Successful Intracytoplasmic Sperm Injection Research Articles

Intracytoplasmic sperm injection in the bovine induces abnormal [Ca2+]i responses and oocyte activation

Fertilisation by intracytoplasmic sperm injection (ICSI), a technique that bypasses the membrane fusion of the gametes, has been widely used to produce offspring in humans and mice. Success with this technique has lent support to the hypothesis that in mammalian fertilisation, a factor from the sperm, the so-called sperm factor, is responsible for oocyte activation and that the fusion process is not involved in the generation of the hallmark [Ca2+]i signalling seen following fertilisation. However, the success of ICSI has largely eluded large domestic species, such as the bovine, porcine and equine, casting doubt on the current model of oocyte activation at fertilisation in these species. Using Ca2+ imagery and a series of treatments to manipulate the chemical structure of the sperm, we have investigated the early events of oocyte activation in response to ICSI in the bovine. Our results demonstrate, for the first time, that following ICSI, the majority of bovine oocytes are unable to mount [Ca2+]i oscillations, although, in few cases, the initiation of [Ca2+]i oscillations can occur in a manner indistinguishable from in vitro fertilisation. We also show that bull sperm possess a full complement of sperm factor. However, either the release and/or activation of the sperm factor are compromised after ICSI, leading to the delivery of a defective Ca2+ stimulus, which results in premature termination of embryo development.

The Current Social Status of Embryologists

With the rapid development of advanced reproductive technology (ART), embryologists have risen in stature. The basic ART techniques were promoted in Japan by a number of physicians who studied this new technology abroad; however, since the first successful intracytoplasmic sperm injection in 1992, the performance of these techniques abruptly changed from physicians to embryologists. In Japan, although we recognize that embryologists are essential members of an ART team, their aspects of background is limited. Therefore, here is described some states for embryologists in regard to factors such as regulation, certification, and salary.

The Impact of Motile Sperm Concentration on the Outcome of ICSI in Natural Cycles

Efficacy of intracytoplasmic sperm injection (ICSI) in natural cycles has been demonstrated. In the literature we found no study evaluating the success of ICSI in natural cycle according to sperm quality and different indications for ICSI. The aim of our study was to establish the impact of motile sperm concentration on the outcome of natural ICSI cycle.

Success of Testicular Sperm Injection and Intracytoplasmic Sperm Injection in Men with Klinefelter Syndrome

The aim of this study was to report the successful fertility treatment of men with Klinefelter syndrome using testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). A total of 42 men with Klinefelter syndrome who underwent 54 TESE procedures were identified. Before TESE, patients with serum testosterone levels less than 15.6 nmol/liter were treated with an aromatase inhibitor. Sperm retrieval rates and results of ICSI, including fertilization and clinical pregnancy, were collected. Mean pretreatment FSH and testosterone levels were 33.2 IU/liter and 9.8 nmol/liter. During medical therapy, the mean testosterone level rose to 17.0 nmol/liter (P < 0.01). Spermatozoa were found during 39 microdissection TESE procedures, on the day before, or day of oocyte retrieval during a programmed in vitro fertilization cycle. The sperm retrieval rate was 72% (39 of 54) per TESE attempt, and 29 of the 42 different men (69%) had adequate sperm found for ICSI. Thirty-three in vitro fertilization cycles yielded embryos for transfer in the 39 (85%) cycles with sperm retrieved. Eighteen clinical pregnancies have resulted in 21 live births [18 of 39 (46%)]. All children had a normal karyotype. TESE/ICSI is a successful intervention for the majority of patients with azoospermia and Klinefelter syndrome. Sperm retrieval and ICSI success in men with Klinefelter syndrome are comparable with other men with nonobstructive azoospermia treated at our center.

Use of a laser to detect viable but immotile spermatozoa

A prerequisite for the success of intracytoplasmic sperm injection (ICSI) is the use of viable sperm that will support oocyte activation and embryo development. Usually, spermatozoa are selected according to motility, which is a clear indication for viability. However, in cases with complete asthenozoospermia, it is impossible to select viable sperm by morphological means. Here we report for the first time on the use of a laser to discriminate between viable and dead immotile spermatozoa. The application of a single laser shot to the far end of the sperm tail causes a curling of the sperm tail only in viable sperm, similar to the reaction observed in the hypo-osmotic swelling test. Using this method for sperm selection prior to ICSI we achieved high fertilization and cleavage rates in cases with immotile sperm in fresh testicular biopsy material as well in cases with ejaculated immotile sperm. It is concluded that laser sperm interaction allows for the identification of viable but immotile spermatozoa.

Intra-cytoplasmic sperm injection in a marsupial

Here we report the first use of intra-cytoplasmic sperm injection (ICSI) in a marsupial, the tammar wallaby (Macropus eugenii), to achieve in vitro fertilization and cleavage. A single epididymal spermatozoon was injected into the cytoplasm of each mature oocyte collected from Graafian follicles or from the oviduct within hours of ovulation. The day after sperm injection, oocytes were assessed for the presence of pronuclei and polar body extrusion and in vitro development was monitored for up to 4 days. After ICSI, three of four (75%) follicular and four of eight (50%) tubal oocytes underwent cleavage. The cleavage pattern was similar to that previously reported for in vivo fertilized oocytes placed in culture, where development also halted at the 4- to 8-cell stage. One-third of injected oocytes completed the second cleavage division, but only a single embryo reached the 8-cell stage. The success of ICSI in the tammar wallaby provided an opportunity to examine the influence of the mucoid coat that is deposited around oocytes passing through the oviduct after fertilization. The presence of a mucoid coat in tubal oocytes did not prevent fertilization by ICSI and the oocytes cleaved in vitro to a similar stage as follicular oocytes lacking a mucoid coat. Cell-zona and cell-cell adhesion occurred in embryos from follicular oocytes, suggesting that the mucoid coat is not essential for these processes. However, blastomeres were more closely apposed in embryos from tubal oocytes and cell-cell adhesion was more pronounced, indicating that the mucoid coat may be involved in maintaining the integrity of the conceptus during cleavage.

Timing of MAP kinase inactivation effects on emission of polar body in porcine oocytes activated by Ca2+ ionophore

Artificial activation is required for successful intracytoplasmic sperm injection (ICSI) to induce haploidy pronuclear formation with extraction of second polar body. The present study showed that an additional treatment with Phorbol 12-myristate 13-acetate (PMA) followed by Ca(2+) ionophore treatment improved the rate of pronuclear formation, however, these oocytes had more than two pronuclei because of the suppression of polar body emission. The cultivation with MEK inhibitor U0126 followed by Ca(2+) ionophore also increased the rate of pronuclear formation but suppressed the emission of second polar body. These results suggested that the decrease of MAP kinase activity at early stage of artificial activation, concomitantly with decreasing p34(cdc2) kinase activity, prevented the second polar body extraction. We investigated that the timing of MAP kinase inactivation affected the extraction of the polar body and pronuclear formation rate. The addition of PMA 8 hr after Ca(2+) ionophore treatment induced the delay of MAP kinase inactivation, which resulted in haploidy pronuclear formation with emission of polar body. These results demonstrated for the first time that the delay of MAP kinase inactivation induced by PMA improved pronuclear formation with the extraction of second polar body in porcine oocytes activated by Ca(2+) ionophore. This method can be available for successfully ICSI in low response species of oocyte activation to Ca(2+) ionophore including pig.

Time- and media-dependent improvements in motility of testicular spermatozoa retrieved from non-obstructive azoospermic men

Testicular-derived spermatozoa motility, reflecting viability, is key to intracytoplasmic sperm injection success. Experiments were conducted to compare number of motile sperm isolated from testicular aspirations incubated in different media over time.

Effect of time since vasectomy and maternal age on intracytoplasmic sperm injection success in men with obstructive azoospermia after vasectomy

Objective To determine the effects of time since vasectomy and maternal age on intracytoplasmic sperm injection (ICSI) outcome in azoospermic men after vasectomy. Design Retrospective analysis. Setting Assisted conception unit. Patient(s) Thirty-seven azoospermic men (after vasectomy) who were undergoing 56 cycles of ICSI. Intervention(s) Surgical sperm retrieval and standard ICSI protocol. The ICSI cycles were analyzed in four groups, according to years since vasectomy, and were reanalyzed in three groups, according to maternal age. Main outcome measure(s) Fertilization rate, implantation rate, clinical pregnancy rate, and live-birth rate (LBR) per ET. Result(s) No effect of time since vasectomy was seen on any outcome. The highest fertilization rate and LBR were found in the group with the longest time interval. These findings could not be explained by differences in either patient characteristics or stimulation regimes. When reanalyzed by maternal age, there was an improvement in implantation rate and LBR with decreasing maternal age. Live birth rates of 38.5%, 22.7%, and 11.8% were achieved for maternal ages of <32, 32–37, and >38 years, respectively. Logistic regression confirmed a statistically significant effect on outcome of maternal age but not time since vasectomy. Conclusion(s) Our data suggest that maternal age, and not interval since vasectomy, remains the principal determinant of ICSI success in men with obstructive azoospermia after vasectomy.

Removal of endometriomas before in vitro fertilization does not improve fertility outcomes: a matched, case–control study

Objective To investigate whether conservative surgery on ovarian endometriomas before an IVF cycle improves fertility outcomes. Design Retrospective, matched case–control study. Setting Two academic IVF programs. Patient(s) One hundred eighty-nine women with endometriomas who underwent IVF treatment: 56 women proceeded directly to IVF, and 133 first underwent conservative ovarian surgery. Intervention(s) Controlled ovarian hyperstimulation and IVF-ET. Main outcome measure(s) Response to gonadotropins, fertilization, implantation, and pregnancy rates. Result(s) Aside from lower peak E 2 levels on the day of hCG and a higher total FSH dose in women previously operated for an endometrioma, no significant differences were found between the two groups in the different IVF variables analyzed. Conclusion(s) Laparoscopic cystectomy for endometriomas before commencing an IVF cycle does not improve fertility outcomes. Proceeding directly to controlled ovarian hyperstimulation in women with asymptomatic ovarian endometriomas might reduce the time to pregnancy, the costs of treatment, and the hypothetical complications of laparoscopic surgery. Conversely, conservative surgical treatment of ovarian endometriomas in symptomatic women does not impair IVF or intracytoplasmic sperm injection success rates.

Successful ICSI depends on DNA stability and disulfide status of sperm nuclei

Relation of successful ICSI (intracytoplasmic sperm injection) with DNA stability and thiol-disulfide status of rat spermatozoa nuclei was investigated. Testicular, caput and cauda epididymal spermatozoa have been used for this research. The nuclei of these spermatozoa were stained with aeridine orange for DNA stability test and labeled with mBBr for thiol-disulfide status before injesting into oocytes. Cauda epididymal spermatozoa were very stable and contained high disulfide than caput epididymal and testicular spermatozoa. Results from spermatozoa injection into oocytes show that they were capable to fertilize oocytes. However, testicular spermatozoa can not develop up to blastocyst stage. These results indicated that successful ICSI at least in the rat depends on DNA stability and disulfide status of sperm nuclei. Key words: ICSI, DNA stability, disulfide status, acridine orange, mBBr

Pregnancy after ICSI with ejaculated immotile spermatozoa from a patient with immotile cilia syndrome: a case report and review of the literature

This study presents a case of intracytoplasmic sperm injection (ICSI) with ejaculated immotile spermatozoa from a patient with immotile cilia syndrome. Semen analysis of the patient suffering from immotile cilia syndrome revealed an extreme oligoasthenoteratozoospermia (OAT: count <1.4 × 10 6/ml, 0% motility and 3% normal morphology). Electron microscopy of sperm flagella showed the absence of inner and outer dynein arms. During the ICSI cycle, the hypo-osmotic swelling test (HOS) was used for the identification of viable spermatozoa in the pool of immotile spermatozoa for ICSI. A normal fertilization rate was found in eight out of the 12 oocytes. A first fresh double embryo transfer resulted in a late miscarriage at 21 weeks. A second healthy singleton pregnancy occurred after transfer of two frozen–thawed embryos from the same ICSI procedure. Although only one successful ICSI case of the immotile cilia syndrome combined with HOS is described here, HOS might be a simple but valuable tool to obtain normal fertilization and pregnancy for patients suffering from immotile spermatozoa.

Patterns of intracellular calcium oscillations in horse oocytes fertilized by intracytoplasmic sperm injection: possible explanations for the low success of this assisted reproduction technique in the horse.

In all species studied, fertilization induces intracellular Ca2+ ([Ca2+]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca2+]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single motile stallion sperm. Few oocytes displayed [Ca2+]i responses regardless of oocyte source and we hypothesized that this may result from insufficient release of the sperm-borne active molecule (sperm factor) into the oocyte. However, permeabilization of sperm membranes with Triton-X or by sonication did not alleviate the deficient [Ca2+]i responses in mare oocytes. Thus, we hypothesized that a step downstream of release, possibly required for sperm factor function, is not appropriately accomplished in horse oocytes. To test this, ICSI-fertilized horse oocytes were fused to unfertilized mouse oocytes, which are known to respond with [Ca2+]i oscillations to injection of stallion sperm, and [Ca2+]i monitoring was performed. Such pairs consistently displayed [Ca2+]i responses demonstrating that the sperm factor is appropriately released into the ooplasm of horse oocytes, but that these are unable to activate and/or provide the appropriate substrate that is required for the sperm factor delivered by ICSI to initiate oscillations. These findings may have implications to improve the success of ICSI in the equine and other livestock species.

Fertilization following intracytoplasmic sperm injection of in vivo and in vitro matured oocytes from an Australian marsupial, the tammar wallaby (Macropus eugenii).

Conventional in vitro fertilization (IVF) techniques have been unable to produce normal embryos in any Australian marsupial, largely owing to problems with the early stages of sperm-oocyte binding. This study has used intracytoplasmic sperm injection (ICSI) of in vivo and in vitro matured tammar wallaby oocytes to bypass these processes and achieve fertilization in vitro. The fertilization rate (i.e. development to the two-pronuclei stage) of in vivo and in vitro matured oocytes following ICSI and sham injection was assessed at 17-19 h after injection. Fertilization occurred in 48% (45/93) of in vivo matured oocytes that were injected with spermatozoa. Significantly fewer sham-injected oocytes (6/82, P < 0.005) and uninjected control oocytes (5/84, P < 0.005) formed two pronuclei. In a direct comparison, the numbers of in vivo and in vitro matured oocytes that formed two pronuclei after ICSI were 22/28 (78.6%) and 23/40 (57.6%), respectively, which are not significantly different. There was also no significant difference in the nuclear response of in vivo and in vitro matured oocytes to sham injection. The numbers of oocytes forming a single pronucleus after sham injection were 10/24 (41.7%) and 24/37 (64.9) for in vivo and in vitro matured oocytes, respectively. Immature germinal-vesicle-stage oocytes were unable to decondense sperm injected during ICSI or to form pronuclei. These results demonstrate that both in vitro and in vivo matured tammar wallaby oocytes can be fertilized by ICSI. The success of ICSI not only offers the opportunity for fundamental analysis of marsupial fertilization but could, in conjunction with development of appropriate culture conditions and embryo transfer technologies, contribute to increased production of offspring from rare or valuable marsupials.

Successful intracytoplasmic sperm injection after failed in vitro fertilization due to multipronuclear oocytes

Objective To report a case of IVF with apparently normal female and male gametes that resulted in the development of only multipronuclear oocytes (instead of oocytes containing two pronuclei) and was successfully treated with ICSI. Design Case report. Setting A university hospital. Patient(s) A 35-year-old woman with unexplained infertility and her partner, a 38-year-old man with normozoospermia. Intervention(s) Intracytoplasmic sperm injection. Main outcome measure(s) Oocyte fertilization and pregnancy. Result(s) Normal fertilization and an ongoing pregnancy occurred after transfer of two embryos. Conclusion(s) Although the usual indication for ICSI is male subfertility, it can also be used to successfully treat oocyte defects.

Male smokers have a decreased success rate for in vitro fertilization and intracytoplasmic sperm injection

Objective Smoking by one or both partners can adversely affect IVF outcome. We investigated whether smoking may also play a role in the success rate of intracytoplasmic sperm injection (ICSI), in which initial steps of fertilization are bypassed. Design Three hundred one couples (ICSI: 153, IVF: 148) participated in 415 treatment cycles (ICSI: 202, IVF: 213). One hundred thirty-nine men were habitual smokers (ICSI: 71, IVF: 68). Seventy-seven women were smokers (ICSI: 41, IVF: 36). Multiple nominal regression analyses of various steps of assisted reproduction included smoking status, age, semen parameters, and number of embryos transferred. Setting Reproductive and andrology unit of the university. Patient(s) Three hundred one couples seeking fertility treatment. Intervention(s) Assisted reproduction by in vitro fertilization (IVF) or ICSI. Main outcome measure(s) Clinical pregnancy. Result(s) Intracytoplasmic sperm injection success (clinical pregnancy) in women with smoking male partners was 22% and was 38% with nonsmoking partners. Similar results were seen for IVF, with 18% vs. 32%. Multinominal logistic regression analysis revealed smoking in men to be a significant predictor of ICSI outcome, along with female age and the number of embryos transferred, whereas clinical pregnancies after IVF were dependent on smoking in men, number of embryos transferred, sperm motility, and female age. Female smoking influenced the number of oocytes retrieved and the fertilization rate of oocytes in IVF but not in ICSI. The odds ratio for failure of ICSI for male smokers in comparison to male nonsmokers was 2.95 (IVF: 2.65). Conclusion(s) Smoking by males decreases the success rates of assisted reproduction procedures, not only in IVF, but also in ICSI. Apart from putative adverse effects during fertilization, altered DNA in spermatozoa might hamper development of the embryo.

Successful ICSI in a case of severe asthenozoospermia due to 93% non-specific axonemal alterations and 90% abnormal or absent mitochondrial sheaths

The use of IVF and intracytoplasmic sperm injection (ICSI) to treat male infertility due to severe asthenozoospermia in cases of electron microscopically confirmed axonemal abnormalities has been reported. However, even with these sophisticated technologies, very few pregnancies and births have been achieved with ejaculated spermatozoa in infertility due to this condition. The existence of sperm-derived defects preclude normal embryo development, and another reason for low success rates may be that the most commonly used method for selecting viable spermatozoa for ICSI is sperm motility, which is absent in affected men. Consequently, the likelihood of selecting non-viable spermatozoa for ICSI is higher in these cases. This report describes a case of a pregnancy achieved by ICSI with ejaculated spermatozoa containing 93% non-specific axonemal alterations and 90% abnormal or absent mitochondrial sheaths. A total of 14 oocytes was obtained from the 31-year-old patient in her first IVF treatment cycle. Three of the 13 matured (metaphase II) oocytes were inseminated conventionally, and the other 10 metaphase II oocytes were submitted to ICSI. None of the oocytes inseminated conventionally were fertilized. Of the 10 oocytes submitted to ICSI, four zygotes and cleavage embryos resulted. Three embryos were transferred on day 3, and a successful pregnancy was achieved. One gestational sac, together with regular heart activity, was recorded by ultrasonography at 8 weeks of gestation. A pregnancy is currently ongoing.

Importance of mitochondrial and nuclear sperm DNA in sperm quality assessment and assisted reproduction outcome

Intracytoplasmic sperm injection (ICSI) has made irrelevant the conventional criteria of concentration, motility and morphology for assessment of sperm quality and so we urgently need new assays by which to gauge sperm ‘health’. ICSI may be facilitating the transfer of genetic disorders to future generations by bypassing all the natural hurdles for sperm selection without imposing more pertinent criteria of selection. Sperm DNA quality is vital to the future offspring irrespective of whether the child is conceived naturally, by in vitro fertilization (IVF) or by ICSI. The DNA integrity of sperm can be determined quickly and accurately using a range of techniques that also have strong prognostic power in predicting successful IVF and ICSI outcomes with ejaculated sperm. Moreover, there is a close correlation between testicular nuclear DNA integrity and pregnancy rates in ICSI. Mitochondrial DNA can be measured using long PCR in ejaculated and testicular sperm and is also useful for predicting success in assisted conception. This review discusses how the integrity of both nuclear and mitochondrial affect the choice of sperm for assisted conception.

Successful intra-cytoplasmic sperm injection treatments in identical twin brothers with severe oligozoospermia

Successful intra-cytoplasmic sperm injection treatments in identical twin brothers with severe oligozoospermia

Effect of injected spermatozoa morphology on the outcome of intracytoplasmic sperm injection in humans

Effect of injected spermatozoa morphology on the outcome of intracytoplasmic sperm injection in humans