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Biomedical subjects

K Lundin

Publications and source records attributed to K Lundin.

At least 19 recordsLinked to original sources
PubMed2001

Development of a time-resolved fluorescence resonance energy transfer assay (cell TR-FRET) for protein detection on intact cells.

An assay named Cell TR-FRET based on time-resolved fluorescence resonance energy transfer, here utilized for detection of receptor proteins on intact cells, is described. In this assay, intact membrane-biotinylated Sf9 cells expressing human interleukin-2Ralpha due to infection with a recombinant baculovirus were prelabeled with a streptavidin-europium (Eu(3+)) chelate, the donor. These prelabeled cells were used in a homogeneous assay by addition of a fluorochrome-labeled anti-hIL-2Ralpha-specific antibody, 7G7B6-Cy5, the acceptor. Binding of 7G7B6-Cy5 to hIL-2Ralpha expressed on the cell surface and europium-labeled streptavidin to surface biotin esters brings the donor and the acceptor in close proximity, allowing transfer of energy from the excited state donor to the acceptor. This energy transfer was specifically inhibited by unlabeled antibody and by free biotin. The described assay constitutes a general method since no specific component of the cell membrane is labeled, thereby allowing a number of binding studies on the cell membrane, including receptor density determinations, to be performed. In addition, due to the rapid fashion in which the Cell TR-FRET assay is accomplished, it can be a valuable method not only for identifying novel membrane-associated proteins, but also for drug screening of large samples in high-throughput format.

Animals↗
PubMed2001

Acrosome index is not an absolute predictor of the outcome following conventional in vitro fertilization and intracytoplasmic sperm injection.

PURPOSE: To investigate if including evaluation of acrosome index (AI) in the semen analysis of teratozoospermic samples could help to predict for which patients intracytoplasmic sperm injection (ICSI) is necessary. METHODS: The fertilization rate, pregnancy rate, and percentage of good quality embryos were compared after performing conventional in vitro fertilization (IVF) and ICSI, respectively, using sibling oocytes. The role of AI was evaluated by dividing patients into two groups; Group A (AI < 7%) and Group B (AI > or = 7%). RESULTS: A significant difference in fertilization rate was observed between Group A and B after conventional IVF. In Group A, the fertilization rate, embryo transfer rate, and percentage of good quality embryos were higher after ICSI than after IVF. In Group B, the fertilization and pregnancy rates were numerically but not significantly higher after IVF compared to ICSI. CONCLUSION: Evaluation of acrosome index will not accurately predict fertilization, although this study shows that a sperm sample with less than 5% normal forms and an AI greater than 7% may achieve a mean fertilization rate > 70% after conventional IVF.

Acrosome Reaction↗
PubMed2001

Production and partial characterization of mouse monoclonal antibodies recognizing common cytokine receptor gamma chain (gammac) of human, mouse and primate origin.

Monoclonal antibodies specific for the common cytokine receptor gamma chain, gammac, were produced using traditional hybridoma technology. Fusion of P3X63-Ag8.653 myeloma cells with splenocytes from Balb/c mice immunized with Spodoptera frugiperda insect cells infected with the recombinant baculovirus VL1392-hIL-2Rgamma resulted in several hybridoma cell clones producing monoclonal gammac-specific antibodies. Four of these antibody-producing clones, IIIC3, IIIE8, IG3 and IF10C5, were further characterized by immunoblotting, flow cytometry and ELISA. Data are presented demonstrating that the generated monoclonal antibodies can identify the extracellular domain of the common cytokine receptor gamma chain of human and mouse origin, and two of the antibodies recognize gammac of primate origin as well.

Animals↗
PubMed2001

Early embryo cleavage is a strong indicator of embryo quality in human IVF.

BACKGROUND: In order to decrease multiple birth rates without decreasing birth rates overall, it is important to increase the capability of selecting the most optimal embryos for transfer. It has been shown that human embryos which cleave early, i.e. complete the first mitotic division within 25-27 h post insemination, provide higher pregnancy and implantation rates. METHODS AND RESULTS: In this prospective study, an evaluation of 10 798 scored embryos showed that early cleavage resulted in a significantly higher proportion of good quality embryos compared with late cleavage (62.5 versus 33.4%, P < 0.0001). When examining both day 2 and day 3 transfers together, early-cleaving embryos (306 transfers) gave rise to significantly higher rates of pregnancy/transfer (40.5 versus 31.3%, P = 0.0049), implantation (28.0 versus 19.5%, P = 0.0001) and birth/ongoing pregnancy (34.3 versus 24.0%, P = 0.0009) than did late-cleaving embryos (521 transfers). A stepwise logistic regression of all data showed that the total number of good quality embryos and female age were independent predictors of both pregnancies and birth. For intracytoplasmic sperm injection (ICSI) embryos, early cleavage was found to be an independent predictor of birth. CONCLUSIONS: Early embryo cleavage is a strong biological indicator of embryo potential, and may be used as an additional embryo selection factor for ICSI embryos.

Adult↗
PubMed2001

Human embryos with unevenly sized blastomeres have lower pregnancy and implantation rates: indications for aneuploidy and multinucleation.

Uneven blastomere cleavage in human embryos of 'good morphology', i.e. those normally used for transfer, is a phenomenon which has been poorly investigated. The main objective in this study was to probe deeper into the aetiology behind previous findings that embryos with uneven cell cleavage have a lower developmental capacity in comparison with evenly cleaved embryos. Our hypothesis was that uneven cleavage may result in embryos with a higher degree of aneuploidy and/or multinuclear rate, which in turn might help to explain their low implantation rate. In the first part of the study, 378 embryo transfers performed over a 3-year period were analysed retrospectively, where all the transferred embryos in each cycle were of identical morphology score and cleavage stage. In the second part of the study, multicolour fluorescence in-situ hybridization (FISH) analyses on good quality embryos, representing the uneven (n = 11) and even (n = 13) study groups were performed. When comparing day 2 transfers between 4-cell embryos, it was found that unevenly cleaved embryos had significantly lower implantation (23.9 and 36.4%) and pregnancy rates (37.6 and 52.9%) compared with evenly cleaved embryos. A significantly higher degree of aneuploidy (29.4 and 8.5%) and multinuclear rate (21.1 and 2.1%) in blastomeres from uneven embryos was also found. It is concluded that uneven blastomere cleavage has a negative effect on both pregnancy and implantation rates in human IVF, and that this can partly be explained by a higher degree of aneuploidy/multinuclear rate. In the light of the results obtained, a new approach in the current embryo scoring system, placing more emphasis on blastomere size, is recommended.

Adult↗
PubMed2001

Preimplantation genetic diagnosis (PGD): the Gothenburg experience.

BACKGROUND: A program for preimplantation genetic diagnosis of pre-embryos from patients with hereditary disorders was set up in our unit at Sahlgrenska University Hospital in 1994. The majority of the patients were carriers of X-chromosome linked disorders; a few patients were translocation carriers. In this paper we describe our experiences of our first 36 cycles, 30 gender determinations and six analyses of embryos with possible translocations. METHODS: Conventional hormone replacement treatment with intracytoplasmic sperm injection to fertilize the eggs followed by blastomere biopsy and fluorescent in situ hybridization at the eight cell stage was used for sexing as well as detection of translocations. RESULTS: Out of the 30 cycles in 13 patients for gender determination, blastomere biopsies could be carried out in 25 cycles. Transfer of normal female embryos (XX) was performed in 18 cycles, resulting in five pregnancies (pregnancy rate 27.8%) and an implantation rate of 20% per transfer. Three girls have been born. Hence the take home baby rate was 16.7% per transfer and 10% per started cycle. Six cycles (three patients) for detection of translocations in embryos were performed. Diagnosis was possible in four cycles. Transfer of normal embryos was carried out in one cycle. No pregnancy was achieved. CONCLUSION: Successful PGD in its clinical application demands close collaboration between a large group of specialists. Even so, the success rate is considerably lower than after conventional IVF or ICSI procedures. Taking into account the stress caused to the parents facing late interruption of pregnancy following conventional prenatal diagnosis we are convinced that this technique is well worthwhile continuing and refining.

Cytogenetic Analysis↗
PubMed2000

Selection of patients suitable for one-embryo transfer may reduce the rate of multiple births by half without impairment of overall birth rates.

The aim of the present study was to identify a subset of patients at high risk of multiple birth after IVF and hence suitable for one-embryo transfer, which undoubtedly would reduce the multiple birth rate. This retrospective study included 2107 IVF cycles in which two embryos were transferred. Factors with possible correlation to multiple birth were studied in a multivariate analysis. The factors included background data (female age, previous pregnancies and births, previous IVF cycles, indication for IVF) and IVF cycle characteristics. The following factors were independently predictive of multiple birth: female age expressed a negative correlation while number of good quality embryos transferred was positively correlated. A subset of patients was identified as being at high risk of multiple birth by including age, cycle number and presence of tubal infertility in a model derived from a logistic regression analysis. The rate of multiple births can be reduced from 26% to 13% of all births if one-embryo transfer is performed in selected cases. The total birth rate will decrease from 29% to 25% but may be completely restored by performing one additional one-embryo freeze transfer in high risk patients who do not achieve a term pregnancy.

Adult↗
PubMed2000

The use of silane-coated silica particles for density gradient centrifugation in in-vitro fertilization.

Silane-coated silica particles (PureSperm) were evaluated as an alternative to Percoll for gradient separation of spermatozoa, for use in assisted reproduction. Recovery of motile and morphologically normal spermatozoa after using a four-layer Percoll and a two- and four-layer PureSperm gradient respectively was recorded. In-vitro fertilization (IVF) results after using PureSperm for the sperm preparation were also evaluated. No difference in sperm recovery or sperm motility was found when comparing the use of Percoll and the four-layer gradient of PureSperm. When using a two-layer PureSperm gradient, motility was significantly decreased (P < 0.05) compared to Percoll. Normal sperm morphology increased from 8-17.2% after using Percoll and to 12.7% and 11.4% after using a four-layer and a two-layer PureSperm gradient respectively. All gradient preparations showed a significant decrease in the teratozoospermia index compared to the ejaculate (P < 0.01). No significant differences in IVF results regarding fertilization and pregnancy rates were found when PureSperm or the swim-up technique were used for the sperm preparation. PureSperm seems to be an acceptable alternative to Percoll but although the percentage of sperm recovery was higher after PureSperm we still recommend the swim-up technique to be the first choice, as a higher percentage of progressive motile spermatozoa is obtained without using other chemicals than IVF culture medium.

Cell Separation↗
PubMed2000

Incidence of congenital malformations in children born after ICSI.

The aim of this study was to determine the incidence of congenital malformations in a complete cohort of children born after intracytoplasmic sperm injection (ICSI). The medical records were retrieved for 1139 infants, 736 singletons, 200 sets of twins and one set of triplets. The total number of infants with an identified anomaly was 87 (7.6%), 40 of which were minor. The incidence of malformations in children born after ICSI was also compared with all births in Sweden using data from the Swedish Medical Birth Registry and the Registry of Congenital Malformations. For ICSI children, the odds ratio (OR) for having any major or minor malformation was 1.75 [95% confidence interval (CI) 1.19-2.58] after stratification for delivery hospital, year of birth and maternal age. If stratification for singletons/twins was also done, the OR was reduced to 1.19 (95% CI 0.79-1.81). The increased rate of congenital malformations is thus mainly a result of a high rate of multiple births. The only specific malformation which was found to occur in excess in children born after ICSI was hypospadias (relative risk 3.0, exact 95% CI 1. 09-6.50) which may be related to paternal subfertility.

Chromosome Aberrations↗
PubMed2000

The position of the metaphase II spindle cannot be predicted by the location of the first polar body in the human oocyte.

When performing intracytoplasmic sperm injection (ICSI) on human oocytes, the injection is traditionally made at the 3 o'clock position, with the first polar body (PB) at the 12 or 6 o'clock position. This has been based on the assumption that the second meiotic metaphase II (MII) spindle lies in close proximity to the first PB. The objective of this study was to document the actual spatial relationship between the first PB and the MII spindle both in in-vivo matured (fresh) human MII oocytes and in oocytes matured in vitro. We found that the MII spindle was, on average, not located directly adjacent to the PB. The in-vivo group (n = 54) showed a mean deviation of the MII spindle from the position of the PB of 41. 7 degrees and the in-vitro group 26.6 degrees (n = 43). The difference between the angle of the two groups was statistically significant (P = 0.005), indicating that the lateral displacement of the first PB is only partly due to the denuding procedure during ICSI, because the in-vitro matured oocytes were denuded before extrusion of the first PB. The majority of the MII spindles in both groups were found in the same hemisphere as the first PB, suggesting that care should be taken to avoid damaging the MII spindle by inserting the ICSI needle in the other half of the oocyte.

Cellular Senescence↗
PubMed1999

Diagnostic epididymal and testicular sperm recovery and genetic aspects in azoospermic men.

Various procedures for sperm recovery in azoospermic men have been described, from open testicular biopsy to simple needle aspiration from the epididymis and the testis. Fifty-one obstructive and 86 non-obstructive azoospermic men were treated to compare the recovery of spermatozoa obtained by percutaneous aspiration from the epididymis (PESA) and aspiration/extraction from the testis (TESA, TESE) with histopathology. If TESA failed, the work up proceeded with TESE. All patients were karyotyped. Spermatozoa were recovered by PESA or TESA in all obstructive men (51/51 patients). In 22 out of 86 patients with non-obstructive azoospermia, testicular spermatozoa could be successfully recovered by TESA. In five additional patients TESE was successful in recovering spermatozoa where TESA had failed. In 43 patients, neither TESA nor TESE was successful. Sixteen patients chose not to proceed with TESE. Seven out of 86 patients had an abnormal karyotype in the non-obstructive group (8%), none in the obstructive group. In the non-obstructive patient group testicular histopathology showed hypospermatogenesis, incomplete maturation arrest and germ cell aplasia with focal spermatogenesis in cases where spermatozoa were recovered and complete germ cell aplasia, complete maturation arrest and fibrosis in cases where no spermatozoa were found. Spermatozoa were recovered by PESA or TESA from all patients with obstructive azoospermia and from approximately 40% of patients with non-obstructive azoospermia by TESA or TESE. Retrieval of viable spermatozoa in the infertility work-up was highly predictable for sperm recovery in subsequent ICSI cycles. TESA performed under local anaesthesia seems almost as effective as more invasive procedures in recovering testicular spermatozoa, both in obstructive and non-obstructive azoospermic men.

Epididymis↗
PubMed1998

Sperm retrieval and fertilization in repeated percutaneous epididymal sperm aspiration.

Percutaneous epididymal sperm aspiration (PESA) for retrieval of spermatozoa for intracytoplasmic sperm injection (ICSI) is a new simplified technique in the treatment of men with obstructive azoospermia. There has been a fear that the PESA procedure, being blind, could cause damage to the epididymal duct system and make it impossible to retrieve spermatozoa if a repeated procedure is required. We report here on repeated PESA procedures from the same unilateral epididymis. Twenty-seven men with obstructive azoospermia were investigated retrospectively regarding sufficiency of the number of motile spermatozoa for ICSI, fertilization rate (FR) and possibility of collecting spermatozoa for cryopreservation in repeated PESA procedures. Sufficient motile spermatozoa for ICSI were found in a similar proportion of men at the first two attempts: 91 and 89% respectively. Fertilization rate and the possibility of collecting spermatozoa for cryopreservation were also similar at the first two PESA procedures: 62 versus 67% and 33 versus 33% respectively. At the third procedure, motile spermatozoa for ICSI were retrieved in 86% (6/7), FR was 47% and spermatozoa were cryopreserved in one case. Two men underwent a fourth PESA. In both cases, a sufficient number of motile spermatozoa for ICSI was found and FR was 62%. This study shows that in men with obstructive azoospermia, PESA can be repeated on the same unilateral epididymis up to three times, with good opportunity of retrieving sufficient motile spermatozoa for ICSI.

Adult↗
PubMed1998

Comparison of fertilization, cleavage and pregnancy rates of oocytes from large and small follicles.

Ovarian stimulation for in-vitro fertilization (IVF) causes development of several cohorts of follicles. At the time of oocyte collection, oocytes are thus retrieved from a wide range of follicles of different sizes and developmental stages. A relationship between size of follicles and pregnancy rates has earlier been demonstrated. The aim of the present study was to compare fertilization, cleavage and pregnancy rates between oocytes retrieved from large and small follicles in conventional IVF and intracytoplasmic sperm injection (ICSI). A total of 200 conventional IVF patients and 175 ICSI patients underwent oocyte retrieval where oocytes from both large and small follicles were collected. A follicle with a volume of > or = 2 ml, corresponding to a follicular diameter > or = 16 mm as determined by ultrasound, was regarded as a large follicle. Only one cycle from each patient was included. Fertilization and cleavage rates were calculated per patient for oocytes from large and small follicles. The mean fertilization and cleavage rates for conventional IVF and ICSI cycles were calculated. Comparison of pregnancy rates was performed for patients receiving embryos derived from oocytes of only large or only small follicles. For conventional IVF patients, fertilization rates were 71.4 and 58.1% (P < 0.01, Wilcoxon paired test) for oocytes of large and small follicles respectively. The corresponding cleavage rates were 95.4 and 93.9% respectively. The pregnancy rate for the two groups was 47% (60/127) and 15% (2/13) (P < 0.05, chi2 test). For ICSI patients the fertilization rate was 72.0 and 71.1% for oocytes of large and small follicles respectively. The corresponding cleavage rate was 93.0 and 91.1%. The pregnancy rate in the two groups was 41% (46/113) and 42% (5/12). The results show that oocytes from smaller follicles also yield fertilization and pregnancies, although in conventional IVF to a lesser extent than oocytes from larger follicles. For IVF cycles, a higher proportion of immature oocytes (which are normally not included in the ICSI procedure) in the group of oocytes from small follicles is most probably the explanation for the lower fertilization rate. The decrease in pregnancy rate with oocytes from small follicles in the IVF cycles was not observed in the ICSI cycles. The possibility of evaluating the degree of oocyte maturation prior to fertilization may be an advantage of the ICSI technique. This suggests that the disadvantages of oocytes from small follicles might be overcome by means of ICSI.

Adult↗
PubMed1998

Indications for intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is the latest of several microfertilization techniques that have been utilized predominantly to overcome severe male subfertility, giving fertilization and term pregnancy rates similar to conventional in-vitro fertilization (IVF) (but for other indications). Even though available data on children born after ICSI are very encouraging, the procedure must still be considered as novel and the safety aspect to a great extent unexplored. In our opinion, therefore, ICSI should only be used for specific indications, and in this communication the non-existent, relative and absolute indications for performing ICSI are outlined and discussed. With an apparently normal sperm sample, ICSI should not be used in a first cycle even if only few oocytes are obtained. When there is reason to suspect poor fertilization, ICSI can be used in combination with conventional IVF in a split cycle. This includes cases of 'subnormal' sperm samples, high titres of antisperm antibodies, or following a single cycle of poor fertilization using conventional IVF. Absolute indications for ICSI include two previous fertilization failures with conventional IVF, use of epidiymal or testicular sperm samples, or when only acrosomeless or immotile spermatozoa are available. The fertilization of oocytes prior to preimplantation genetic diagnosis is another absolute indication. It is, however, important to keep in mind that for this novel technique, indications should not be rigid, but remain variable with respect to new findings.

Ciliary Motility Disorders↗
PubMed1997

S1 nuclease hybrid analysis of mitochondrial DNA amplified by long-distance PCR: rapid screening for small-scale rearrangements.

We report on a method suitable for screening large regions (>3 kb) of mtDNA for structural changes of <500 bp and their localization. Heteroduplexes consisting of a wild-type and a mutant strand are cleaved by S1nuclease when single-stranded loops are present due to deletions or duplications/insertions. This strategy was successfully applied to screen the muscle mtDNA of 20 patients with mitochondrial encephalomyopathies. In three of them, an altered cleavage pattern was observed caused by a homoplasmic 9 bp deletion as shown by subsequent mapping and sequencing studies.

Base Sequence↗
PubMed1997

The relationship between sperm morphology and rates of fertilization, pregnancy and spontaneous abortion in an in-vitro fertilization/intracytoplasmic sperm injection programme.

The morphological normality of a spermatozoon is considered to be an important factor in relation to its ability to fertilize an oocyte. We examined the influence of morphology (strict criteria) on the rates of fertilization, pregnancy and spontaneous abortion obtained following conventional in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in our clinical programme. We found our fertilization cut-off values for conventional IVF to be slightly different from those of the Kruger group (10 and 5%, compared to 14 and 5%). We also found the pregnancy rate per transfer to be as good or better in the groups with < 5% normal forms: 36% of these men had a fertilization rate > 50% using conventional IVF, showing that fertilization capacity is not necessarily impaired even in this 'poor prognosis' group. With the exception of the ICSI group with 5-9% normal forms, the rate of spontaneous abortion in this study was similar to or lower than in our IVF/ICSI programme overall. When the 5-9% normal spermatozoa group was divided into those with teratozoospermia as the only factor and those with additional sperm factors, the increased abortion rate was found in the group with multiple sperm factors (67% spontaneous abortions).

Abortion, Spontaneous↗