this patient.     

Use of Mammalian eggs for assessment of human sperm function: molecular and cellular analyses of fertilization by intracytoplasmic sperm injection

PROBLEMS: Intracytoplasmic sperm injection (ICSI ) has been described as the 'cure' for male sterility because a single sperm can now be directly introduced into an egg with some chance of pregnancy. While ICSI has revolutionized the practice of assisted reproductive techniques (ART), there are few molecular and cellular studies about its safety and efficacy. Even by using ICSI, fertilization in humans succeeds only if the sperm effectively accomplishes a number of tasks including 'post-ICSI events' in fertilization. To assess the function of human sperm after ICSI, we used heterologous ICSI with human sperm into animal eggs. Egg activation, sperm decondensation and sperm centorosomal function were examined in sperm from fertile men and infertile patients. METHODS: Sperm from fertile men and infertile patients were injected into hamster, rabbit and bovine eggs by Piezo micromanipulator, and studied in decondensation of sperm nuclei, egg activation and microtubule organization. RESULTS: Decondensation human sperm head following ICSI into hamster eggs occurred initially form basal lesion, and apical portion of sperm nuclei which is surrounded by acrosome and perinuclear theca, still condensed in early pronuclear stage. Radial array of microtubules from sperm centrosome 'sperm aster' which is essential for pronuclear movement was observed in 30% rabbit eggs following ICSI with human sperm. By heterologous ICSI system with fertile human sperm and bovine eggs, 83.3% of eggs was activated and 60% eggs had sperm aster, indicating that bovine Piezo ICSI system is appropriate for assessing human sperm oocyte activation ability and human sperm centrosomal function. Oocyte activation and sperm centrosomal function were significantly low in sperm from men with globozoospermia and men with dysplasia of fibrous sheath. CONCLUSION: These assays indicate differences of the process of fertilization between in vitro fertilization and ICSI, and reflect the human sperm function especially for the 'post-ICSI events' in fertilization. More molecular and cellular analyses in fertilization by ICSI are needed for improvement of ART.     

A novel method for chromosome analysis of human sperm using enucleated mouse oocytes

BACKGROUND: Mouse oocytes can be used in conjunction with intracytoplasmic sperm injection (ICSI) as a technique to permit chromosomal analysis of human sperm. However, chromosomes derived both from the human sperm and the mouse oocyte appear simultaneously following ICSI. The present study focused on evaluating whether or not previously enucleated mouse oocytes are usable for the analysis of human sperm chromosomes. METHODS: The metaphase chromosome-spindle complex was removed from a mouse oocyte. Human sperm from a donor with proven fertility were injected into mouse enucleated oocytes or intact oocytes. The presence of pronuclei in the oocytes was confirmed approximately 7-11 h after ICSI, and the oocytes were then fixed so that the nuclei could be observed as chromosome samples at 15-16 h after ICSI. RESULTS: The formation rate of one pronucleus in enucleated oocytes after ICSI was 93.9% (186/198) while that of two pronuclei in intact oocytes after ICSI was 85.4% (88/103). The appearance rate of metaphase chromosomes of human sperm in the enucleated oocytes, 89.4% (160/179), was significantly higher than that in intact oocytes, 78.7% (74/94) (P = 0.017). CONCLUSIONS: An efficient ICSI method using enucleated mouse oocytes was established to allow the visualization of the human sperm chromosome complement without the risk of confusion with mouse oocyte chromosomes.     

The Implications of a Paternally Derived Centrosome During Human Fertilization: Consequences for Reproduction and the Treatment of Male Factor Infertility

PROBLEM: Successful fertilization in humans follows a complex series of events, including the completion of meiotic maturation of the oocyte with the extrusion of the second polar body, the decondensation of the sperm nucleus and the maternal chromosomes into male and female pronuclei, the restoration of the sperm centrosome, and the nucleation of microtubule-mediated motility necessary to bring the male and female pronuclei into close apposition. These events occur after both fertilization in vitro and after intracytoplasmic sperm injection (ICSI), a new technique which is currently being applied in many clinics to overcome severe male infertility. Defects in any of the events leading to fertilization can be lethal to the zygote and may prove to be causes of infertility. METHODS: Imaging of inseminated human and rhesus oocytes using immunohistochemical techniques reveals several phases at which fertilization arrests. RESULTS: Oocytes from some infertile patients failed to complete fertilization due to failure of the sperm aster microtubules in uniting the sperm and egg nuclei. The rate of sperm aster formation, size, and organization during fertilization has been used as a measurement of bovine sperm quality. The development of an assay using Xenopus laevis oocyte extract can also be used to test sperm from various species for their ability to form asters and perform other centrosomal functions in vitro, as well as another indicator of sperm quality. Semen from men with questionable fertility was found to contain sperm which are generally incapable of producing sperm asters. In addition, the activity of centrosomal proteins such as γ-tubulin and centrin have been detected in mammalian eggs and sperm. The levels of γ-tubulin increase markedly after exposure to X. laevis egg extract. CONCLUSION: Defects in either male or female nucleus decondensation also resulted in the arrest of fertilization and was found to occur in both inseminated human oocytes and in rhesus oocytes fertilized by ICSI. These discoveries on the molecular basis of infertility in humans have important implications for infertility diagnosis and managing reproduction.     

The effects of meiosis activating sterol on in-vitro maturation and fertilization of human oocytes from stimulated and unstimulated ovaries

The object of this study was to assess functional maturation in vitro by obtaining data on the fertilization and embryonic competence of human oocytes with or without exposure to meiosis activating sterol (MAS) during maturation in vitro. Immature oocytes were either collected from unstimulated patients with polycystic ovaries (PCO) during gynaecological surgery, or were donated by patients undergoing a cycle of intracytoplasmic sperm injection (ICSI) treatment including ovarian stimulation with gonadotrophins. PCO oocytes had variable cumulus cover, which was retained during culture while those from ICSI patients were cultured without cumulus. The study included 119 oocytes from PCO patients and 72 from ICSI patients. The oocytes were allowed to mature in vitro for up to 46 h in the presence or absence of MAS. Mature oocytes were inseminated by ICSI with fertile donor spermatozoa and embryo development was monitored in vitro. MAS (30 microg/ml) significantly increased the survival of oocytes from PCO patients (P < 0.01) but did not significantly affect the proportion completing maturation in vitro. For the ICSI patients, >90% of oocytes survived in all culture groups, regardless of MAS addition, however MAS (10 or 30 microg/ml) significantly increased the proportion of oocytes maturing in vitro (P < 0.05). The apparent tendency towards improved subsequent development in vitro will require larger numbers of oocytes for evaluation. Oocytes from ICSI patients matured more rapidly in vitro than those from PCO patients. Our results show positive effects of MAS on human oocytes, confirming previous data in mice. This work may have implications for the future clinical application of IVM.     

Andrology: Analysis of the oocyte activating capacity and chromosomal complement of round-headed human spermatozoa by their injection into mouse oocytes

Intracytoplasmic sperm injection (ICSI) in the human is a veryeffective procedure which allows the fertilization of the majorityof oocytes even in cases of extreme oligoasthenoteratozoospermia.Round-headed acrosomeless human spermatozoa, however, form anexception to this rule, because in about half of the coupleswith globozoo-spermia all oocytes remain unfertilized afterinjection. The incapacity of the spermatozoon to activate theoocyte following injection of round-headed spermatozoa couldbe the underlying mechanism. To investigate this hypothesis,activation rates of mouse oocytes injected with spermatozoafrom a patient with globozoospermia were compared with thoseobtained after injection with normal spermatozoa. Of mouse oocytessurviving the injection with donor spermatozoa, 95% underwentactivation, compared to none of the 88 mouse oocytes survivingthe injection with round-headed spermatozoa. After fixation,prematurely condensed sperm chromosomes were found in theseoocytes. Partheno-genetic activation of mouse oocytes (8% ethanolat 40 min after injection) injected with round-headed spermatozoaled to the activation of 96% of oocytes. These oocytes developednormally to the first mitosis and were fixed for analysis ofthe sperm karyotypes. The incidence of chromosomal abnormalitiesof round-headed spermatozoa (6%) was similar to that in spermatozoafrom a fertile donor (9%). These data provide further informationon the basic defect in cases of globozoospermia and demonstratethat globozoospermia is not associated with sperm karyotypeabnormalities.     

A pathology of the sperm centriole responsible for defective sperm aster formation,syngamy and cleavage

BACKGROUND: In the present report we analyse the structural and functional features of sperm from a patient with severe asthenoteratozoospermia and failure of cleavage after ICSI. METHODS: Sperm were studied by phase contrast and transmission electron microscopy and microinjected into bovine oocytes to examine aster formation using antibodies against acetylated alpha- and beta-tubulins. RESULTS: Acephalic sperm, headless tails and abnormal alignments of the head-tail junction were observed. Flagella evidenced the features of dysplasia of the fibrous sheath. Bovine oocytes injected with patient's sperm showed male and female pronuclei but a faulty development of microtubules from the sperm-derived centrosome. The first ICSI attempt using conventional sperm selection methods resulted in fertilized two pronuclei zygotes, but no syngamy or cleavage. Three more ICSI attempts were performed, carefully avoiding sperm with obvious anomalies of the connecting piece. Fertilization and cleavage took place in all cycles, and in two of them positive betahCG plasma levels were detected but preclinical abortions ensued. CONCLUSIONS: We propose that the alterations in the head-tail junction and attachment, responsible for the observed sperm phenotype, result from centriolar dysfunctions that cause insufficient sperm aster formation, lack of syngamy and cleavage or defective embryos leading to early abortions.     

Andrology: Intracytoplasmic injection of human spermatozoa into mouse oocytes: a useful model to investigate the oocyte-activating capacity and the karyotype of human spermatozoa

When intracytoplasmic sperm injection (ICSI) is performed, itis important to know the capacity of sperm cells to activatethe oocytes, although knowledge of their ability to fuse withthe oocytes is not vital. Hamster oocytes are not suitable forthis purpose because they are easily activated by the injectionprocedure itself. We therefore investigated whether mouse oocytescould be used to assess the activation properties of human spermatozoa.Mouse oocytes were randomized for injection with initially motilespermatozoa, medium, heat-treated or salt-extracted spermmatozoa,and the survival and activation rates were examined. About halfof the mouse oocytes survived the intracytoplasmic injectionof a human sperm cell. Unlike hamster oocytes, the rate of activationprovoked by the injection procedure itself was acceptably low(20%), resembling in this respect the behaviour of human oocytes.Following the injection of initially motile human spermatozoa,all mouse oocytes were activated. The injection of heat-treatedor salt-extracted human spermatozoa resulted in activation ratesof 14 and 15% respectively, comparable with the results followingsham ICSI. These data support the hypothesis of a sperm-associatedoocyte activation factor. In most activated oocytes, the humansperm nucleus decondensed to form a male pronucleus. Cytogeneticanalysis at the first metaphase revealed that human sperm chromosomeswere able to undergo replication in a heterologous environment.From our data we concluded that human spermatozoa can be injectedsuccessfully into mouse oocytes, resulting in a reasonable survivalrate, and that mouse oocytes provide a useful model for boththe assessment of the sperm-associated oocyte activation factorand the cytogenetic analysis of human spermatozoa.     

A cytogenetic study of in-vitro matured murine oocytes after ICSI by human sperm

BACKGROUND: The purpose of this study was to investigate the chromosomal complement and developmental potential of in-vitro matured murine oocytes following ICSI by human sperm. METHODS: Heterologous ICSI fertilization between mouse oocytes and human sperm was employed in order to overcome the reduced fertilization rates observed after conventional IVF due to zona hardening during in-vitro maturation, and to assess separately maternal and paternal chromosome complements. Cytogenetic analyses were performed in four types of oocytes: (i) in-vitro matured metaphase II (MII) oocytes; (ii) in-vivo matured MII oocytes; (iii) in-vitro matured oocytes after ICSI; (iv) in-vivo matured oocytes after ICSI. RESULTS: Activation rates after ICSI of in-vitro matured oocytes was lower than that of in-vivo matured oocytes (69.9 versus 97.2%, P < 0.01), and premature chromosomal condensation was only observed in in-vitro matured oocytes. However, there were no significant differences in developmental rates after successful activation between in-vivo and in-vitro matured ICSI oocytes (69.7 versus 76.6%). The incidences of aneuploidy and structural aberrations were similar between the ICSI embryos and non-ICSI (MII) oocytes. Furthermore, the frequency of chromosomal aberrations was not associated with in-vitro or in-vivo maturation. Similar analyses of paternal chromosomes indicated that there were no significant differences in the incidence of chromosomal aberrations between the embryos derived from in-vitro and in-vivo matured oocytes. CONCLUSIONS: These results suggest that in-vitro matured oocytes following ICSI do not lead to an increase in the frequency of aneuploidy and structural aberrations when human sperm are injected into mouse oocytes.     

Paternal effects acting during the first cell cycle of human preimplantation development after ICSI.

BACKGROUND: The ability of human embryos to undergo normal development has been shown previously to be subject to strong paternal (sperm-derived) effects. This study was undertaken to determine whether paternal influences on human embryo quality are detectable as early as the first cell cycle after fertilization. METHODS: The quality of zygotes and cleaving embryos resulting from sibling donor oocytes fertilized by sperm from different patients were compared in a donor oocyte-sharing programme. RESULTS: Fertilizations with sperm from certain individuals repeatedly resulted in the formation of high proportions of zygotes with abnormal pronuclear morphology that subsequently tended to cleave slowly and to show extensive fragmentation and blastomere irregularities. This phenomenon was observed with oocytes from two different donors for each of these individuals and contrasted with normal developmental performance of embryos resulting from sibling oocytes fertilized by sperm from other men with similar basic sperm characteristics. Fertilization rates were not related to these differences. CONCLUSIONS: These data point to a very early onset of paternal effects that condition human embryo development. These effects may be both of genetic (related to the minor gene activity of the male pronucleus) or epigenetic (related to the sperm-derived oocyte-activating factor or sperm centrosome) origin.     

Fertilization and embryo development to blastocysts after intracytoplasmic sperm injection in the rhesus monkey

Notwithstanding the thousands of seemingly healthy children born after intracytoplasmic sperm injection (ICSI), it is not yet possible to conclude absolutely that the ICSI procedure might induce some altered development or that the ICSI protocol might not be improved even further. To address this in a clinically relevant system, the developmental potential of rhesus monkey embryos produced by ICSI is reported. Oocytes collected by laparoscopy from gonadotrophin- stimulated fertile females were fertilized by ICSI using spermatozoa obtained from fertile males by electro-ejaculation. Neither sperm immobilization prior to injection nor an additional chemical stimulus were necessary to achieve oocyte activation and pronuclear formation. Survival and activation of the injected oocytes were judged by the extrusion of the second polar body. Successful fertilization was confirmed by the presence of two pronuclei within 12 h post-ICSI. Some oocytes were fixed and processed for the detection of microtubules and chromatin. Fluorescent labelling revealed that by 12 h post-ICSI the male and female pronuclei were closely apposed and eccentrically positioned within a large microtubule aster. ICSI resulted in a 76.6 +/- 14.9% fertilization rate. First cleavage was completed within 24 h post- ICSI. Two-cell ICSI embryos were co-cultured in CMRL medium on a buffalo rat liver cell monolayer until the hatched blastocyst stage. Oocytes collected laparoscopically from stimulated monkeys can be fertilized by ICSI and will complete preimplantation embryo development in vitro demonstrating that the rhesus monkey is an excellent preclinical model for examining and understanding many aspects of human ICSI.      

Factors influencing the outcome of ICSI in patients with obstructive and non-obstructive azoospermia: a comparative study

BACKGROUND: Factors influencing success of sperm retrieval in azoospermic patients and outcome of ICSI were evaluated. METHODS AND RESULTS: Uni- and multifactorial analysis were performed using logistic and stepwise analysis, following surgical sperm retrieval by percutaneous epididymal sperm aspiration (55 cycles) or testicular sperm extraction (142 cycles) in 52 and 123 patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) respectively. ICSI cycles using fresh or cryopreserved-thawed sperm were included. Sperm were retrieved to allow ICSI in 100 and 41% of OA and NOA patients, with no significant correlation with patients' age or FSH level. Occurrence of pregnancy was significantly correlated with female age (90th quantile: 38 years), number of oocytes retrieved (10th quantile: five oocytes) and number of oocytes injected (10th quantile: four oocytes). Sperm origin (epididymal versus testicular), status (fresh or thawed), male partner's age, and serum FSH had no significant effect upon implantation rate, pregnancy rate per embryo transfer or spontaneous miscarriage rate. CONCLUSIONS: In OA patients ICSI should be planned in conjunction with surgical sperm retrieval. In contrast, the lack of efficient non-invasive parameters to predict sperm retrieval in NOA suggests that elective surgical sperm retrieval may be offered to these patients prior to ovarian stimulation of their partners, especially when donor back-up is not an alternative. Female factors such as age and ovarian reserve have significant impact upon clinical success rates.     

Reduced oocyte activation and first cleavage rate after ICSI with spermatozoa from a sterile mouse chromosome mutant

BACKGROUND: Male mice, heterozygous for two semi-identical reciprocal translocations T(1;13)70H and T(1;13)1Wa are usually sterile. We have investigated this oligoasthenoteratozoospermic mouse model using ICSI. METHODS: B6D2F1 oocytes were injected with epididymal or testicular sperm from fertile or sterile translocation carriers and from chromosomally normal fertile controls. ICSI efficiency was determined by pronucleus formation and first cleavage rates. For arrested zygotes, cell cycle progression was evaluated by BrdU incorporation and incubation with okadaic acid. RESULTS: Epididymal sperm from infertile translocation carriers showed a slightly lower fertilization rate (70% vs. 92%, 95% and 95% for fertile translocation carriers and two groups of normal fertile control males, respectively) and a severely reduced cleavage rate (33% vs. 87%, 96% and 89% for the same control groups). However, the use of testicular sperm significantly improved the cleavage rate (62% vs. 83% for normal fertile controls). Development of arrested zygotes was delayed or blocked during S- and G2-phase. CONCLUSIONS: Whereas control testicular and epididymal sperm performed equally well, the use of testicular sperm from oligospermic T/T' males significantly increased first cleavage rates when compared to the low rates with epididymal sperm. Epididymal storage in oligospermics may negatively influence zygote division.     

Centrosome and microtubule dynamics during early stages of meiosis in mouse oocytes

Centrosomes, major regulatory sites for the microtubule (MT) nucleation, are regulated in a dynamic manner throughout the process of meiotic maturation. Recently, centrosome orientation in mouse oocytes has been demonstrated in metaphase I through metaphase II. However, centrosomal protein expression in concordance with MT polymerization in earlier stages of oocyte maturation from germinal vesicle stage (GV) to prometaphase I still remains unclear. The present study aims to assess the centrosome-microtubule remodelling during the onset of meiosis based on strict criteria of nuclear maturation. Six consecutive stages were determined for scoring the oocytes as unrimmed nucleolus (UR), partially rimmed nucleolus (PR), fully rimmed nucleolus (FR), nuclear lamina dissolution (NLD), disappearance of nucleolus (DON), and chromatin condensation (CC). A centrosomal protein, pericentrin, was found tightly localized adjacent to nuclear lamina in UR, lacking any MT nucleation activity. In concordance with the competency to resume meiosis, an increase in the amount and nucleation capacity of pericentrin is noted. In FR, cytoplasmic MT almost disappeared while de-novo microtubule polymerization was found in small aggregates of pericentrin localized around the nucleus. Towards the end of DON and CC, a sudden burst of pericentrin was noted with an extreme MT nucleation activity in an organized fashion that is essential for the rapid formation of first meiotic spindle. The results show that centrosomes display precisely controlled spatio-temporal changes during the onset of meiotic maturation. Accumulation of centrosomal proteins to a single locus followed by a sequestration to several spots might be evidence of a mechanism by which the proper distribution of centrosomal material during nuclear breakdown and subsequently formation of spindle are regulated in concordance with the nuclear maturation.     

Chemically and mechanically induced membrane fusion: non-activating methods for nuclear transfer in mature human oocytes

Most current studies of nuclear transfer in mammalian oocytes have used electrofusion to incorporate donor cell nuclei into enucleated oocyte cytoplasts. However, the application of electrofusion to human oocytes is hampered by the relative ease with which this procedure induces oocyte activation. Here we tested a previously described chemical fusion technique and an original mechanical fusion procedure in this application. Enucleated metaphase II oocytes were first agglutinated with karyoplasts originating from other metaphase II oocytes and then induced to fuse with the use of polyethylene glycol or by micromanipulation with an intracytoplasmic sperm injection (ICSI) micropipette. Both techniques yielded a high frequency of fusion and did not cause oocyte activation. Moreover, the reconstructed oocytes were easily activated by subsequent treatment with ionophore A23187 and 6-dimethylaminopurine. These techniques may be used in attempts to alleviate female infertility due to insufficiency of ooplasmic factors by nuclear transfer from patients' oocytes to enucleated donor oocyte cytoplasts. For eventual future use in human cloning, they would ensure prolonged exposure of transferred nuclei to metaphase promoting factor, which appears to be required for optimal nuclear reprogramming.     

Cell cycle regulation: Changes in centrosomal domains during meiotic maturation in the human oocyte

The distribution of microtubule organizing centres (MTOC) inthe human oocyte was examined using the microtubule-active drug,taxol, to promote polymerization. Oocytes were obtained fromgonadotrophin stimulated in-vitro fertilization (IVF) patientsand examined during various phases of meiotic maturation usingconfocal fluorescence microscopy. During the prophase of meiosisI, taxol failed to stimulate microtubule nucleation in any regionof the cells. Only a few microtubules were visible in the oocytecortex. As the transition from prophase to metaphase began,during germinal vesicle breakdown, taxol stimulated the appearanceof a small number of isolated aster-like arrays of microtubulesin the cortex, predominantly in regions adjacent to the nucleus.Oocytes treated with taxol when they had reached the first meioticmetaphase exhibited a large number of aster-like microtubulearrays. These structures were predominantly located in the corticalregion of the oocyte, but smaller arrays were also visible inthe endoplasmic areas. There did not appear to be any increaseddensity of asters in any particular cortical or endoplasmicregion. Oocytes treated with taxol during the second metaphaseof meiosis exhibited a similar response to the drug as seenduring metaphase I. Again, the microtubule asters were mostprevalent in the cortex, with smaller, less dense arrays inthe endoplasm. The metaphase spindle was also affected by taxolas revealed by increased density and hyperelongation of microtubulesat the poles of the spindle as compared to untreated oocytes.The metaphase plate of chromosomes was highly disrupted by taxoltreatment, most likely due to the forces generated by microtubuleelongation. We conclude that the human oocyte develops MTOCas meiotic maturation proceeds beyond the prophase I arrest.The first MTOCs are perinuclear, but the number and distributionincreases widely as the oocytes enter metaphase. We hypothesizethat the centrosome of the human oocyte recruits several MTOCdomains for the assembly of the meiotic spindles in both meioticdivisions. It is speculated that one or more of the non-spindle-associatedMTOCs may combine with sperm centrosomal material during fertilizationto create the complete centrosome needed for embryonic mitosis.The widespread distribution of MTOC foci throughout the cortexmay ensure this recombination regardless of the point of spermincorporation into the oocyte. centrosomal domain/meiosis/microtubule organizing centres/oocyte/taxol     

Does ICSI require acrosomal disruption? An ultrastructural study

BACKGROUND: Aggressive immobilization of sperm prior to ICSI significantly improves fertilization rates, but the mechanism of this effect is not yet clear. This study was performed in order to assess the characteristics of mechanically immobilized human sperm by transmission electron microscope (TEM). METHODS: Sperm obtained from ejaculated semen samples from three different donors were immobilized in a standard manner for ICSI. They were then injected into the perivitelline space of mouse oocytes in order to be able to locate them by TEM. Intact motile sperm injected subzonally served as controls (n=160). Finally, the 'carrier' oocytes were fixed and processed for TEM. RESULTS: A total of 300 sperm were mechanically immobilized and inserted into the perivitelline space of mouse oocytes. Ultrathin sections revealed consistent alterations in the acrosomal region including disruption of the plasma membrane, and disruption, vesiculation or even loss of the acrosome. Thus, all of the sperm assessed had undergone some disorganization of the head, in contrast to a majority of control sperm. CONCLUSIONS: Immobilization of sperm for ICSI by compressing and rolling the sperm tails induces a variable disruption and sometimes loss of the acrosome. This could well be a reason for the higher success rates when ICSI is performed using immobilized sperm.     

A detailed cytogenetic analysis of large numbers of fresh and frozen-thawed human sperm after ICSI into mouse oocytes

BACKGROUND: Since information about chromosome aberrations in micro-manipulated sperm is still inadequate, cytogenetic analysis was performed on large numbers of fresh and frozen-thawed (FT) human sperm after injection into mouse oocytes. The effects of the ICSI procedure on oocytes are also discussed based on analysis of the mouse chromosome complements. METHODS: After the injection of fresh and FT human sperm into mouse oocytes, chromosomes of the hybrid oocytes were analysed at first cleavage metaphase. RESULTS: Incidences of the hybrid oocytes at the first cleavage metaphase were significantly different between fresh (71.5%) and FT sperm groups (80.1%) (P < 0.05). The chromosome analysis of 477 fresh and 141 FT sperm showed no difference in the incidences of aneuploidy (1.6/0.7%), structural aberrations (8.8/7.8%) or diploidy (0.0/0.0%) between these categories. The cytogenetic result did not differ from our previous result using IVF between human sperm and hamster oocytes. In an additional cytogenetic study on 615 mouse chromosome complements, the incidence of diploidy (5.4%) was significantly higher than those (0.3-2.8%) in the previous mouse cytogenetic studies, and the hybrid oocytes with no mouse chromosomes (2.0%) existed. CONCLUSIONS: This result suggests that the ICSI procedure induces no sperm chromosome aberrations but increases numerical aberrations in oocyte chromosome complements.     

Different fertilization rates between immotile testicular spermatozoa and immotile ejaculated spermatozoa for ICSI in men with Kartagener's syndrome: case reports

We report two cases of infertility treatment in couples where males suffered from Kartagener's syndrome (KS) and a total absence of motile sperm in the ejaculate. A total of three ICSI cycles was carried out. In all cycles, viable ejaculated or testicular spermatozoa were selected using the hypo-osmotic swelling (HOS) test. Case 1: In the first ICSI cycle total fertilization failure occurred after using ejaculated spermatozoa. In the following cycle testicular spermatozoa were used for ICSI, resulting in 75% fertilized oocytes and a pregnancy. Case 2: In the same ICSI cycle 50% of the oocytes were injected with ejaculated and 50% with testicular spermatozoa. The fertilization rates were 44 and 56% respectively and high quality embryos were achieved in both groups. One single embryo derived from testicular sperm was transferred with a resulting singleton pregnancy. In conclusion, testicular sperm for ICSI seem to have reliable fertilization capacity in men with KS, while ejaculated sperm, even if tested viable, seem more unpredictable. HOS test for selection of viable sperm for ICSI is recommended when ejaculated as well as testicular sperm are used for ICSI.     

Fertilization and embryo development in a mouse ICSI model using human and mouse sperm after immobilization in polyvinylpyrrolidone

BACKGROUND: For human ICSI, sperm are normally immobilized immediately prior to injection. However, there are some situations when only sperm of questionable viability are available. There are few evaluations of fertilization or developmental problems in human or animal models using sperm having known intervals between immobilization and injection. METHODS: Immobilized human sperm were maintained for 1-24 h in 10% polyvinylpyrrolidone (PVP) before injection into mouse oocytes. Mouse sperm heads were similarly maintained in either PVP or a high potassium-containing 'nucleus isolation medium' (NIM) before ICSI and embryo development to the blastocyst stage. RESULTS: Immobilized human sperm activated mouse oocytes comparably to controls even 24 h after immobilization. However, mouse sperm heads showed a decrease in activating ability 6 h after isolation, either in PVP or NIM. A significant reduction in blastocyst development occurred if mouse sperm heads were maintained for even 1 h in PVP. After 6 h, no blastocysts formed, with arrest occurring at the morula stage. NIM provided partial protection for up to 3 h. CONCLUSIONS: Immobilized human sperm maintained oocyte activating activity for 24 h. However, mouse sperm are susceptible to alterations that affect both fertilization and development.