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Biomedical subjects

M Montag

Publications and source records attributed to M Montag.

At least 19 recordsLinked to original sources
PubMed2006

The effect of nitric oxide inhibition and temporal expression patterns of the mRNA and protein products of nitric oxide synthase genes during in vitro development of bovine pre-implantation embryos.

This study was conducted to determine the effect of Nitric oxide (NO) inhibition in bovine in vitro development and expression analysis of the three Nitric oxide synthase (NOS) isoforms: endothelial (eNOS), neuronal (nNOS) and inducible (iNOS), mRNA and protein in bovine oocytes and embryos. Selective inhibitor of NOS, N-omega-nitro-l-arginine methyl ester (l-NAME) was applied at different doses (0, 0.1, 1 and 10 mm) in maturation (experiment 1A), culture medium (experiment 1B) and in both maturation and culture media (experiment 1C). No significant differences were observed in cleavage and blastocyst rates when oocytes were matured in the presence of l-NAME as long as the inhibitor was omitted during fertilization and culture. However, significantly lower blastocyst rates were observed when l-NAME was present at higher level (10 mm) in culture medium alone and in both maturation and culture media. In experiment 2, mRNA isolated from triplicate pools of oocytes and embryos (n = 15-20) was subjected to quantitative real time reverse transcription polymerase chain reaction to investigate the expression of eNOS, iNOS and nNOS mRNA in normal IVP bovine oocytes and embryos. While eNOS and iNOS transcripts were detected at higher level in oocytes (immature and mature), two-cell and four-cell stage embryos, the nNOS was detected only in immature oocyte, two-cell and morula stages. In experiment 3, eNOS and iNOS protein expression analysis was performed in IVP oocytes and embryos and both proteins were detected in the cytoplasm and the nuclei (weak) of oocytes and embryos. These data provide the first evidence for the role of NO production and the presence of mRNA and protein products of NOS isoforms during bovine embryogenesis.

Animals↗
PubMed2004

Aseptic technology of vitrification of human pronuclear oocytes using open-pulled straws.

BACKGROUND: The aim of this study was to compare the viability of human pronuclear oocytes subjected to vitrification using cooling by direct submerging of open-pulled straws in liquid nitrogen versus vitrification by cooling of open-pulled straws located inside a closed 0.5 ml straw (aseptic system). METHODS: Two- and three-pronuclei stage oocytes (n=114) were cryopreserved in super-open-pulled straws by vitrification in 20% ethylene glycol +20% dimethylsulphoxide (DMSO) + osmotic active and neutral non-permeable cryoprotectants with a four-step exposure in 20, 33, 50 and 100% vitrification solution for 2, 1 and 1 min, and 30-50 s, respectively at room temperature, and plunging into liquid nitrogen. Oocytes of group 1 (n=42) were rapidly cooled at a speed of 20,000 degrees C/min by direct plunging of open-pulled straws into liquid nitrogen. Oocytes of group 2 (n=44) were first located in 0.5 ml straws, which were closed at both sides by metal balls, and then plunged into liquid nitrogen. This method resulted in a cooling speed of 200 degrees C/min. For both groups, oocytes were thawed rapidly at a speed of 20 000 degrees C/min using an identical protocol. Oocytes subsequently were expelled into a graded series of sucrose solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) at 2.5 min intervals. RESULTS: Oocyte development up to expanded blastocyst stage after in vitro culture was 15% in group 1, 14% in group 2 and 29% in an untreated control group. CONCLUSION: The deposition of human pronuclear oocytes in open-pulled straws which are placed inside a hermetically closed container guarantees a complete isolation of oocytes from liquid nitrogen and avoids potential contamination by pathogenic microorganisms. The combination of direct plunging of this container into liquid nitrogen and rapid warming makes this process as efficient as conventional vitrification.

Asepsis↗
PubMed2004

A European multicentre prospective randomized study to assess the use of assisted hatching with a diode laser and the benefit of an immunosuppressive/antibiotic treatment in different patient populations.

BACKGROUND: Assisted hatching (AH) techniques, designed for facilitating the embryo escape out of the zona pellucida (ZP) have been used in IVF centres since 1992. The initial indications for AH were patient's age, ZP thickness, high basal FSH and repeated IVF failures. Several retrospective and prospective studies assessing AH in these indications have given disparate results. Our aims were to evaluate the benefits of AH and immunosuppressive/antibiotic treatment (IA) in patients with either a poor prognosis of success, previous implantation failures or transfers of cryopreserved embryos. METHODS: Four IVF centres allocated 426 patients, randomized for AH and IA, into four groups of AH indications between 1997 and 1999. AH was performed with a diode laser. ZP thickness, opening size and embryo score were recorded. Outcome measures were implantation and delivery rates. RESULTS: Patients coming for a first or third transfer of cryopreserved embryos and poor prognosis patients admitted for a first trial did not benefit from AH. Even patients with repeated implantation failures of fresh embryos did not gain significantly from AH. CONCLUSIONS: Among AH indications, absence of implantation after several transfers of good quality embryos remains the strongest patient selection criterion. Prescription of an immunosuppressive/antibiotic treatment is essential.

Adult↗
PubMed2004

Previous miscarriages influence IVF and intracytoplasmatic sperm injection pregnancy outcome.

Previous conceptions are one predictor for the outcome of assisted reproductive technology procedures. Approximately 18-34% of clinical pregnancies following assisted reproduction procedures result in spontaneous abortion. The risk of such pregnancy loss is believed to increase with women's age, previous miscarriages and use of frozen-thawed embryos. This study analyses German IVF Registry data to examine the impact of previous miscarriages on the outcome of assisted reproduction procedures. The data set consists of a total of 174,909 assisted reproduction procedures performed between January 1998 and December 2000. Multiple logistic regression is used to assess the correlation between women's age, spousal/partner change, and infertility diagnosis. It is demonstrated that any previous miscarriage will increase the treatment-dependent miscarriage rate in assisted reproduction procedures. A significantly higher impact is shown for one previous miscarriage achieved by assisted reproduction procedures compared with spontaneous conception. Partner change is shown to have no specific impact on the treatment dependent miscarriage rate, whereas a statistically significant increase in miscarriages in all assisted reproduction procedures was found among women older than 34 years of age. Overall, the highest rate of treatment-dependent miscarriages was seen in assisted reproduction procedures with cryopreserved embryo transfer.

Abortion, Spontaneous↗
PubMed2004

[Interventional occlusion of the aneurysm of the pulmonary artery: treatment of a rare complication when applying the Swan-Ganz catheter].

Dissecting aneurysm of the pulmonary artery is a rare (1 : 3000) but life threatening complication of the use of Swan-Ganz catheters. The most typical clinical manifestation is hemoptysis, even a asymptomatic aneurysm causes pathognomonic X-ray findings. The eventually long free intervall between catheterisation and formation of the dissecting aneurysm requires thorough follow up of the concerning patients, e. g. chest roentgenograms and thorax CT-scans. The endovascular therapy of this lesion has replaced surgery due to a much better risk profile. The aim of the endovascular treatment is a coil-occlusion of the parent artery immediately proximal to the aneurysm. The success of this procedure will be instantaneously evident.

Aged↗
PubMed2004

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.

Cleavage Stage, Ovum↗
PubMed2003

[A "late" scimitar syndrome. Diagnostic contribution of cardiac computed tomography].

A 51-year-old female hospitalized with a non-specific colitis, presented a crescent-like shadow in the right lower lung accompanied by a reduced right lung volume on a routine chest x-ray. There was no family history of congenital heart disease. The initially performed, noninvasive, contrast enhanced cardiac CT (electron-beam tomography [EBT]) proved the suspected diagnosis of a partial, anomalous pulmonary, transdiaphragmatic vein drainage (APVD) in combination with a hypoplastic right lower lobe and dextrocardia. These findings are in accordance with scimitar syndrome. Regarding to the clinical situation with symptoms like slowly progressive dyspnea on exertion and low exercise tolerance for the last 2 years and an invasively documented left-to-right shunt ratio >50% (Qp:Qs = 2.6 : 1) surgical repair was recommended. The anomalous vein was connected to the left atrium creating a "neo-septum". On a postoperative checkup after 9 months the patient is without any medication, symptoms during moderate activity are relieved, exercise tolerance was substantially better and noninvasive imaging visualized the corrected drainage of the anomalous vein to the left atrium.

Anastomosis, Surgical↗
PubMed2002

[Diagnosis and embryogenesis of partial anomalous pulmonary venous connection].

In Anomalous Pulmonary Venous Drainage one or more pulmonary veins are not connected with left atrium, but drain into systemic circulation or right atrium. The clinical signs of the shunt between pulmonary and systemic circulation increase during lifetime, thus the abnormality gets late or not diagnosed. Partial Anomalous Pulmonary Venous Drainage is a developmental disorder, according to embryogenesis we recommend a classification of this abnormality. Two case reports are given to illustrate difficulties in diagnosis of this relatively common abnormality. A diagnostic standard of assessment of pulmonary venous disorders is discussed. The "Gold Standard" of selective pulmonary Angiography in combination with oxygen measurement is widely used for diagnosis of Partial Anomalous Pulmonary Venous Drainage. In the future improvement and common use of modern cross sectional imaging techniques will redefine the value of conventional Angiography.

Adolescent↗
PubMed2001

Comparison of steric and electronic requirements for C-C and C-H bond activation. Chelating vs nonchelating case.

C-H bond activation was observed in a novel PCO ligand 1 (C(6)H(CH(3))(3)(CH(2)OCH(3))(CH(2)P(t-Bu)(2))) at room temperature in THF, acetone, and methanol upon reaction with the cationic rhodium precursor, [Rh(coe)(2)(solv)(n)()]BF(4) (solv = solvent; coe = cyclooctene). The products in acetone (complexes 3a and 3b) and methanol (complexes 4a and 4b) were fully characterized spectroscopically. Two products were formed in each case, namely those containing uncoordinated (3a and 4a) and coordinated (3b and 4b) methoxy arms, respectively. Upon heating of the C-H activation products in methanol at 70 degrees C, C-C bond activation takes place. Solvent evaporation under vacuum at room temperature for 3-4 days also results in C-C activation. The C-C activation product, ((CH(3))Rh(C(6)H(CH(3))(2)(CH(2)OCH(3))(CH(2)P(t-Bu)(2))BF(4)), was characterized by X-ray crystallography, which revealed a square pyramidal geometry with the BF(4)(-) anion coordinated to the metal. Comparison to the structurally similar and isoelectronic nonchelating Rh-PC complex system and computational studies provide insight into the reaction mechanism. The reaction mechanism was studied computationally by means of a two-layer ONIOM model, using both the B3LYP and mPW1K exchange-correlation functionals and a variety of basis sets. Polarization functions significantly affect relative energetics, and the mPW1K profile appears to be more reliable than its B3LYP counterpart. The calculations reveal that the electronic requirements for both C-C and C-H activation are essentially the same (14e intermediates are the key ones). On the other hand, the steric requirements differ significantly, and chelation appears to play an important role in C-C bond activation.

Journal Article↗
PubMed2001

Testicular sperm extraction in a patient with metachronous bilateral testicular cancer.

A new indication for testicular tissue cryopreservation is demonstrated in a patient with metachronous bilateral testicular tumours and azoospermia. At the age of 18 (1982) the patient underwent left orchidectomy and radical retroperitoneal lymphadenectomy for a testicular teratoma (pT1N0M0). Semen samples were not cryopreserved because of absence of motile spermatozoa after thawing. Seventeen years after the primary testicular cancer, a seminoma of the contralateral right testis was diagnosed (pT1N0M0). Since the patient was azoospermic, no semen samples could be cryopreserved. However, spermatozoa were detected in testicular biopsy material of the right testis and were cryopreserved for ICSI. Since all spermatozoa were dead after thawing, testicular sperm extraction (TESE) was performed in the remaining tissue samples at the time of ICSI treatment. Only spermatids could be extracted from frozen-thawed samples due to the inhomogeneous distribution of spermatogenic activity in the testicular tissue. Although one oocyte was fertilized with these spermatids, a clinical pregnancy was not achieved. Despite the disappointing results of ICSI in the couple presented here, this case report demonstrates that cryopreservation of testicular tissue and TESE should be considered in patients with bilateral testicular tumours and azoospermia, if frozen semen samples are not available.

Adolescent↗
PubMed2001

Evaluation of pronuclear morphology as the only selection criterion for further embryo culture and transfer: results of a prospective multicentre study.

BACKGROUND: The aim was to study zygote evaluation based on pronuclear morphology under the German embryo protection law, according to which only a maximum of three zygotes are allowed to be cultured for embryo transfer. METHODS: In this prospective multicentre study, a total of 512 treatment cycles was performed at 10 centres, between November 1999 and October 2000. Zygotes were classified into seven patterns (0A, 0B and 1-5). Pattern 0A and 0B zygotes were preferentially used for further culture and transfer. RESULTS: Cycles with transfer of at least one embryo derived from pattern 0B, but not pattern 0A, resulted in significantly higher pregnancy (37.9%) and implantation rates (20.5%) compared with non-pattern 0B cycles (26.4 and 15.7%; P < 0.05 and P < 0.01 respectively). In younger patients (aged < or =35 years), significantly more 0B zygotes were available for transfer than in older patients (34.2 versus 25.8%; P < 0.005). CONCLUSIONS: From these data, it is concluded that evaluation of pronuclear morphology is beneficial, especially for countries with legal restrictions regarding embryo selection.

Adult↗
PubMed2000

Activation of mouse oocytes requires multiple sperm factors but not sperm PLCgamma1.

A sperm cytosolic factor is responsible for oocyte activation at fertilization in mammals. The molecular identity of this factor is not yet known, although a sperm phospholipase Cgamma (PLCgamma) is a potential candidate. In this study, cation-exchange chromatography with a Heparin column was used for the fractionation of porcine sperm cytosolic extracts. Oocyte activation potential of the resulting fractions was tested and active fractions were subjected to Western blot analysis using antibodies specific to PLCgamma1. PLCgamma1 was detected in fractions other than those supporting oocyte activation (Ca(2+)-release and pronuclear formation). The active Heparin fraction was then purified on a Mono Q anion-exchange column. One of the resulting fractions still contained Ca(2+)-releasing activity, but pronuclear formation did not occur. We conclude that sperm PLCgamma1 is not involved in oocyte activation and that Ca(2+)-release and pronuclear formation requires multiple factors from sperm cytosol.

Animals↗
PubMed2000

A new method to produce equally sized hemizonae pellucidae for the hemizona assay.

The hemizona assay (HZA) is a valuable tool to study the binding potential and interaction of spermatozoa with the zona pellucida. Its accuracy strongly depends on the use of equally sized hemizonae. Usually, manipulator-guided microblades are used for cutting the zona pellucida. Recently, lasers were introduced for precise local thermolysis of the zona. The use of a 1.48 microm diode laser for the generation of hemizonae from human oocytes was investigated. This laser allowed drilling of equally sized hemizonae which were used for hemizona binding assays. It is concluded that the 1.48 microm diode laser system can be applied for the production of hemizonae. The method is easy to perform and offers a fast and efficient access to hemizonae of identical size.

Female↗
PubMed2000

Laser-induced immobilization and plasma membrane permeabilization in human spermatozoa.

We evaluated the potential use of a non-contact, 1.48 microm wavelength diode laser for immobilization of human spermatozoa and permeabilization of the sperm membrane in different culture media. When we applied a single laser shot near to the middle region of the sperm tail, spermatozoa could be immobilized either temporarily or permanently, depending on the energy used. Above an energy of 2 mJ in polyvinylpyrrolidone and 2-3 mJ in culture medium, a reliable permanent immobilization was achieved by permeabilization of the sperm tail membrane. We then explored the use of a double laser shot technique. Spermatozoa were temporarily immobilized by a first laser shot applied near to the sperm tail followed by permeabilization with a second laser shot aimed directly at the sperm tail. This sequential approach yielded permanent immobilization at much lower energy values compared with the single shot technique. Following the injection of laser-treated spermatozoa, mouse oocytes underwent normal activation and pronuclear formation. We conclude that a non-contact 1.48 microm diode laser system can be used for immobilization of spermatozoa and for permeabilization of the sperm tail membrane. This laser procedure may offer an alternative to currently used sperm pretreatment prior to intracytoplasmic sperm injection.

Animals↗
PubMed2000

Significance of the number of embryonic cells and the state of the zona pellucida for hatching of mouse blastocysts in vitro versus in vivo.

We investigated the course of mouse blastocyst hatching in vitro after experimental modulation of the hatching process by growth hormone or by laser treatment and compared it to embryos grown in vivo. When embryos were grown in vitro, successful hatching was dependent on blastocyst expansion and was based on a minimum number of embryonic cells. Embryos grown in the presence of growth hormone were more advanced in their development and hatched earlier. When an artificial opening was laser-drilled into the zona pellucida, hatching occurred at lower numbers of embryonic cells. In vivo, escape from the zona pellucida occurred earlier and independent of blastocyst expansion. However, when we isolated in vivo-grown blastocysts with intact zonae that had developed in vivo and then cultured them in vitro, blastocysts started to expand and hatched the following day when a sufficiently high number of embryonic cells was present. Our data show that successful hatching in vitro is dependent on a sufficiently high number of embryonic cells, which enables blastocyst expansion and zona shedding. In vivo, the lower number of embryonic cells detected in zona-free blastocysts indicates that the underlying mechanism of zona escape is different, does not depend on blastocyst expansion, and presumably involves lytic factors from the uterus.

Animals↗