Zona Dissection Research Articles

Effect of IVF and laser zona dissection on DNA methylation pattern of mouse zygotes

In vitro fertilization (IVF) and zona pellucida laser microdissection-facilitated IVF (Laser-IVF) are presently routine procedures in human assisted reproduction. The safety of these methods at the epigenetic level is not fully understood. Studies on mouse Laser-IVF embryos provide evidence that the use of Laser-IVF leads to reduced birth rate, indicating a potential harm of this technique for the embryo. Hence, the aim of this study was to examine the difference in DNA methylation pattern between IVF- and Laser-IVF-derived mouse zygotes. We examined two experimental groups of C3HeB/FeJ oocytes: (1) zona-intact and (2) laser-microdissected oocytes that were fertilized in vitro with freshly collected spermatozoa. Zygotes were fixed 5, 8, and 12 h after fertilization, and indirect immunofluorescence staining was studied using an anti-5-methylcytidine (5-MeC) antibody. The fluorescence intensities of paternal and maternal pronuclei were evaluated using the computer-assisted analysis of digital images. In addition, we performed a semiquantitative RT-PCR analysis of the presence of transcripts of three developmental marker genes, Oct4, Dab2, and Dnmt3b, in IVF- and Laser-IVF-derived blastocysts. We observed no significant differences in methylation status of the paternal genome and in the transcripts of the developmental marker genes after IVF and Laser-IVF. In conclusion, epigenetic patterns and early embryonic development are not altered by laser-assisted IVF techniques and another explanation must be sought for the poor implantation rates observed in mice.

Reduced Oocyte Damage Rates After ICSI With Blunted Needle

Reduced Oocyte Damage Rates After ICSI With Blunted Needle

High survival and developmental rates of vitrified mouse zygotes following polar body biopsy

The effect of two different methods of polar body biopsy followed by vitrification on the survival and development to blastocyst stage of mouse zygotes was examined. Prior to vitrification, a total of 119 and 124 mouse zygotes were subjected to polar body biopsy using either laser-assisted or partial zona dissection (PZD) techniques, respectively. Vitrification was also applied to 122 zona-intact zygotes that served as a control group. Following vitrification, no differences in the rate of zygote survival (95.8%, 91.9% and 94.3%) or in the rate of development to expanded blastocyst stage (82.3%, 79.8% and 82.0%) were observed between the two groups of biopsied zygotes, or between the biopsied zygotes and control zygotes. The mean total number of cells comprising the blastocysts of controls (77.1 ± 4.7) was comparable to the mean cell number recorded in the laser (66.4 ± 4.7) and PZD (69.7 ± 5.3) groups. Blastocysts developed from laser-treated zygotes hatched much earlier than blastocysts developed from the control and PZD groups of zygotes ( P < 0.001). The data obtained in the present study demonstrate that, irrespective of the biopsy method used prior to vitrification, mouse zygotes survive and develop to blastocysts upon warming in proportions similar to those of non-biopsied zygotes.

Beneficial effect of long zona dissection on frozen-thawed blastocysts at a young age.

Aim: To assess the appropriateness of assisted hatching using long zona dissection of human frozen-thawed blastocysts at the time of warming, especially in women over 35years of age or with repeated implantation failures. Methods: Of 177 frozen-thawed blastocyst transfer cycles, 89 control cycles had an intact zona and 88 cycles had assisted hatching using long zona dissection of human thawed blastocyst at the time of warming. These two groups were further subdivided by age to a total of four subgroups: ≤34years (assisted hatching, n=39; controls, n=39) and ≥35years (assisted hatching, n=49; controls, n=50). Twenty-seven cycles in the control group and 28 cycles in the assisted-hatching group had repeated implantation failures. The clinical and ongoing pregnancy rates and the implantation rate between the two groups were analyzed retrospectively. Results: The clinical pregnancy and implantation rates in women ≤34years were significantly higher after the application of assisted hatching compared with the control group (87.2% and 71.2%vs 56.4% and 46.6%, P<0.001). The clinical pregnancy and implantation rates of women with repeated implantation failures were higher after the application of assisted hatching compared with the control group (64.3% and 46.3%vs 48.1% and 34.1%), but this difference was not statistically significant. Conclusions: Routine assisted hatching using long zona dissection at the time of warming on frozen-thawed blastocysts is a safe and easy method to perform and is extremely beneficial for increasing the pregnancy rate in young women ≤34years of age, but not in women ≥35years of age. Despite increased pregnancy and implantation rates in patients with repeated implantation failures, statistical significance was not achieved. (Reprod Med Biol 2007; 6: 211-218).

IMPACT OF RECONSTRUCTION BY GERMINAL VESICLE TRANSFER ON NUCLEAR MATURATION OF DOMESTIC CAT OOCYTES

Cytoplasmic defects as a result of aging or cryopreservation can be circumvented in immature oocytes by transferring the germinal vesicle (GV) into a previously enucleated good quality oocyte. We are interested in oocyte reconstruction by GV transfer in felids to provide options for propagating rare domestic cat biomedical models as well as endangered species. Objectives of this study were to assess the impact of (1) cumulus cell removal, (2) treatments that allow viewing the GV, and (3) enucleation and fusion on subsequent oocyte meiotic resumption. In Experiment 1 (3 replicates), grade I immature cumulus-oocytes complexes (COC) collected from adult cat ovaries were denuded in 0.2% hyaluronidase and matured in vitro (28 h) alone (n = 56 total denuded oocytes; DO) or in co-culture (n = 52 DO with equal number of COC). Nuclear status of DO and intact COC was assessed by fixation in ethanol and Hoechst 33341 staining. The proportions of metaphase II stage (MII) oocytes was less (P < 0.05; ANOVA) when oocytes were denuded and matured alone (23.8 ± 2.7%; Mean ± SD) compared to the co-cultured DO (76.7 ± 4.3%) or intact COC (82.3 ± 3.1%). In Experiment 2 (3 replicates), oocytes were denuded and either treated to visualize the GV (n = 74; incubation in 7.5 μg/ml Cytochalasin B for 15 min at 38.5°C, centrifugation at 15,000 × g for 10 min, and exposure to 0.1 M sucrose for 10 min at 38.5°C) or not treated (n = 75; control). Nuclear status of treated and control DO was assessed after in vitro maturation (IVM) with intact COC (co-culture; ratio 1:1). Percentages of MII were similar (P > 0.05) between DO treated to visualize the GV (79.7 ± 3.5%) and controls (82.7 ± 4.6%). In Experiment 3 (4 replicates), oocytes were denuded and treated as described above. After partial zona dissection of DO and enucleation, GV surrounded by a small amount of cytoplasm (karyoplasts) were fused by two electrical pulses (1.8 kV/cm DC for 30 μs in 0.15 M sorbitol) with the same or different cytoplasts (n = 93 homoplasmic, 91 heteroplasmic reconstructions, respectively). Control oocytes (n = 85) were denuded, treated to visualize the GV and subjected to electric pulses alone. Nuclear status of reconstructed and control oocytes was assessed after IVM with intact COC (1:1). Percentages of fused oocytes after homo- (71.7 ± 6.3%) and heteroplasmic reconstruction (69.0 ± 4.2%) were similar (P > 0.05). The proportion of fused oocytes reaching MII after homo- (46.5 ± 2.2%) versus heteroplasmic (47.7 ± 1.4%) reconstruction also did not differ (P > 0.05), but was less (P < 0.05) than controls subjected to electric pulses alone (80.0 ± 3.5%). This is the first report in carnivores of successful oocyte reconstruction by GV transfer. Results clearly demonstrate that removing cumulus cells is detrimental to nuclear maturation, but can be circumvented by co-culturing DO with intact COC. Furthermore, treatments that allow viewing the GV have no impact on oocyte nuclear maturation. Although enucleation reduces the overall proportion of oocytes able to reach MII, neither electric pulses nor heteroplasmic reconstruction appear to have any influence on GV-cytoplasm interactions during meiotic resumption. (Supported by the National Institutes of Health K01 RR020045). (platform)

Citation classics in Fertility and Sterility, 1975–2004

Citation classics in Fertility and Sterility, 1975–2004

An improved mechanical technique for assisted hatching

Varied clinical outcomes of assisted hatching (AH) have been reported. We attempt to investigate whether the size of the zona opening created by AH is adequate for blastocyst hatching, and, if not, set up a new method to improve it. A new AH technique, long zona dissection (LZD), was established, and experiments were performed to compare the effects of different sizes of zona opening on complete hatching of blastocysts in mouse and human embryos in vitro. The LZD technique can create a long zona slit on early embryos, even blastocysts, with the slit size beyond two-thirds of zona diameter. Compared with three-dimensional partial zona dissection, LZD can significantly enhance the hatching speed and the rate of complete hatching of mouse blastocysts (93.9%). All (100%) human blastocysts completely hatched following LZD; however, when the slit size after AH was about two-fifths of zona diameter, more of the larger inner cell masses (ICM) became trapped by the zona opening during hatching than the smaller ICM (53.3 versus 12.5%, P = 0.01). Zona opening of moderate size following AH is inadequate for the completion of blastocyst hatching in vitro; in some cases, however, it can be significantly improved by LZD.

Monochorionic triplet gestation after in vitro fertilization using donor oocytes: Case report and review

To report on a patient with a monochorionic triamnionic triplet pregnancy after IVF with donor oocytes. Case report. Academic tertiary care hospital. A 42-year-old woman who underwent IVF with donor oocytes. After failed IVF attempts, the patient chose to undergo treatment with donor oocytes. Her 23-year-old oocyte donor underwent standard controlled ovarian hyperstimulation. Retrieved oocytes were fertilized in vitro, and two embryos were transferred at the blastocyst stage. Intrauterine pregnancy with single gestational sac and three fetal poles with cardiac activity. After extensive counseling with perinatologists about pregnancy complications, the patient elected to terminate at 10 weeks of gestation. Several processes have been suggested to explain the increase in monozygotic twinning after IVF. These factors include advanced maternal age, superovulation, manipulation of the zona pellucida, and prolonged culture. It is possible that other factors may also play a role, especially in high-order monozygotic multiple pregnancies. All patients should be informed of the potential risk of a high-order multiple pregnancy after IVF, even when only one or two embryos are transferred.

Developmental potential of embryos after 1 to 3 biopsy procedures for preimplantation genetic diagnosis (PGD)

To study the potential impact of multiple micromanipulation procedures for PGD on embryo development. Retrospective analysis of 701 PGD cycles (7737 oocytes) over a 15-month period with regard to the type and number of biopsy procedures in relation to fertilization and embryo development as compared to 1695 control cycles (17346 oocytes) during that same period. Intracytoplasmic sperm injection (ICSI) was performed on all oocytes in both control (non-tested) and PGD cycles. Cell sampling for PGD was accomplished by creating an opening in the zona pellucida using the mechanical method of partial zona dissection. The first polar body (PB1) was removed prior to ICSI for maternally derived disorders followed by second polar body (PB2) removal through the same opening after fertilization assessment. Both PB1 and PB2 were simultaneously removed following fertilization assessment for aneuploidy testing. Embryo biopsy was performed on day 3 of embryo development after an additional intersecting slit was made forming a flap type opening in the zona pellucida in order to remove a single blastomere. When PGD was performed for mutation analysis in conjunction with aneuploidy testing, as many as three micromanipulation procedures, in addition to ICSI, were performed on the same oocyte/embryo. Fertilization rates, for cases requiring PB1 removal prior to ICSI, and embryo development to the blastocyst stage for all micromanipulation groups were compared with the control group. Both PGD and control groups were further divided by age, patients ≤ to 34 and ≥ to 35 years. Chi-square analysis was used to determine statistical significance. For the group of patients ≤ 34 years, the fertilization rate for oocytes in which PB1 was removed prior to ICSI was 82.2% (499/607) compared to 79.4% (8413/10613) in the control group. No significant difference was found. Fertilization rates for the group of patients ≥ 35 years were as follows: control group 78.3% (5270/6733) vs. 86.5% (499/607) in the group in which PB1 was removed prior to ICSI. A significant difference was seen (p=0.001) between the control group and test group with a higher fertilization rate achieved in the group of oocytes in which PB1 had been removed prior to ICSI. Comparison of blastocysts rates between 1, 2 and 3 biopsy procedures compared to the control group in patients ages ≤ 34 years were 52.9 % (9/17), 55.2%, (374/677), 56.3% (198/352) and 54.3% (4216/7766) respectively. No significant difference was found. Comparison of blastocysts rates between 1, 2 and 3 biopsy procedures and the control group for patients ages ≥ 35 years were 41.0% (16/39), 47.8% (795/1664), 62.1%, (121/195) and 45.7% (1922/4204) respectively. No significant difference was seen with the exception of the group in which three micromanipulations were performed resulting in a higher blastocyst rate when compared to the control group (p=0.001). Multiple micromanipulations, to include ICSI, sequential PB1 and PB2 removal and embryo biopsy on day 3 can be performed on oocytes and subsequent embryos without detriment to embryo development.

Embryo biopsy medium may hold the key to successful outcomes following preimplantation genetic diagnosis

Objective: Embryo biopsy methods, particularly mechanical partial zona dissection (PZD) and acidified Tyrode’s solution (ATS) have been compared by several authors. These two methods are both believed to be similarly efficient and safe in experienced hands. We believe that the “embryo biopsy medium” (EBM) used in this step is just as important as the method employed for zona opening, and its effect on the embryo may be easily overlooked. This retrospective data analysis study compared the pregnancy rate using two kinds of EBM used at our center from 03/01/2002 to 10/31/2003 for aneuploidy screening cases using Fluorescent in Situ Hybridization (FISH).

Advantages of the use of the non-contact laser in the preimplantation genetic diagnosis

Background: Biopsy of single cells from developing embryos is one of the steps of the Preimplantation Genetic Diagnosis (PGD) procedure. Chemical opening by acidified Tyrode’s solution and mechanical zona dissection (PZD) can lack precision and reproducibility because of the different skills among embryologists. The use of a laser in PGD provides a more precise and controlled opening from embryo to embryo and between operators.

Intra-cytoplasmic sperm injection versus conventional techniques for oocyte insemination during in vitro fertilisation in patients with non-male subfertility.

In vitro fertilisation (IVF) as treatment for male factor subfertility is associated with lower fertilisation and pregnancy rates than for other indications. Since the late 1980s several assisted fertilisation techniques have emerged and have been rapidly developed to try to enhance results for couples with male factor subfertility, or to help couples with severe male factor for whom conventional IVF was not possible. The techniques of partial zona dissection (PZD) and of subzonal microinjection of spermatozoa into the perivitelline space (SUZI) are by far surpassed by the technique of intra-cytoplasmatic sperm injection (ICSI). ICSI has proven to be the therapy of choice for couples with severe male factor subfertility. To investigate whether ICSI improves livebirth rate in comparison to IVF in couples with non-male subfertility. We searched the Cochrane Menstrual Disorders and Subfertility Group trials register (searched 30 May 2002), the Cochrane Controlled Trials Register (Cochrane Library Issue 2, 2002), PubMed (January 1992 to July 2002) and reference lists of articles. Trials were included if they compared the effects of these techniques on livebirths, pregnancy and fertilisation outcomes. Only randomised studies were included in this review. One study met the inclusion criteria for this review. The study compared ICSI with IVF within couples with non-male infertility. Data was extracted independently by two reviewers. There were no randomised data comparing livebirth rates. The single identified study did not find a difference in pregnancy rates (OR 1.4, 95% CI 0.95 to 2.2). There were no randomised data on miscarriage rates, or on other adverse events such as congenital malformations that may be of concern. Whether ICSI should be preferred to IVF for cases of non-male factor subfertility remains an open question. Further research should report livebirth rates and adverse events.

Correct coding for laboratory procedures during assisted reproductive technology cycles

Correct coding for laboratory procedures during assisted reproductive technology cycles

The effect of different embryo biopsy procedures for PGD on blastocyst formation and pregnancy

Objective: To study the effect on blastocyst formation and pregnancy rates of zona opening for PGD using three dimensional partial zona dissection or noncontact diode laser. Design: Prospective randomized study. Materials/Methods: A total of 75 IVF/ICSI-PGD cycles undertaken for repeated implantation failure were studied. Following fertilization of the oocytes by IVF or ICSI patients were randomized on the day of PGD to embryo biopsy performed by three dimensional partial zona dissection (n=37) or a 1.48 mu non-contact diode laser (Hamilton-Thorne Instruments)(n=38). After the biopsy the embryos were individually cultured in G2 medium until transfer. Chromosomes 13,16,18,21,22,X,and Y were analysed by multicolor FISH technique for aneuploidy screening. Blastocyst formation from euploid embryos and pregnancy rates after embryo transfer on day 5 were analysed and compared. Results: Mean age, duration of infertility, mean number of previous implantation failures, number of oocytes, 2PN fertilization rate, and cleavage rate were similar between groups. Number of G1+G2 embryos and 8 cell embryos were likewise similar. A total of 139 embryos were biopsied and 129 analysed in the mechanical and 142 embryos were biopsied and 130 analysed in the laser group. Aneuploidy rates were 60.4% and 62.3% respectively (NS). The rate of progression to the blastocyst stage in normal embryos was 54% and 53% in the two groups (NS). Mean number of blastocysts tranferred was 1.5 in the mechanical and 1.2 in the laser group (NS). Implantation and clinical pregnancy rates per embryo transfer were 15.2 and 23.3% in the mechanical and 16.3 and 21% in the laser group (NS). Conclusions: Zona opening for PGD by either three dimensional partial zona dissection or noncontact diode laser have similar impact on blastocyst formation and the occurrence of pregnancy in women undergoing IVF/ICSI for repeated implantation failures. Supported by: None.

Successful use of 1.48-micron laser in human embryo biopsy for preimplantation genetic diagnosis

Objective: To assess the efficacy and safety of human embryo biopsy using a 1.48-micron diode laser for zona pellucida dissection. Design: Case report. Materials/Methods: The biopsy was performed 68 hours post-insemination. A 30–40-micron hole was made in the ZP with a 1.48-micron diode laser (Hamilton Thorne Research, Beverly Massachusetts). Once the hole was created, a 35-micron blastomere biopsy pipette (Humagen) was inserted and a blastomere with a visible nucleus was removed. The blastomeres were placed in lysis-buffer then transported to a reference laboratory for PCR analysis for detection of Spinal Muscular Atrophy (SMA). Results: Patient A had 13 oocytes retrieved with ICSI performed on 10 mature oocytes, resulting in 9–2PN’s. All 9 embryos were biopsied on Day 3 post-retrieval. The PGD results: 6 unaffected, 1 NR (no result), and 2 affected embryos. Three unaffected embryos at the morula/cavitation stage were transferred on Day 5-post retrieval, resulting in a singleton gestation. The patient’s obstetrical course was uncomplicated and a healthy 7lb 5oz infant was delivered vaginally at 37-weeks. Patient B had 20 oocytes retrieved with ICSI performed on 15 mature oocytes, resulting in 13–2PN’s. All 13 embryos were biopsied on Day 3 post-retrieval. The PGD results: 5 unaffected, 6 NR (no result), and 2 affected embryos. Two unaffected embryos at the morula stage were transferred on Day 5-post retrieval with a resultant dizygotic twin gestation. One twin was lost at 10 weeks but the remainder of the obstetrical course was uncomplicated and the patient is now 38-weeks gestation. Conclusions: Many critics have questioned the potential damage to embryos caused by the thermal energy in the vicinity of the embryo at the time of exposure to laser radiation. Our results appear to demonstrate the efficacy and safety of laser assisted embryo biopsy. With the delivery of 1 healthy offspring and 1 uncomplicated (post miscarriage) 38-weeks singleton gestation, thermal energy did not appear to effect embryo development, hatching ability, and implantation. Conventional zona dissection methods require extensive training, physical contact with the embryo, and exposure to acidified media. In contrast, the laser is likely superior because it requires minimal training, no contact with the embryo, and exposure to low pH. Although the diode laser is currently an alternative to conventional zona dissection methods, it may likely supersede all other conventional methods. Supported by: na.

Increase in the monozygotic twinning rate after intracytoplasmic sperm injection and blastocyst stage embryo transfer

Increase in the monozygotic twinning rate after intracytoplasmic sperm injection and blastocyst stage embryo transfer

PGD in female carriers of balanced Robertsonian and reciprocal translocations by first polar body analysis.

Preimplantation genetic diagnosis (PGD) using the first polar body (1PB) is a modality of PGD that can be used when the woman is the carrier of a genetic disease or of a balanced chromosomal reorganization. PGD using 1PB biopsy in carriers of balanced chromosome reorganizations has not become generalized. Here, we describe our experience based on the analysis of unfertilized or fresh, non-inseminated control oocytes, by fixing separately the 1PB and the corresponding oocyte, and on the study of six clinical cases of PGD using 1PB biopsy (four Robertsonian translocations and two reciprocal translocations). In fresh oocytes, the chromosome morphology of the 1PB was well preserved, and the results were always concordant for each oocyte-1PB pair. This indicates that the 1PB can be reliably used for the diagnosis of chromosome reorganizations. In these studies the technical problems encountered when performing PGD using 1PB biopsies for chromosome studies are also addressed. Three different strategies of 1PB biopsy (laser beam, partial zona dissection and acid Tyrode's) and two different protocols (intracytoplasmic sperm injection before or after 1PB biopsy) and their effect on the percentage of oocytes diagnosed and the fertilization rate, are discussed. In reciprocal translocation cases, published in the literature or studied by us, in which at least nine oocytes had been diagnosed, a correlation has been found between the frequency of nondisjunction observed and the theoretical recombination rate. To date, PGD by 1PB analysis alone or combined with blastomere biopsies in female carriers of chromosomal rearrangements has been used in 18 cases, with a further six cases reported here. A total of 325 cumulus-oocyte complexes have been obtained, of which 294 were biopsied and 224 were diagnosed. A total of 52 embryos was transferred, 19 of which implanted and 17 produced full-term pregnancies.

Aspects of biopsy procedures prior to preimplantation genetic diagnosis.

Today, preimplantation genetic diagnosis (PGD) is offered in over 40 centres worldwide for an expanded range of genetic defects causing disease. This very early form of prenatal diagnosis involves the detection of affected embryos by fluorescent in situ hybridization (FISH) (sex determination or chromosomal defects) or by polymerase chain reaction (PCR) (monogenic diseases) prior to implantation. Genetic analysis of the embryos involves the removal of some cellular mass from the embryos (one or two blastomeres at cleavage-stage or some extra-embryonic trophectoderm cells at the blastocyst stage) by means of an embryo biopsy procedure. Genetic analysis can also be performed preconceptionally by removal of the first polar body. However, additional information is then often gained by removal of the second polar body and/or a blastomere from the embryo. Removal of polar bodies or cellular material from embryos requires an opening in the zona pellucida, which can be created in a mechanical way (partial zona dissection) or chemical way (acidic Tyrode's solution). However, the more recent introduction of laser technology has facilitated this step enormously. Different biopsy procedures at different preimplantation stages are reviewed here, including their pros and cons and their clinical applications. The following aspects will also be discussed: safety of zona drilling by laser, use of Ca2+/Mg2+-free medium for decompaction, and removal of one or two cells from cleavage-stage embryos.

Zona dissection by infrared laser: developmental consequences in the mouse, technical considerations, and controlled clinical trial

Infrared laser systems are currently being marketed for application in clinical zona pellucida dissection. However, these systems have undergone only limited animal testing and minor clinical trials that lacked proper controls. Two of these systems have been evaluated in protocols that addressed potential detrimental effects on embryonic development in the mouse. Exaggerated large openings were made in the zona pellucida of 8–16 cell mouse embryos. Embryonic development and subsequent implantation and viability were assessed. A definite negative effect on these parameters was observed following the use of one of these systems. Following this animal trial, the second system was evaluated in a clinical trial for assisted hatching and embryo biopsy. Laser dissection was directly compared with the standard zona drilling. using acidified Tyrode's solution While no significant difference was evident between the two protocols, it was felt that laser dissection presented some problems in both consistency between operators and in the efficacy of subsequent manipulations such as blastomere biopsy and fragment removal. These results argue that laser zona dissection is far from a simple technique and should be carefully evaluated before any clinical application is made.

Intracytoplasmic sperm injection (ICSI)

IVF has become a well-established treatment for certain types of infertility, including long-standing infertility due to tubal disease, endometriosis, unexplained infertility or infertility involving a male factor. Soon it became clear that certain couples with severe male-factor infertility could not be helped by conventional IVF. Extremely low sperm counts, impaired motility and/or abnormal morphology represent the main causes of failed fertilization in conventional IVE In order to tackle this problem, several procedures of assisted fertilization based on micromanipulation of oocytes and spermatozoa have been established. The evolution of these techniques started with partial zona dissection (PZD), followed by subzonal insemination (SUZI) and finally led to the procedure of intracytoplasmic sperm injection (ICSI). ICSI represents the injection of a single spermatozoon directly into the ooplasm, thereby crossing not only the zona pellucida but also the oolemma. In 1992, the first human pregnancies and births after replacement of embryos generated by this novel procedure of assisted fertilization were reported (Palermo et al., 1992). The use of PZD had become controversial and was subsequently abandoned by many workers. Fertilization rates after ICSI were reported to be significantly better than after SUZI (Van Steirteghem et al., 1993a). Moreover, ICSI resulted in the production of more embryos with higher implantation rates, in comparison with SUZI (Van Steirteghem et al., 1993b). As a result, ICSI has been used worldwide and successfully to treat infertility due to impaired testicular function or obstruction of the excretory ducts resulting in severe oligoasthenoteratozoospermia or even azoospermia in the ejaculate. Successful IVF depends on the presence in the ejaculate of a certain number of spermatozoa with good motility and morphology. Riedel et al. (1989) reported minimum andrological requirements for in-vitro fertility by means of conventional IVF: 5 x 106/ml total count, 30% progressive motility and 30% normal morphology. Men with sperm parmeters below these values were considered to have a poor prognosis. Today, however, the most efficient procedure to treat this type of male infertility is ICSI: only one motile (live) spermatozoon is required per mature metaphase II oocyte to be injected. A