[Assisted hatching of in-vitro fertilized human embryos by micromanipulation of the zona pelucida].
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Animals↗
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[Pregnancy by micromanipulation in a case of excretory azoospermia associated with a natural spermatocele].
Microinjection of capacitated sperm into the perivitelline space of oocytes has been performed in a case of male infertility attributed to excretory azoospermia. In this particular case, a spermatocele detected five years ago was observed, which provide motile spermatozoa. These sperm collected by direct puncture into the cyst, were capacitated using discontinuous Percoll gradient. After ovarian stimulation, seventeen oocytes were collected. Nine of them were used for classical IVF whereas the other eight were submitted to microinjection. Only one oocyte of the second group fertilized and cleaved 48 hours later. A clinical pregnancy was achieved after embryo transfer and a normal, healthy boy delivered at term.
Adult↗
[How good is spatial orientation with rigid endoscopic optics? Study of micromanipulation in the nasal cavity].
Rigid endoscopes are used as an alternative to the operating microscope for visual control during paranasal sinus surgery. Since the view through the endoscope is monocular, perception of depth (stereopsis) through the operating microscope must be expected to be superior due to the binocular view afforded. To investigate whether monocular visualization through an endoscope is a disadvantage in paranasal sinus surgery, we compared stereoacuity in a model of the nasal cavity, using a head lamp, an operating microscope and a 0 degree Hopkin's endoscope. Twenty volunteers had to touch defined points in a spatial model of the nasal cavity. Under binocular vision with a head lamp, performance was significantly better than with the microscope or the endoscope. Manipulations were performed with the same speed under microscopic and endoscopic control, although under microscopic guidance more faults in point sequence were made than with the endoscope. Various monocular phenomena seem to allow sufficient spatial orientation through the endoscope, so that monocular visualization does not appear to be a disadvantage for quick and safe manipulations during functional endoscopic sinus surgery.
Depth Perception↗
Assisted hatching of embryos by micromanipulation for human in vitro fertilization: UAMS experience.
In vitro fertilization and embryo transfer (IVF) is utilized as a treatment for infertile couples who cannot conceive with standard therapy. Assisted hatching (AH) is a procedure whereby an opening is made in the zona pellucida of the embryos, thereby increasing the probability of implantation and pregnancy. AH is beneficial in patients with elevated FSH levels, older than age 38 or those who failed IVF repeatedly. Success rates after IVF with AH at the University of Arkansas for Medical Sciences (UAMS) compares favorably with rates achieved by other centers in the USA. Pregnancy rates after IVF with AH in patients older than 38 years is approximately 20% compared to a pregnancy rate of 10% in patients who did not have AH. This report summarizes the UAMS experience with IVF and AH.
Adult↗
[A micromanipulator for two-channel recording of the neuronal activity with high-impedance electrodes in freely moving animals].
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Animals↗
Strategies for the isolation and detection of fetal cells in transcervical samples.
OBJECTIVE: The aim of the study was to evaluate the detection of fetal cells from transcervical samples, collected in early pregnancy, by means of different molecular techniques. The value of the isolation of trophoblasts using an inverted microscope, also referred to as micromanipulation, is discussed. METHODS: All the 89 specimens were obtained by intrauterine lavages before termination of pregnancy (TOP), between 7 and 12 weeks of gestation. Micromanipulation was carried out in a subgroup of 57 for the isolation of fetal material. Fetal sexing was achieved by FISH (fluorescent in situ hybridisation) using fluorescently labelled probes for X and Y chromosomes and by polymerase chain reaction (PCR). Male samples were also investigated for aneuploidy of the chromosome 21. Quantitative fluorescent (QF)-PCR using two short tandem repeat (STR) markers for chromosome 21 was carried out in 26 micromanipulated samples. RESULTS: FISH analysis revealed that 45/89 placental samples derived from pregnancies with male fetuses. Correct sexing of the lavage samples from male pregnancies was achieved in 41/45 (91%) using dual-FISH technique, and in 43/45 (95.5%) with PCR. All the samples derived from male pregnancies tested for chromosome 21 were normal. From 57 samples subjected to micromanipulation, 51 (89.5%) showed discernible chorionic villous filaments or cell clumps of possible trophoblastic origin. One case of tetraploidy and two cases of monosomy were recorded. The rate of fetal cells, in the non-micromanipulated samples, was between 4% and 97% (mean 54.3%). In micromanipulated specimens, maternal contaminant cells were absent or extremely rare (1-2%). The efficiency of the QF-PCR analysis in detecting paternal peaks in all lavage samples was only 61.5%. CONCLUSION: The present study confirms the presence of fetal cells in a very high proportion of both whole and micromanipulated intrauterine lavage samples. The isolation of trophoblastic elements can be achieved in most cases by micromanipulation. FISH and PCR techniques allowed the analysis of the most common fetal aneuploidies, confirming the power of this minimally invasive method.
Adult↗
Assisted hatching in an in vitro fertilization programme.
Assisted hatching was performed on 37 women (IVF, n = 12; micromanipulation, n = 25), who were at risk of poor implantation after in vitro fertilization and micromanipulation, and who had previous cycles of failed implantation. A total of 292 oocytes was retrieved (7.9 per cycle) of which 90 were subjected to IVF and 202 were micromanipulated. Assisted hatching was performed on day 3 of culture, on six- to eight-cell embryos, by the technique of mechanical partial zona dissection. All the patients undergoing the procedure of assisted hatching had embryos with thick zonae, greater than 15 microns. Assisted hatching was performed on 39 embryos (55.7%) in the IVF group and 104 embryos (77.6%) in the micromanipulation group. The damage rate after hatching was 1.4%. The pregnancy rate per cycle was 50% in the IVF group and 44% in the micromanipulation group, compared with 10% (IVF) and 19.2% (micromanipulation) in the control groups. Assisted hatching, by partial zona dissection, results in significant improvement in pregnancy rates in women more than 38 years of age, who have a thick zona and with previous poor implantation results. This paper reports the first pregnancy in India by the technique of assisted hatching (partial zona dissection).
Adult↗
Blastocyst formation and hatching in vitro following zona drilling of mouse and human embryos.
Hatching in vitro was studied following zona drilling of 507 two-cell mouse embryos using three methods: 1) acidic Tyrode's (AT), 2) partial zona dissection (PZD) using a sharp microneedle, and 3) zona chiseling (CH), using a large beveled needle. PZD and CH were performed while the embryos were kept in a sucrose/PBS solution. Hatching was compared to 191 unmicromanipulated controls. The incidences of cavitation and completion of hatching did not differ between groups, however more micromanipulated embryos (20-25%) hatched partially than controls (9%). The zona pellucida thinned in 59/59 (100%) control blastocysts during expansion, but in only 3/205 (2%) micromanipulated blastocysts. The hatching gap was wide in all control embryos, but smaller in 96/129 (75%) micromanipulated embryos. Partially hatched blastocysts with a "figure-8" shape were found in 59/129 (46%) micromanipulated embryos and in none of the 39 hatching controls. Hatching usually occurred a day earlier in micromanipulated embryos as 214/218 (98%) had started extruding on day 5 as compared to 20/59 (27%) control blastocysts. Fifty percent of 1-day-old human oocytes were fertilized following PZD and reinsemination and 15/31 (48%) were monospermic. Thirteen monospermic embryos cleaved, six compacted and four cavitated--of these, three extruded through the PZD incision upon expansion. The zonae did not thin and one blastocyst twinned spontaneously as it was caught between the thick ridges of the PZD hole. Results indicate that the hatching process is abnormal following zona drilling; more embryos start hatching, extrusion occurs earlier, and many become trapped which may lead to artificial twinning or the formation of trophoblastic vesicles.
Animals↗
Partial zona dissection of human oocytes when failure of zona pellucida penetration is anticipated.
Partial zona dissection (PZD) of human oocytes facilitates sperm penetration through mechanically made holes in the zona pellucida. Only 1 of 69 eggs was damaged when sucrose was used to shrink the ooplasm during micromanipulation. The fertilization rate of micromanipulated oocytes in 18 couples with male factor infertility was 68% (34/50), which compared favourably with insemination of non-micromanipulated controls (21/45, 47%). PZD was advantageous in oligozoospermic patients, but not in cases of asthenozoospermia, combined semen problems or immunological infertility. Three twin and two singleton pregnancies resulted following replacement of 23 micromanipulated and eight control embryos in 14 patients. No differences in embryo morphology and development rates were found between the micromanipulated and control groups. The incidence of polyspermy in couples with abnormal semen analyses was relatively low (less than 20%) possibly due to partial activation of the oocytes following exposure to sucrose. Polyspermy was high (57%) in normozoospermic patients with either immunological infertility (n = 3) or failure of fertilization in previous cycles (n = 4). In the three immunological patients, nine of 11 hyaluronidase and sucrose-exposed control embryos fertilized and six implanted, possibly indicating that cumulus and corona cells are contributing factors inhibiting fertilization in such cases.
Embryo Transfer↗
Effects of the size and number of zona pellucida openings on hatching and trophoblast outgrowth in the mouse embryo.
The present studies were performed to establish the effects of the size and number of artificial holes produced in the zona pellucida (ZP) on hatching and trophoblast outgrowth in vitro. Limited partial zona dissection (PZD) produced small, narrow incisions, and zona drilling with acidic Tyrode's (AT) across a larger area in the ZP was used to produce bigger round holes. Some embryos were micromanipulated once; others were micromanipulated several times. Blastocysts hatched preferentially through the artificial gaps, but completion of hatching was dependent on the size of the hole. Only 16% (26/167) of PZD embryos migrating through narrow holes hatched completely; the remainder were trapped in a typical figure-eight shape. Seventy-two percent (43/60) of those migrating through larger PZD holes hatched, but trophoblast outgrowth was not observed. Significantly (P less than 0.001) more AT-blastocysts hatched (248/270; 92%) and showed trophoblast outgrowth (176/248; 70%). Simultaneous hatching through several openings was rarely observed in AT-embryos (14/167; 8%), but this did occur in 36% (73/201) of the PZD embryos. Trapping of PZD-embryos could be almost entirely avoided by drilling with AT elsewhere on the ZP. Embryos with multiple holes in their zonae preferentially hatched through the largest opening. The results suggest that the ability of microsurgically treated human embryos to fully hatch in vitro, should be carefully (re)assessed prior to application of clinical micromanipulation systems. Micromanipulated embryos with small holes in their zonae may be rescued by performing an additional more aggressive opening procedure elsewhere on the ZP.
Animals↗
Effects of ultraviolet exposure and near infrared laser tweezers on human spermatozoa.
Photostress has to be considered during optical micromanipulation of gametes. Ultraviolet light, including low-energy UVA (32-400 nm) radiation, as well as high-intensity near infrared (NIR) laser radiation may induce cell damage. A total number of 580 light-exposed sperm cells were studied in single-cell photostress experiments. Low-power (1.5 mW, 5.3 W/cm2) UVA exposure with 365 nm radiation of a standard mercury microscopy lamp to human spermatozoa resulted within 109 +/- 30 s in paralysis and within 310 +/- 110 s in cell death. Cytotoxic effects during cell manipulation with laser microbeams were found to be partly based on non-linear excitation phenomena, in particular two-photon absorption by endogenous cell chromophores. Two-photon absorption will be more intense in the case of pulsed laser microradiation, but occur also during micromanipulation with highly focused continuous wave (cw) microbeams used as laser tweezers ('optical traps'). In particular, short-wavelength NIR traps < 800 nm induce UVA-like biological effects (oxidative stress). For example, sperm trapping with 760 nm microbeams resulted in UVA-like autofluorescence modifications, paralysis within 35 +/- 20 s and cell death within 65 +/- 20 s. In contrast, laser microbeams at 800-1064 nm may act as relatively safe micromanipulation tools. In most optical traps multifrequency cw lasers are employed. Radiation of these lasers can magnify cytotoxic effects. Therefore, single-frequency laser operation should be preferred. In general, laser assisted cell micromanipulation requires a new understanding of microbeam-cell interaction, including aspects of non-linear optics.
Cell Survival↗
Production of live offspring with predicted genotypes using PCR-RFLP analysis of polar bodies from mouse oocytes.
Accurate identification of genotypes in gametes and early embryos could facilitate the efficient production of offspring with desirable traits. This study demonstrates the feasibility of producing offspring with predictable genotypes from micromanipulated mouse oocytes. The Polymerase Chain Reaction (PCR) was used to amplify genes in the IA subregion of the major histocompatibility complex of the mouse. The validity of the approach was demonstrated in experiment 1 with IA haplotypes of unfertilized mouse ova amplified via PCR and distinguished by restriction fragment length polymorphism (RFLP) analysis. In experiment 2, fertilized oocytes were micromanipulated to remove the first and second polar bodies, which were then genotyped by validated PCR-RFLP procedures. Primary oocytes of heterozygous females contain two copies of each of the different alleles. Following meiosis I and II, the genotype of the ovum was predicted by subtracting the alleles observed in micromanipulated polar body samples. Sixty-two fertilized ova were micromanipulated and transferred to recipient females resulting in 27 live offspring (44%). The correct maternal contribution to the embryonic genotype was predicted in 19 of 27 (71%) offspring as confirmed by PCR-RFLP analysis of DNA from pup tails. Predicted genotypes of two pups were not confirmed (7%), whereas no prediction could be made in six cases (22%).
Animals↗
Microsurgery on bovine embryos at the morula stage to produce monozygotic twin calves.
Mature Brangus donor cows were superovulated with follicle stim-ulating hormone administered twice daily in intramuscular injections. On day 6.5 to 7 post-estrus, embryos were collected non-surgically using a phosphate-buffered saline medium. A total of 37 ova was collected, of which 28 were advanced morulae and early blastocysts. Twenty of these embryos were selected for micromanipulation with a radial-type Leitz micromanipulator. While the embryos were in a holding medium containing 10% fetal calf serum, three glass microinstruments were used to open the zona pellucida, remove the mass of blastomeres and bisect the embryo on a vertical plane. Halved embryos were inserted into bovine zonae and placed either as single half-embryos or twin half-embryos in 0.25 ml French straws with fresh holding medium. The micromanipulated embryos (demi-embryos) were then non-surgically transplanted, either as a single demi-embryo or as a twin demi-embryo pair, into the uterine horn of day 6.5 to 8 recipient beef females ipsilateral to the existing corpus luteum. Of the 14 micromanipulated embryos that were transplanted to recipients, pregnancy rates were 16.6% for the single demi-embryos and 62.5% for the twin demi-embryos. No pregnancies resulted from bisected blastocysts.
Journal Article↗
Partial zona dissection or subzonal sperm insertion: microsurgical fertilization alternatives based on evaluation of sperm and embryo morphology.
OBJECTIVE: To establish guidelines for application of partial zona dissection, subzonal sperm insertion, and regular in vitro fertilization (IVF) in severe male factor patients. DESIGN: Two studies were performed: partial zona dissection and IVF was applied in 57 couples during the first period, and subzonal sperm insertion was also applied in a second group of 47 couples. SETTING: Procedures were performed in an academic research environment. PATIENTS, PARTICIPANTS: Couples who failed fertilization previously, others not acceptable for IVF, and a third group in whom IVF was expected to fail. INTERVENTIONS: Oocytes were micromanipulated with either partial zona dissection or subzonal sperm insertion, or the zona pellucida was left intact. Embryos were replaced in patients prophylactically treated with methylprednisolone and antibiotics. MAIN OUTCOME MEASURES: Because several microsurgical fertilization techniques are now available, this study was performed to compare sperm parameters, embryo morphology, fertilization, and implantation rates after application of two successful micromanipulation procedures. RESULTS: Twenty-one pregnancies were established in 104 patients, 5 definitely from subzonal sperm insertion and 4 from partial zona dissection. Patients who failed IVF before had a similar chance of pregnancy after the use of micromanipulation, as first time patients (9/53 versus 12/51). In a subgroup of 15 patients who failed IVF with insufficient numbers of motile sperm, fertilization was significantly higher after subzonal sperm insertion. Partially zona-dissected embryos from couples with severe teratozoospermia (less than or equal to 5% normal forms; strict criteria) had significantly more morphological abnormalities than those from patients with moderate teratozoospermia (6% to 10% normal forms). In severely teratozoospermic patients, significantly fewer partially zona-dissected than subzonally inserted embryos implanted. CONCLUSIONS: The decision of which micromanipulation method to perform can possibly be based on careful analysis of sperm morphology.
Embryo Implantation↗
A simple technique for determining stereotaxic coordinates for brain implantation of probes at rotated angles in one or two planes.
A simple method for deriving stereotaxic coordinates for angular approaches to brain target sites is described. This method requires the use of a stereotaxic instrument with two micromanipulators and is based on the relative measurement of stereotaxic zero and the target site. This is achieved using a conventional (perpendicular) angle of approach with a probe held in one micromanipulator to visually guide the placement of the angled micromanipulator which holds a second probe. The technique may be used to derive angular approach coordinates for any angle in the coronal and/or sagittal planes. Once the coordinates are derived, they may be used with a single micromanipulator, as in the case of conventional placements. The derivation of the angular approach coordinates involves simple subtraction; no trigonometrical calculations are used. This method is simple and efficient.
Animals↗
Nuclear transfer of adult and genetically modified fetal cells of the rat.
The present study examines the handling, activation, and micromanipulation of rat eggs in an attempt to produce live young using nuclear transfer (NT) of adult and genetically modified rat fetal cells. Mature rat eggs cultured in calcium-free medium showed reduced rates (24%) of chromosomal dispersion ("spontaneous activation" characteristic of this species) compared with eggs cultured in calcium-containing medium (47%), but failed to survive micromanipulation procedures. High rates of parthenogenetic cleavage were obtained with chemical activation using ethanol/cycloheximide (65%) compared with other standard chemical activation methods (4-28%). This type of activation was also effective in reestablishing cleavage capability (19-71%), in a time-dependent manner, of spontaneously activated eggs arrested at a second prophase-like state. At most, two of four tested micromanipulation procedures were effective in producing NT embryos capable of morula or blastocyst development (14-16%) in vivo following transfer to mouse oviducts. NT blastocysts produced from cumulus cells and transfected rat fetal fibroblasts appeared morphologically and karyotypically normal (2n = 42). Nocodazole-assisted metaphase enucleation and piezoelectric-assisted donor cell injection produced significant and equivocal effects on survival and cleavage rates of reconstructed embryos but failed to significantly improve in vivo morula/blastocyst development rates (16-28%) compared with unassisted micromanipulation (16%). Live births have not yet been obtained from early cleavage stage embryos (n = 269) transferred to pseudopregnant recipient rat oviducts. Improvements in reconstituted NT embryo culture and transfer are required for these methods to be an effective means of transgenic rat production.
Animals↗
Roentgen stereophotogrammetric analysis: an accurate tool to assess stent-graft migration.
PURPOSE: To evaluate in an in vitro model the feasibility and accuracy of Roentgen stereophotogrammetric analysis (RSA) versus computed tomography (CT) for the ability to detect stent-graft migration. METHODS: An aortic model was constructed from a 22-mm-diameter Plexiglas tube with 6-mm polytetrafluoroethylene inlays to mimic the renal arteries. Six tantalum markers were placed in the wall of the aortic tube proximal to the renal arteries. Another 6 markers were added to a Gianturco stent, which was cast in Plexiglas and placed inside the aorta and fixed to a micromanipulator to precisely control displacement of the stent along the longitudinal axis. Sixteen migrations were analyzed with RSA software and compared to the micromanipulator. Thirty-two migrations were measured by 3 observers from CT images acquired with 16x0.5-mm beam collimation and reconstructed with a 0.5-mm slice thickness and a 0.4-mm reconstruction interval. Measurements were made with Vitrea postprocessing software using a standard clinical protocol and central lumen line reconstruction. Results of CT were also compared to the micromanipulator. RESULTS: The mean RSA measurement error compared to the micromanipulator was 0.002+/-0.044 mm, and the maximum error was 0.10 mm. There was no statistically significant interobserver variability for CT (p=0.17). The pooled mean (maximum) measurement error of CT was 0.14+/-0.29 (1.00) mm, which was significantly different from the RSA measurement error (p<0.0001). CONCLUSION: Detection of endograft migration by RSA is feasible and was significantly more accurate than CT in this nonpulsatile in vitro model.
Aortography↗
IVF: the new era.
In-vitro fertilization (IVF) has made great strides in the past 25 years including the development of micromanipulation techniques. Intracytoplasmic sperm injection (ICSI) for treatment of severe male factor infertility paved the way for the use of the micromanipulator. Micromanipulation techniques are now an integral part of an IVF program and are now also employed to biopsy embryos from patients harboring genetic abnormalities. Micromanipulation has also been used in the development of nuclear transfer techniques--a potential solution for creating "younger" oocytes for women of advanced maternal age and spermatozoa for azoospermic men. Other recent developments include the sequential media enabling the embryo to survive to blastocyst stage and also successful oocyte vitrification to preserve future fertility. This paper describes the latest technologies in in-vitro fertilization and relates them to the total problem of infertility, age-related and otherwise.
Embryo Transfer↗