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At least 1,153 records · Page 64Linked to original sources
PubMed1998

The effect of multiple cryopreservation procedures and blastomere biopsy on the in-vitro development of mouse embryos.

Preimplantation genetic diagnosis is currently used in clinical practice. In experienced hands the biopsy procedure alone does not affect the further in-vitro and in-vivo developmental potential of animal and human embryos. No data exist on the combination of cryopreservation of embryos at the pronuclear and/or 8-cell stage and/or biopsy at the 8-cell stage. Pronuclear stages of mouse F1 hybrids (C57Bl/jxCBA) were harvested and divided into several experimental groups. The developmental rates of zygotes, which were neither biopsied nor cryopreserved were used as data control. Others were only cryopreserved at the pronuclear stage (C-PN), or at the cleavage stage (C-CS), or both. Each of these groups was also combined with or without a biopsy. Only the hatched blastocyst rate (HBR), but not the 'simple' blastocyst rate, showed significant differences between groups. Neither C-PN (HBR = 60.42%), nor C-CS (63.16%), nor a combination of both (59.46%) had an impact on the hatched blastocyst rate when compared with that of the control group (67.46%). The biopsy procedure (55.93%) also proved not to be harmful for the embryos. The embryos, which were C-PN and C-CS, and subsequently biopsied, showed a significantly lower hatched blastocyst rate (39.62%) than that of the control, C-PN, C-CS, and C-PN/C-CS groups (P < 0.05). The combination of C-PN and cleavage-stage biopsy also lead to a lower hatched blastocyst rate (42.22%), compared with that of the control group (P < 0.05). It was concluded that couples must be advised that an effect on embryos which have undergone a combined cryopreservation and micromanipulation procedure cannot be ruled out. However, cryopreservation at the pronuclear or at the 8-cell stage alone, or in combination with a biopsy procedure, does not influence the further development of the embryo.

Animals↗
PubMed1998

Comparison of microscopic epididymal sperm aspiration and intracytoplasmic sperm injection/in-vitro fertilization with repeat microscopic reconstruction following vasectomy: is second attempt vas reversal worth the effort?

Since 1986, we have performed microscopic reconstruction in 18 men following failed microscopic vasectomy reversal. Between 1994 and 1996, nine couples have undergone microscopic epididymal sperm aspiration (MESA)/ intracytoplasmic sperm injection (ICSI) treatment for male infertility due either to congenital absence of the vas deferens (CAVD) or inoperable excurrent duct obstruction. We compared the cost efficiency of repeat vasectomy reversal to that for MESA combined with ICSI/in-vitro fertilization (ICSI/IVF). The cost of male partner procedures (vasectomy reversal, MESA) was based on physician and hospital charges, while the cost of ICSI/IVF included preparation of the female partner (medications and physician charges) and procedures (physician and hospital charges including oocyte retrieval, micromanipulation, and embryo transfer). Our cost examination does not include charges related to follow-up visits, prenatal monitoring, complications of pregnancy (i.e. miscarriage) or delivery in either group. Overall patency and pregnancy rates in the repeat vasectomy reversal group were 78 and 44% respectively. The cost per delivered baby (including multiple metachronos deliveries per couple) was $14892. Fertilization of oocytes has been achieved in 37/72 (51%) and pregnancies have occurred in 6/9 (67%) attempts and 5/9 (56%) report delivery. The average cost per pregnancy was $25637 and the average cost per delivered baby (or ongoing pregnancy) was $35570. The cost per delivery by MESA/ ICSI/IVF is 2.4 times the charges per delivery obtained through repeat vasectomy repair. Couples attempting to overcome infertility caused by vasal obstruction should be informed that vas reconstruction remains a cost effective means of re-establishing fertility even in men who have previously failed vasectomy reversal.

Cost-Benefit Analysis↗
PubMed1998

Comparison of two methods of assisted fertilization in cynomolgus monkeys (Macaca fascicularis): intracytoplasmic sperm injection and partial zona dissection followed by insemination.

Intracytoplasmic sperm injection (ICSI) and partial zona dissection followed by insemination (PZD-I) were used to establish a microinjection system in cynomolgus monkeys (Macaca fascicularis), which are potential models for human reproduction. Two experimental systems were studied, in which either hamster oocytes or cynomolgus monkey oocytes were used as the vehicle. When hamster oocytes were used, 66 out of 81 ICSI-treated oocytes (82%) showed sperm head swelling or pronucleus formation. Following PZD-I of hamster oocytes the rates of spermatozoa penetration (85/114; 75%) and fertilization (71/114; 62%) were relatively high. When cynomolgus monkey oocytes were used, 19 out of 31 (61%) were fertilized by ICSI with cynomolgus monkey spermatozoa and, subsequently, two embryos (7%) developed to the morula stage. In total, 94% (15/16) of the PZD-I treated oocytes were penetrated by spermatozoa and 63% (10/16) were fertilized. These results demonstrate that both micromanipulation techniques can be used in assisted fertilization with cynomolgus monkeys.

Animals↗
PubMed1998

Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes.

Human embryo cryopreservation represents an indispensable extension of in-vitro fertilization (IVF) programmes as long as they are based upon the recovery of a large number of oocytes. The most widely used procedures include the cryopreservation of human zygotes or embryos in early cleavage, using 1,2-propanediol and sucrose as cryoprotectants. Our results over a 10 year period (1986-1995) on 5032 thawed cycles involving 14 222 stored embryos make it possible to appraise the results and the contribution of embryo freezing to assisted reproduction. Embryos survived the freeze-thaw process in 73% of cases leading to 4590 transfers of 2.2 embryos (91% of thawed cycles). The clinical pregnancy rate per transfer was 16%, the live birth rate 12%, and the rate of babies born alive per transferred embryo was 6%. Embryo freezing monitored 10 years later produced an average of 8% of additional births. By then, 86% of stored embryos had been thawed for transfer to patients. Destruction or donation were required for only 8% of all frozen embryos and there was no news from the parental couple in relation to almost 6% of embryos. The fate of the vast majority of embryos was decided during the first 5 years of storage. Blastocyst cryopreservation is making new strides, thanks to co-culture systems and embryo selection. Micromanipulation procedures seem to have little impact on the outcome of embryo freezing. Human oocyte freezing is again clinically applied. Indeed, much of the concern about injuries to the oocyte structures through the freeze-thaw process do not seem to be justified, and the problems with frozen-thawed oocyte fertilization has been overcome using intracytoplasmic sperm injection (ICSI). As long as oocyte in-vitro maturation is not well controlled, better results will probably be obtained with mature oocyte cryopreservation. Emerging methods include the freezing of immature oocytes, follicles and ovarian tissue.

Cryopreservation↗
PubMed1999

Morphology comparison of individually selected hyperactivated and non-hyperactivated human spermatozoa.

The objective of this study was to compare the morphology of human spermatozoa undergoing hyperactivated motility in vitro with those that were non-hyperactivated (non-hyp). Hyperactivation criteria were established by the Hobson Sperm Tracker (HST), sampling at 25 Hz, as curvilinear velocity (VCL) > or = 70 microns/s, amplitude of lateral head displacement (ALH) > or = 7 microns, linearity (LIN) < or = 30% and straight-line velocity (VSL) < or = 30 microns/s. Specially developed software incorporated in the HST produced a white computer-generated overlay for spermatozoa satisfying hyperactivation criteria. These spermatozoa, visually identified on a tracking monitor, were individually removed with micromanipulation equipment using a 12 microns-diameter needle. Fifty-six patient ejaculates were examined comprising a total morphological analysis of 1886 non-hyp spermatozoa and 1051 hyperactivated spermatozoa. Hyperactivated spermatozoa had a significantly higher mean percentage of normal heads and small acrosomes (P < 0.0001 and < 0.0001 respectively) and a significantly lower percentage of large and round heads, midpieces and tail defects (P = 0.002, < 0.0001, 0.02 and < 0.0001 respectively) when compared with non-hyp spermatozoa. These data demonstrate, for the first time, that a homogeneous live population of human hyperactivated spermatozoa, selected in vitro from patients with highly variable degrees of teratozoospermia, is comprised predominantly of cells with normal morphology (P < 0.0001).

Fertilization↗
PubMed1999

Failure of fertilization after intracytoplasmic sperm injection in a patient with Kartagener's syndrome and totally immotile spermatozoa: case report.

Patients with Kartagener's syndrome (KS) are invariably infertile with totally immotile spermatozoa. Intracytoplasmic sperm injection (ICSI) is considered to be the treatment of choice for patients with immotile spermatozoa. We report the second KS case in the literature from whom immotile spermatozoa from the ejaculate failed to fertilize mature oocytes after ICSI. The role of micromanipulation in the treatment of KS patients is discussed.

Adult↗
PubMed1999

Easily decapitated spermatozoa defect: a possible cause of unexplained infertility.

We report the first 16 cases of a new sperm abnormality which we call 'easily decapitated spermatozoa defect'. This was discovered during intracytoplasmic sperm injection (ICSI) in couples with unexplained infertility. Semen analysis was normal, but minimal micromanipulation for ICSI resulted in decapitation of the spermatozoon during immobilization. For some oocytes the head and tail were injected separately, in others the intact sperm was injected after minimal immobilization. A fertilization rate of 47.5% was obtained using ICSI. Conventional in-vitro fertilization (IVF) on sibling oocytes (three cases) or in a previous cycle (three cases) resulted in total failure of fertilization. All patients reached the embryo transfer stage and three pregnancies resulted. Findings on electron microscopy in four cases included spermatozoa with degeneration or absence of the basal plate, abnormalities of the proximal centriole and degeneration of the midpiece with a large cytoplasmic droplet. We conclude that an occult sperm abnormality presenting as easily decapitated spermatozoa during ICSI could be a cause of unexplained infertility, as it resulted in total failure of fertilization in conventional IVF. Further research is necessary to investigate this sperm abnormality.

Adult↗
PubMed1999

Reconstruction of mouse oocytes by germinal vesicle transfer: maturity of host oocyte cytoplasm determines meiosis.

We evaluated the maturational competence of mouse oocytes reconstructed by the transfer and electrofusion of germinal vesicles (GV) into anuclear cytoplasts of GV stage oocytes (both auto- and hetero-transfers), metaphase II stage oocytes or zygotes. Following in-vitro culture, the maturation rates of the reconstructed oocytes to metaphase II did not significantly differ between auto- and hetero-transfers (40/70 versus 95/144 respectively); these rates also did not differ from those of control oocytes (57/97) which were matured in vitro without micromanipulation and electrofusion. In contrast, when a GV was transferred into an enucleated metaphase II oocyte or a zygote, only a few reconstructed oocytes underwent germinal vesicle breakdown (5/30 and 2/21 respectively); moreover, none reached metaphase II stage. Cytogenetic and immunofluorescence analyses were conducted on hetero-GV oocytes that extruded a first polar body. Each oocyte showed two groups of chromosomes, one in the cytoplast and one in the polar body, as well as a bipolar spindle with twenty univalent chromosomes. Our findings suggest that oocytes reconstructed by GV transfer into a cytoplast of the same developmental stage mature normally in vitro through metaphase II. Such oocytes may be a useful research model to elucidate the cytoplasmic and nuclear mechanisms regulating meiosis and the relationships between meiotic errors and age-related changes in the oocyte.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Pregnancies, growth and development of children conceived by subzonal injection of spermatozoa.

Subzonal injection of spermatozoa (SUZI) was one of the first micromanipulation techniques efficient in treating male factor infertility and unexplained in-vitro fertilization failures. The aim of this retrospective study was to evaluate the in-vitro development of embryos conceived by SUZI, the obstetric outcome, the rate of congenital malformations and subsequent follow-up in children. Fifty-five pregnancies were obtained between 1991 and 1994 (54 after fresh embryos were transferred and one after cryopreserved embryos were transferred). Among the 50 clinical pregnancies, there were seven miscarriages (14%) and two ectopic pregnancies (4%). Among the 41 resulting evolutive pregnancies, the discovery of one anencephaly led to a medical abortion. Forty deliveries including six twin pregnancies occurred, leading to the births of 45 live neonates and one stillbirth. The gender distribution of the offspring included 17 males and 29 females (ratio 0.59:1). Birth weight, length and head circumference were within the expected ranges. Two children presented a malformation: the first one had one thumb with congenital shelf and the second a polymalformative neurological syndrome. Growth curves were normal for all these children except one (weight above the 2 SD curve). Medical follow-up detected no pathological features in these children apart from a physical disability in one girl. In this small series a 4.2% rate of malformation was observed, particularly affecting the neural tube, in SUZI offspring. However, no firm conclusions can be drawn since the study was carried out on a small cohort. SUZI is no longer performed but these observations suggest that it is necessary to collect extensive data about children conceived by microfertilization.

Abortion, Spontaneous↗
PubMed1999

Possible applications of lasers in assisted reproductive technologies.

Laser systems seem to be the most promising technical tools to be introduced in assisted reproduction treatment in recent years. The 1.48 microm diode laser in particular is on course to supersede conventional mechanical and chemical methods for opening the zona pellucida in assisted hatching, and for biopsy of polar bodies or blastomeres. This is because it works without physically touching the cells, has no traceable toxic effects on living cells and is easy to handle. There will be manifold future possibilities for micromanipulation with lasers, as already demonstrated by the immobilization of spermatozoa prior to intracytoplasmic sperm injection, and the successful dissection of the zona pellucida into two halves with equal diameter for the hemizona assay.

Animals↗
PubMed2000

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

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

Adenine↗
PubMed2002

Dizygotic twin boys born after ICSI with maternal meiosis I-derived free trisomy 21 in the first and multiple congenital anomalies in the second: chance or common aetiology?

We report on a pair of dizygotic twin boys born after ICSI. One twin was affected with maternal meiosis I-derived free trisomy 21. The other had multiple congenital malformations including a complex heart defect and oesophageal atresia. The advanced maternal age of 37 years predisposed for chromosome 21 meiosis I non-disjunction in twin A. Each of the multiple congenital anomalies in twin B has been described in trisomy 21. However, due to dizygosity demonstrated by a panel of molecular markers mapped on chromosome 21 as well as the results of investigations with 16 short tandem repeat markers localized on various other chromosomes, low level mosaicism or chimerism for this aneuploidy in twin B is unlikely. In addition, the twinning process, which by itself is associated with an increased rate of congenital malformations particularly affecting heart and oesophagus, might be responsible for the multiple congenital anomalies in twin B. Thus, in agreement with the results of several population-based studies from the literature, it appears unlikely that the micromanipulation of ICSI is causally responsible for the different anomalies found in these two boys.

Abnormalities, Multiple↗
PubMed2002

Adenovirus gene transfer vector toxicity to mouse embryos: implications for human IVF.

BACKGROUND: The promulgation and diversification of micromanipulation procedures which open the zona pellucida of the oocyte or early embryo is steadily increasing the chance that zygotes will encounter infectious viral agents or gene transfer vectors derived from these agents. Such interactions could lead to toxic effects on the embryo or to insertion of foreign genes into the germ line. Adenovirus is a ubiquitous human viral pathogen that is commonly used as a gene therapy vector. However, the toxicity of this virus or its vector derivatives to embryos has not been extensively investigated. METHODS: In the present study, a mouse model was used to investigate the time of appearance of embryo toxicity, the manifestations of that toxicity, and the mechanism by which adenoviruses exert toxicity. The effects of exposure to adenovirus on in-vivo embryo development was also examined. RESULTS: These vectors exerted no deleterious effects until after the 2-cell stage, where they caused developmental delay and disorganized cleavage. Toxicity was associated with expression of a lacZ reporter gene cloned into the vectors, and with the proportion of infectious particles within the virus preparations: preparations with a high proportion of 'empty capsids', including a preparation of wild-type virus, were less toxic than a replication-defective vector with a high proportion of functional, infectious particles. Subzonal insertion of adenovirus vector followed by embryo transfer led to a dramatic reduction in the number of embryos which developed to term-findings which establish the physiological significance of the findings from in-vitro culture. CONCLUSIONS: These results indicate that adenoviruses and their vector derivatives are not likely to insert genetic material into the germ line, but they may pose a significant threat to the viability of human embryos undergoing opening of the zona pellucida during IVF.

Adenoviridae↗
PubMed1992

Preimplantation diagnosis.

Although healthy babies have been born after preimplantation diagnosis for sex determination, this technique is still in an experimental phase. To date, removal by micromanipulation of one or two blastomeres from an eight-cell embryo seems an acceptable procedure. Since a reasonable number of biopsied embryos will implant and develop in utero, the methodology for the analysis of the blastomeres at the chromosomal, gene product or DNA level, needs to be improved before further clinical application. In the meanwhile, more information about early embryo development will become available.

Animals↗
PubMed2003

Monozygotic twin delivery following reduction from quadramniotic-dichorionic gestation established after ICSI and embryo transfer: Case report.

We report the delivery of healthy monozygous (MZ) twins in a 31 year-old nulligravida following gonadotrophin ovulation induction, ICSI, assisted hatching and fresh embryo transfer. Although a sonogram on day 35 confirmed that two of four transferred embryos had implanted, a second transvaginal sonogram 1 week later showed each gestational sac had two conceptuses-all four were associated with distinct amniotic compartments. Cardiac activity was observed in all four embryos. At 12 weeks and 5 days gestation, chorionic villus sampling was performed on fetuses 1 and 2 which were euploid for chromosomes 13, 18, 21, X and Y via fluorescence in-situ hybridization analysis. Subsequent KCl injection into sacs 3 and 4 resulted in asystole for these fetuses, while cardiac activity in sacs 1 and 2 remained unchanged after reduction. A twin vaginal delivery occurred at 36 weeks gestation, resulting in the birth of two male infants and one placenta. This case represents the first known report of human quadruplet pregnancy consisting of two MZ twin sets conceived by assisted reproductive techniques. Our report reviews proposed mechanisms for explaining twinning, with special emphasis on zona pellucida micromanipulation and subsequent MZ twin induction.

Adult↗
PubMed2005

Transfer of germinal vesicle to ooplasm of young mice could not rescue ageing-associated chromosome misalignment in meiosis of oocytes from aged mice.

BACKGROUND: [corrected] Transferring a germinal vesicle (GV) from an aged woman's oocyte into ooplasm from a younger woman has been proposed as a possible way to overcome the problem of age-related decline in female fertility. Here we assessed this possibility by determining whether ooplasts derived from young mice could rescue ageing-associated chromosome misalignment in meiosis of oocytes from aged mice. METHODS: Three groups of reconstructed oocytes, young GV-young cytoplast (group YY), aged GV-young cytoplast (group AY), and young GV-aged cytoplast (group YA), were created by micromanipulation and electrofusion. RESULTS: Nuclear transplantation was successful in 89.8-94.4% of GV-ooplast complexes, and maturation rate of the reconstructed oocytes was 93.5-97.9%. Confocal microscopy analysis showed a significantly higher rate (49.2%) of chromosome misalignment in ageing mice than in young mice (16.9%), and 57.1% of oocytes in group AY exhibited chromosome misalignment, while the abnormality rate in groups YY and YA was 16.3 and 16.7% respectively. Calcium imaging showed that the three groups of reconstructed oocytes exhibited a similar pattern of calcium oscillations upon stimulation with bovine sperm extracts. Fertilization rate and developmental capacity to 2-cell embryos were also similar among the three groups of oocytes. CONCLUSIONS: Our findings suggest that: (i) the ooplasm from young mice could not rescue ageing-associated chromosome misalignment in meiosis of GV from aged mice; and (ii) behaviour of chromosome alignment over metaphase spindle is predominantly determined by GV material.

Age Factors↗
PubMed2005

Nucleocytoplasmic ratio of fully grown germinal vesicle oocytes is essential for mouse meiotic chromosome segregation and alignment, spindle shape and early embryonic development.

BACKGROUND: This study examined the effect of nucleocytoplasmic ratio of fully grown germinal vesicle (GV) oocytes on meiotic chromosome segregation and alignment, spindle shape, Ca(2+) oscillations and capacity of early embryonic development in mouse. METHODS: GV oocytes with reduced volume (equal to 1/5 to 4/5 of an intact oocyte) were made by micromanipulation to remove different amounts of cytoplasm, and then matured and fertilized in vitro. RESULTS: When >1/2 of GV oocyte cytoplasm was removed, the time-course of GV breakdown (GVBD) was delayed and oocyte maturation rate decreased significantly. Abnormal chromosome segregation rate increased if >1/2 of the cytoplasm was removed from the oocyte. Length and structure of meiotic spindle and chromosome alignment were also impaired by the reduction of cytoplasmic volume. Once matured in vitro, the oocytes could undergo Sr(2+)-induced Ca(2+) oscillations and form pronuclei in a manner independent of nucleocytoplasmic ratio, but their ability to develop to 2-cell embryos was affected if >1/2 of their cytoplasm was removed from the GV oocytes. CONCLUSIONS: These results suggest that nucleocytoplasmic ratio is essential for normal meiotic chromosome segregation, spindle formation and chromosome alignment over the metaphase spindle, and development to 2-cell stage, for which 1/2 of the volume of the GV oocyte appears to be a threshold.

Animals↗
PubMed2006

A comparative approach to somatic cell nuclear transfer in the rhesus monkey.

BACKGROUND: Despite the potential utility of primate somatic cell nuclear transfer (SCNT) to biomedical research and to the production of autologous embryonic stem (ES) cells for cell- or tissue-based therapy, a reliable method for SCNT is not yet available. Employing the rhesus monkey as a clinically relevant animal model, we have compared a conventional electrofusion method for SCNT with a one-step micromanipulation (OSM) method. METHODS: A prospective, randomized trial was conducted using only oocytes that were mature [metaphase II (MII)] at collection and a fibroblast-like cell line as nuclear donor cells (fetal fibroblasts). The embryos produced were characterized for in vitro developmental potential, cell number, karyotype and expression of nuclear mitotic apparatus (NuMA) and OCT-4. RESULTS: An in vitro blastocyst development rate of 24.4% was achieved with the OSM method, significantly higher than the 12.2% obtained following electrofusion. SCNT-produced embryos expressed normal karyotypes, cell numbers and NuMA and OCT-4 proteins in most cases. SCNT with male nuclear donor cells resulted in the production of male, SCNT blastocysts, eliminating the possibility of a parthenogenetic origin. Of the four fibroblast cell lines tested as nuclear donor cells, two supported the routine production of blastocysts following SCNT. CONCLUSIONS: The application of a modified SCNT technique (OSM) followed by embryo culture in hamster embryo culture medium-10 (HECM-10) allows, for the first time, the routine production of SCNT blastocysts, most of which appear normal by immunochemical, cytochemical and in vitro developmental criteria. These embryos will provide a resource for isolating ES cells and for studies of nuclear reprogramming by monkey cytoplasts.

Animals↗