Frequent structural chromosome aberrations in immotile human sperm exposed to culture media

BACKGROUND: The influence of culture media or centrifugation on chromosomes of immotile human sperm was examined using ICSI into mouse oocytes. METHODS: In experiment 1, immotile and motile human sperm retrieved directly from ejaculates were injected into mouse oocytes. In experiment 2, immotile human sperm were exposed to seminal plasma or one of four kinds of culture media (HEPES-BWW, modified-BWW, modified-human tubal fluid (HTF) and Dulbecco's phosphate-buffered saline) for 1.5-2.5 h at 18 degrees C in air before microinjection. In experiment 3, immotile human sperm were centrifuged along with HEPES-BWW before microinjection. In experiment 4, frozen-thawed immotile human sperm washed with seminal plasma or HEPES-BWW were injected into mouse oocytes. The hybrid oocytes were prepared for chromosome slides at first cleavage metaphase and were then examined cytogenetically. RESULTS: In experiment 1, there was no significant difference in the incidences of structural chromosome aberrations between motile and immotile sperm (4.3% versus 5.8%). In experiment 2, culture media caused more frequent structural chromosome aberrations (14.3-32.6%) in immotile sperm than did seminal plasma (5.4%). In experiment 3, structural chromosome aberrations were found in 48.1% of the centrifuged immotile sperm, and a live/dead sperm viability test intimated that the aberrant sperm were probably dead. In experiment 4, the incidence of structural chromosome aberrations in frozen-thawed immotile sperm was significantly higher in HEPES-BWW (62.2%) than in seminal plasma (17.2%). CONCLUSIONS: The results indicate that immotile sperm do not have significantly more DNA lesions than motile sperm, although DNA of immotile sperm appears to be vulnerable to damage caused by different culture media.     

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

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

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.     

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

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

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.     

Human fertilization by micro-injection of immotile spermatozoa

Microfertilization of human oocytes with spermatozoa from aman with immotile cilia syndrome is reported, confirming a preliminaryinvestigation where a zona-free donor oocyte was fertilizedwith spermatozoa from the same patient. Oocytes from his spousewere obtained by laparoscopy after routine stimulation withclomiphene citrate, human menopausal and chorionic gonadotrophins,and were cultured for 4–6 h in Whittingham's T 6 medium,supplemented with 10% of her serum. The spermatozoa were washedand processed in the same medium and capacitated for 6–8h before micromanipulation. Three of five mature oocytes werefertilized by micro-injection of a single immotile spermatozooninto the perivitelline space. One oocyte produced a two-pronuclearovum assessed 19 h after injection, while the other two produced2-cell embryos with blastomeres of equal size, 22h after injection.These embryos cleaved to 3–8-cell stages in culture beforeembryo replacement. No pregnancy resulted from embryo transfer.The results conclusively demonstrate that human oocytes canbe fertilized successfully with immotile spermatozoa by micro-injectionand the work has profound implications in the treatment of severemale infertility     

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.     

Chromatin decondensation, pronucleus formation, metaphase entry and chromosome complements of human spermatozoa after intracytoplasmic sperm injection into hamster oocytes

Obtaining karyotypes from human spermatozoa after microinjection into Syrian golden hamster oocytes is difficult and the hitherto reported results are unsatisfactory. This may be related to the injection and culture technique or to the high susceptibility of the hamster oocytes to undergo parthenogenetic activation or both. Therefore, we investigated the hamster oocyte-human sperm microinjection model using the following two approaches: (i) application of contemporary techniques for injection (touching the sperm tail) and culture (hamster embryo culture medium, HECM-3, 10% CO2) and (ii) omission of Ca2+ from the injection medium. Thus, in the first series of experiments, 252 hamster oocytes were injected with human spermatozoa. Among the 219 (87%) oocytes that survived the injection procedure, the mean percentages of male pronucleus formation [two pronuclei (2PN), two polar bodies (PB)], mitotic metaphase entry and sperm chromosome spreads were 41.4, 27.8 and 18.2% respectively. Analysis of the oocytes which failed to develop the male pronucleus following injection revealed that most of them had developed only the hamster female PN while the sperm nuclei were either intact or swollen (partially decondensed), indicating that failure of oocyte activation was not the likely reason for the failure of male PN formation in these oocytes. In the next series of experiments, sibling oocytes were alternately injected with spermatozoa suspended either in the regular (1.9 mM Ca2+) or Ca2+-free injection medium (experiment set 2, n=278). A significant improvement was noted in the mean percentages of oocytes with 2PN, 2PB, metaphase entry and sperm chromosome spreads in the Ca2+-free group versus the regular group (2PN, 2PB: 51 versus 36.6%, metaphase entry: 36.3 versus 26.9% and sperm chromosome spreads: 28 versus 20.4%; all P < 0.04). Thus, parthenogenetic activation appears to be one of the contributing factors for the failure of male PN formation after heterospecific hamster ICSI. From these experiments it can be concluded that application of the advanced injection and culture techniques and omission of Ca2+ from the injection medium are promising for the routine application of the hamster oocyte microinjection for karyotyping of human spermatozoa with poor fertilizing capacity.      

Cytogenetic abnormalities of unfertilized oocytes generated from in- vitro fertilization and intracytoplasmic sperm injection: a double- blind study

In the present study we have assessed the cytogenetic abnormalities of unfertilized oocytes from in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) programmes during a one year period (July 1995 to July 1996) with the cytogenetic analysis being carried out in a double-blind manner. A total of 88 unfertilized ICSI and 85 unfertilized IVF oocytes were used for the study and of these 51 and 62 oocytes, in each respective group, were suitable for analysis. The haploidy, diploidy and aneuploidy rates between ICSI (62.7, 7.8 and 5.9%) and IVF (61.3, 9.7 and 14.5%) groups were similar. A significant inter-patient variation in the incidence of hypohaploidy was observed within the IVF group. Chromosomal fragmentation or breakage was observed at a similar rate in both groups of unfertilized oocytes (23.5 and 14.5% for ICSI and IVF respectively). A significantly higher proportion of ICSI oocytes contained sperm nuclei (27/51, 52.9%) than did IVF oocytes (20/62, 32.3%, P < 0.01). The distribution and state of sperm head chromatin in relation to oocyte chromosomal complement was studied in both groups. ICSI oocytes contained decondensed or swollen sperm nuclei in association with haploid oocyte chromosomes (12/27, 44.4%) or condensed sperm heads in oocytes showing no chromosomal complements (7/27, 25.9%). In IVF oocytes sperm heads were either arrested in the condensed state (5/20, 25%), metaphase stage (3/20, 15%) or had undergone premature chromosome condensation (PCC; 6/20, 30%) in association with haploid oocyte chromosomes. The incidence of PCC was similar in the two groups. A marked variation in the incidence of total chromosomal abnormality was observed between patients within both ICSI (0-75%) and IVF (0-71%) groups indicating a possible similarity in oocyte quality between the majority of male factor and tubal infertility patients. The type of sperm used in the two fertilization procedures showed an increased incidence of chromosomal breakage with ICSI-MESA (microepididymal sperm aspiration) spermatozoa (4/6, 67%) compared to the ICSI-ejaculated (6/35, 17.1%; P < 0.05), ICSI-testicular biopsy (2/10, 20%) and IVF-normospermic (9/62, 14.5%; P < 0.01) spermatozoa. Chromosomal fragmentation may be associated with the degree of difficulty experienced at sperm injection, especially with sperm retrieved from the reproductive tract. Thus chromosomal fragmentation in ICSI may need further investigation using a larger sample size in order to assess the possible causative factors.      

Developmental capacity of damaged spermatozoa.

We report the first detailed and systematic study in a mammalian system to unravel the mystery of the beginnings of life. The fertilizing ability of damaged spermatozoa at various levels of disintegration (cellular and molecular) has been investigated in homologous (mouse) and heterologous (human spermatozoon, hamster oocyte) models. Live pups were produced after destruction of spermatozoa at various cellular and molecular levels followed by injection into oocytes. We demonstrate that with damaged spermatozoa, the key point in the fertilization process is the activation of the oocyte by injection of cytosolic sperm factor. A similar fertilization rate as that using live intact spermatozoa can be achieved following activation. However, the integrity of the genetic material influenced in-vitro development of the embryos and live fetuses. This study contributes to a better understanding of the fertilizing ability of damaged spermatozoa. These findings can be applied clinically to patients with necrozoospermia or very severe oligozoospermia and in wildlife research where damaged spermatozoa from rare species can be used to regenerate young, and hence propagate the species. Also implied is the possible contribution of sperm DNA strand breakage to early pregnancy loss.     

The use of a modified hypo-osmotic swelling test for the selection of viable ejaculated and testicular immotile spermatozoa in ICSI

A modified hypo-osmotic solution was used to select viable ejaculated and testicular spermatozoa to perform intracytoplasmic sperm injection (ICSI) in 27 treatment cycles from patients with total absence of sperm motility. The treatment cycles consisted of 15 cycles in which ejaculated spermatozoa were used and 12 cycles in which testicular spermatozoa were used. The hypo-osmotic solution consisted of 50% culture medium and 50% deionized water and was shown in previous in-vitro studies to be superior to the original solution used in the classical hypo-osmotic swelling test. Fertilization was achieved in 37.3% of the oocytes injected. Embryos were replaced in 70.4% of the cycles with a mean of 2.0 embryos per cycle. There were no statistically significant differences between the ejaculated sperm group and the testicular sperm group in the fertilization rate (42.7 versus 30.1%) or in the cleavage rate (92.7 versus 77.3%). Four pregnancies resulted, two in the ejaculated sperm group and two in the testicular sperm group, a pregnancy rate of 14.8%. All pregnancies were singletons but one pregnancy in each group had an early miscarriage. There were no statistically significant differences between both groups in the pregnancy rates (13.3 versus 16.7%), in the implantation rates (5.3 versus 11.8%) or in the delivery/ongoing pregnancy rates (6.7 versus 8.3%). It is concluded that the use of this solution to select viable but immotile spermatozoa for ICSI is a simple and practical method and is associated with acceptable fertilization and pregnancy rates.     

Successful intracytoplasmic sperm injection with spermatozoa from a patient with dysplasia of the fibrous sheath and chronic respiratory disease

The present report describes a successful intracytoplasmic sperm injection (ICSI) procedure performed with immotile spermatozoa from a young man with a combination of dysplasia of the fibrous sheath and dynein deficiency, a recently described variant of the immotile cilia syndrome. This methodology provides the only suitable solution for these patients in whom all other assisted fertilization technologies have previously failed, and opens the possibilities for treatment of male infertility due to severe, irreversible sperm defects such as the one reported here.      

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.     

Patients with absolutely immotile spermatozoa and intracytoplasmic sperm injection

The microinjection of completely immotile spermatozoa may impair the outcome of intracytoplasmic sperm injection (ICSI). Eleven couples underwent an initial ICSI cycle with 100% immotile freshly ejaculated spermatozoa. Two-pronuclear fertilization ensued in 18 of 145 (12.4%) successfully injected oocytes. None of these cycles resulted in a pregnancy. Nine couples underwent ICSI in subsequent cycles (n = 16). Ejaculated spermatozoa were injected in 15 cycles and testicular spermatozoa in one cycle. In 10 of the 15 cycles, motile spermatozoa were available at the time of injection. Motile testicular spermatozoa could also be injected. In the subsequent cycles, 91 of 176 (51.7%) successfully injected oocytes fertilized normally and four patients became pregnant. In the subsequent cycles where again immotile spermatozoa had to be injected no pregnancies occurred. In four subsequent cycles embryo cryopreservation was carried out. After replacement of two frozen-thawed embryos one additional pregnancy was obtained. In all, five healthy infants were born. It has been ascertained that motile spermatozoa can be detected either in repeated ejaculates or after testicular biopsy. The causes of total asthenozoospermia are variable and the problem is a sporadic rather than a permanent condition.      

Successful fertilization and pregnancy following ICSI and electrical oocyte activation.

In a total of 1048 intracytoplasmic sperm injection (ICSI) cycles, motile spermatozoa from four out of 424 patients (0.9%) failed to fertilize oocytes, despite an apparently successful ICSI procedure. No activation was observed in these injected oocytes. The spermatozoa from three of the four patients were injected into unfertilized mouse oocytes by ICSI (mouse test) to evaluate their oocyte activating ability. The oocyte activation rate of the spermatozoa of patients A, B, and C in the mouse test was 46, 100, and 86% respectively (control: 100%). Simultaneous injection of two spermatozoa from patient A into the mouse oocytes increased the oocyte activating rate to 89% (sham control: 29%). 100% fertilization rates were obtained for patients A and B by combining ICSI and electrical stimulation, and this resulted in pregnancy and the birth of healthy twins for the partner of patient A. Thus, it is considered that the spermatozoa of these patients are not lacking sperm factors but that the activity of these factors is depressed. The combination of ICSI and electrical stimulation is effective in these cases.     

Human sperm cytosolic factor triggers Ca2+ oscillations and overcomes activation failure of mammalian oocytes

Among the possible mechanisms of oocyte activation after sperm penetration, it appears most likely that a protein released by the spermatozoon elicits a calcium elevation in the ooplasm. To further test this idea, cytosolic factors obtained from human spermatozoa by two different methods, freezing-thawing and sonication, were injected into mouse oocytes following which intracellular calcium release was measured. Of a total of 42 mouse oocytes, a pattern of calcium oscillations was observed in nine out of 16 oocytes injected with sonicated fraction, in all of eight oocytes with the frozen-thawed fraction and in none of 18 control oocytes. Injection of the frozen- thawed fraction also produced regular calcium oscillations in all of five in-vitro matured human oocytes. To assess the putative factor's ability to support fertilization, human oocytes that were not activated by prior intracytoplasmic injection of spermatozoa (ICSI) and round spermatids were reinjected with the frozen-thawed sperm fraction. Of 23 human oocytes which remained unfertilized after ICSI, 19 became activated after injection with sperm cytosolic factor; eight showed two pronuclei, three one pronucleus and eight showed three or more pronuclei. Of 11 oocytes unfertilized after prior round spermatid injection, two developed two pronuclei, four developed one pronucleus and two had three or more pronuclei. Cytogenetic analysis by fluorescence in-situ hybridization confirmed the existence of a male pronucleus in eight out of nine such zygotes displaying two or more pronuclei. Thus, human sperm extracts activated mouse and human oocytes after injection, as judged by calcium flux patterns in conjunction with male pronucleus formation.      

Epigenetic analysis of human spermatozoa after their injection into ovulated mouse oocytes

BACKGROUND: The epigenetic status of human spermatozoa is difficult to analyse. The method of interspecies fertilization can be used for different purposes. The aim of our work was to adopt this approach for the detailed analysis of epigenetic status of human spermatozoa injected into mouse oocytes. METHODS: Human spermatozoa were injected into ovulated mouse oocytes. When both parental pronuclei were formed, the zygotes were fixed and labeled with antibodies against histones methylated or acetylated at different positions (residues). RESULTS: Our results show that human spermatozoa injected into mouse oocytes fully respond to oocyte cytoplasmic factors and form analysable pronuclei. The labeling of zygotes showed that as in other species, the paternal chromatin is extensively epigenetically remodeled. CONCLUSIONS: The interspecies ICSI may be a powerful tool for the analysis of sperm epigenetic status even with a very low number of spermatozoa available. This analysis could be used as an additional approach for the assessment of certain forms of human infertility, as well as for testing the normality of male gametes obtained from embryonic stem cells.     

Andrology: Fertilizing ability of immotile spermatozoa after intracytoplasmic sperm injection

Sometimes spermatozoa from ejaculate, epididymis or testis showa total absence of motility. For some patients, however, veryfew spermatozoa with very poor motility can be found after severalhours of incubation (initially immotile spermatozoa). Othersamples show no motility at all even after extended culture(totally immotile spermatozoa). Intracytoplasmic sperm injection(ICSI) is the only method available to select and retrieve asingle immotile or initially immotile spermatozoon and injectit into the oocyte. A total of 103 patients with asthenozoospermiaunderwent ICSI in this study. It was shown that initially immotileand totally immotile spermatozoa, whatever their origin, havethe capacity to fertilize an oocyte after ICSI. No significantdifference could be observed between the fertilizing capacityof testicular or epididymal spermatozoa. Totally immotile ejaculatedspermatozoa, however, fertilized significantly fewer oocytesafter ICSI when compared with initially immotile ejaculatedspermatozoa. Embryos of lower quality tended to be producedwhen totally immotile spermatozoa of any origin were used, comparedwith embryos resulting from initially immotile spermatozoa.Ongoing pregnancies were conceived after ICSI with initiallyimmotile spermatozoa from any origin and totally immotile spermatozoaretrieved from testis only. One biochemical pregnancy was theresult of embryo transfer after ICSI with totally immotile ejaculatedspermatozoa. No supernumerary embryos could be cryo-preservedfor patients with totally immotile spermatozoa from ejaculateor epididymis. For a Kartagener patient, subzonal insemination(SUZI) seemed to be a better approach for obtaining fertilizationand pregnancy than ICSI because no fertilization occurred afterICSI on sibling oocytes. Hence a healthy pregnancy was obtainedafter SUZI.     

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.