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Biomedical subjects

H Zech

Publications and source records attributed to H Zech.

At least 19 recordsLinked to original sources
PubMed2006

Apoptosis affects integration frequency: adult stem cells injected in blastocysts show high caspase-3 activity.

Chimeric organisms are commonly generated by injecting stem cells into blastocysts. Embryonic stem cells injected into the blastocoel cavity participate in the further development of the embryo. Adult stem cells have also been used in injection experiments to study their potential plasticity. In this study we focused on the early fate of injected human adult hematopoietic stem cells (HSCs). HSCs were followed immunohistochemically 1-19 h after injection into murine blastocysts. We found that they only rarely attached and integrated into the blastocysts. The high rate of loss of injected cells after prolonged in vitro culture of the chimeras can be explained by apoptosis. Our findings are consistent with previous studies reporting a low rate of integration of adult cells injected to produce chimeric embryos, but this is the first demonstration that the low efficiency of adult stem cell injections into blastocysts is influenced by apoptosis.

Adult↗
PubMed2005

Vitrification of hatching and hatched human blastocysts: effect of an opening in the zona pellucida before vitrification.

Partially or completely hatched blastocysts were obtained from women with at least one previous cycle with failure of implantation. On day 5 after fresh embryo transfers, the remaining vitrification cycles (VC) were divided into three categories according to the types of blastocysts. The first group (20 VC) contained blastocysts (n = 38) with open zona pellucida (ZP), the second group of 34 VC contained blastocysts (n = 100) with both open and intact ZP, while the third group (14 VC) contained blastocysts with intact ZP (n = 39). Blastocysts were exposed to two mixtures of cryoprotectants. At 24 h after warming, survival rates of 82% (31/38), 72% (72/100) and 64% (25/39) were observed in the three groups respectively. Numbers of embryo transfers and blastocysts in the three groups respectively were 20, 31 and 13 transfers, and 31, 60 and 25 blastocysts, resulting in corresponding ongoing pregnancy rates per VC of 35, 26 and 21%. Blastocysts with a larger blastocoelic cavity survived vitrification better when they had partially or completely hatched. Survival rates significantly increased (P < 0.01) from 55 to 81% after warming expanded blastocysts with an intact ZP (n = 42) compared with an open ZP (n = 54) respectively. This study shows that partially or completely hatched blastocysts can be cryopreserved by a simple vitrification procedure using the hemi-straw as embryo carrier.

Adult↗
PubMed2005

Paraffin-embedded manipulated blastocysts: a tool to demonstrate stem cell plasticity?

One of the big question marks in current stem cell research is whether there is true plasticity of adult progenitor cells (APC) or if cell fusion is the principle source of the supposed plasticity. The generation of chimeras by injecting adult progenitor cells into blastocysts is not new. This paper describes an efficient embedding technique for murine blastocysts injected with human APC. This method could help in establishing a novel tool to analyse the process of plasticity, if it truly exists. If this is the case, this technology could be of great help to characterize surface markers of stem cells in great detail. On the other hand, fusion of cells could also be investigated. A system of embedding blastocysts was set up using paraffin for further analysis by means of light microscopy and immunohistochemistry. The embedding of the chimaeras consists of fixing them first with paraformaldehyde in phosphate-buffered saline (PFA/PBS), embedding them in gelatine, fixing the gelatine block with PFA/PBS and finally fixing the gelatine block in a Petri dish by embedding it in paraffin. Using this protocol, the morphology of the blastocysts is well preserved.

Animals↗
PubMed2002

Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification.

BACKGROUND: In 1996, with the introduction of sequential media, we set up a programme of cryopreservation of supernumerary morulae (day 4) and blastocysts (day 5) using a vitrification procedure. Our results showed that the efficiency of the vitrification method was dependent on the stage of embryo development and was negatively correlated with the expansion of the blastocoele. We postulated that a large blastocoele might disturb cryopreservative potential due to ice crystal formation during the cooling step. We analysed therefore the effectiveness of reducing before vitrification the volume of the blastocoelic cavity. METHOD: Day 4 and day 5 embryos were vitrified in 40% ethylene glycol-18% Ficoll and 0.3 mol/l sucrose before plunging the straws directly into liquid nitrogen. Artificial shrinkage of the blastocyst was achieved after pushing a needle into the blastocoele cavity until it contracted. RESULTS: The survival rate post-thawing of day 4 and intact day 5 embryos was correlated with the volume of the blastocoele. In the control group only 20.3% blastocysts or expanded blastocysts survived as compared with 54.5 and 58.5% with morulae and early blastocyst respectively. After puncturing the blastocoelic cavity, an increase in the survival rate of up to 70.6% was noted. The pregnancy rates were improved after the artificial shrinkage procedure (20.5%) compared with the control intact blastocyst group (4.5%) (not significant). After reduction of the blastocoelic cavity, a significant increase in the implantation rate per vitrified blastocyst was observed (12.0 versus 1.4% P < 0.01). CONCLUSIONS: Our results showed that survival rates in cryopreserved expanded blastocysts could be improved by reducing the fluid content. This was presumably because mechanical damage caused by ice crystal formation was avoided. These observations should be considered when establishing a strategy and a protocol for cryopreservation of day 5 embryos.

Adult↗
PubMed2000

Congenital malformations after intracytoplasmic injection of spermatids.

Spermatid microinjection into oocytes was applied in cases of intracytoplasmic sperm injection (ICSI)/testicular sperm extraction (TESE) where no spermatozoa could be found in numerous testicular samples. Although several pregnancies were obtained with this procedure, serious concerns remain regarding its safety. Although the relevance of the injection of spermatids is by no means certain, we wish to report that from four pregnancies obtained after injection of elongated spermatids, two cases of major malformation resulted.

Adult↗
PubMed1999

A prospective, randomized clinical trial comparing 150 IU recombinant follicle stimulating hormone (Puregon((R))) and 225 IU highly purified urinary follicle stimulating hormone (Metrodin-HP((R))) in a fixed-dose regimen in women undergoing ovarian stimulation.

A prospective, randomized, open, multicentre (n = 3) study was conducted to compare the efficacy and efficiency of a fixed daily dose of 150 IU (3x50 IU) recombinant follicle stimulating hormone (recFSH, Puregon((R))) and 225 IU (3x75 IU) highly purified urinary FSH (uFSH-HP, Metrodin-HP((R))) in women undergoing ovarian stimulation prior to in-vitro fertilization treatment. A total of 165 women were treated with FSH, 83 subjects with recFSH and 82 subjects with uFSH-HP. In the recFSH group a mean number of 8.8 oocytes were retrieved, compared with 9.8 in the uFSH-HP group (not statistically significant). In the recFSH group, a significantly lower total dose was required compared to the uFSH-HP group, 1479 versus 2139 IU, respectively (P < 0.0001; 95% confidence interval -747 to -572). Treatment with recFSH resulted in a significantly higher embryo development rate (69.6 versus 56.2%; P = 0.003) and more embryos accessible for the embryo freezing programme (3.3 versus 2.0; P = 0.02) compared to uFSH-HP. The vital pregnancy rate per cycle started was 30.2 versus 28.3% in the recombinant and urinary FSH group, respectively. It is concluded that treatment outcome of a fixed daily dose of 150 IU recFSH is comparable to a fixed daily dose of 225 IU uFSH-HP. However, a significantly lower total dose was needed in the recFSH group (nearly 700 IU less).

Adolescent↗
PubMed1998

Is there a future for spermatid injections?

Microinjection of spermatids into oocytes has proven to be a successful assisted reproduction procedure in the animal model. In the human, low fertilization and cleavage to the 4-cell stage were reported after intracytoplasmic sperm injection (ICSI) with round spermatids. In comparison with a conventional ICSI-testicular sperm extraction (TESE) programme, the implantation rate after round spermatid injection is dramatically low. Different problems have been encountered during the development of the spermatid injection technique and they could be partially responsible for the lower outcome when using round spermatids. Compared with the round spermatid cells, spermatids in the elongation phase are easy to isolate and identify from other round cells present in a wet preparation. The morphological identification does not reveal anything about the viability or the genetic normality of the round spermatids. Severe testicular dysfunction may have consequences on the quality of the few spermatogenic cells present. Others factors, such as the pathology of the patient, play an important role in the successful treatment. Even if the results are extremely low, spermatid injection seems more favourable for men who have already proven their capacity to produce some spermatozoa. A spermatogenic block at the round spermatid level has led to early abortions, increasing the suspicion of the role of a genetic factor. In order for this technique to be safe for use in clinics, more intensive work is needed to improve the selection and handling of cells and to ascertain the genomic imprinting and gene expression necessary for embryonic development. Hence, when using immature cells for conception, the screening of the patient and the follow-up of the pregnancies and babies should be mandatory.

Animals↗
PubMed1997

Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation.

Spermatid microinjection into oocytes has proven to be a successful assisted reproduction procedure in the animal model and in the human species, since in the latter a few full-term pregnancies were actually obtained. Patients entering our spermatid injection study included those with a total absence of spermatozoa in the testicular tissue notwithstanding previous positive biopsies (n = 29): an obstructive problem (n = 3), secretory azoospermia (n = 26), and those with total arrest at the spermatogenesis level in previous explorative biopsies (n = 15). In the latter group, absence of spermatids was recorded in four cases. Mature, elongated, elongating and round spermatids (ROS) were injected in respectively 3, 2, 3, and 32 attempts. A total of 260 metaphase II oocytes were injected with ROS, 36 oocytes with spermatids at other stages of maturity. The rates of oocytes showing two pronuclei (2PN) and two polar bodies reached 22% and 64% respectively after injection of round or elongated-mature spermatids. The fertilization rate after ROS injection was influenced by the percentage of spermatozoa observed in a previous biopsy. Patients with a positive preliminary biopsy had significantly more 2PN (33%) when compared to those with a severe spermatogenic dysfunction and in whom no spermatozoa were found (only 11%) (P < 0.05). Incubation of oocytes in calcium ionophore after ROS injection had a positive effect on the rate of 2PN formation (36 versus 16%). Ninety per cent of all the normally fertilized oocytes cleaved. The percentage of grade A and B embryos depended on the type of injected cells: 12% after ROS and 30% with the other types of haploid cells. A total of 39 transfers resulted in five pregnancies: three full term with healthy babies delivered (one after ROS injection, and two after injection of an elongating and a mature spermatid), one 4 months ongoing (after elongating spermatid injection) and one miscarriage at 4 weeks (after elongated cell injection). Compared to our conventional intracytoplasmic sperm injection-testicular sperm extraction (ICSI-TESE) programme, the implantation rate after ROS injection was very low (5.5 versus 10.5%).

Animals↗
PubMed1993

[Comparison of preparation techniques with reference to final spermatozoa concentration and survival and fertilization rate in vitro in normal and pathologic ejaculates].

Various methods are available in the preparation of normal and pathological ejaculates within the framework of available techniques of assisted reproduction. The authors examined the recovery rate, the long-term motility and the fertilization rate in vitro after both normal and pathological test results, of preparations, using one of the three most commonly used techniques. Centrifugation with its resulting swim up (Z) is a method still widely used because of the good sperm recovery rate it produces. However, in our tests, it achieved the lowest level capacity, compared with sperm separation by migration/sedimentation, with resultant swim up (M) and Percoll gradient centrifugation (P). The statistically highly significant fast decline of motility after preparation by Z, compared with the other two above mentioned techniques, in both normal and pathological ejaculates is a further indication, that Z has a negative influence upon the integrity of the spermatozoa. The results of research submitted here suggest the abandonment of Z in its standard form in favour of other techniques. After normal test results, both M and P might be used, but in the case of pathological ejaculate, P would be recommended.

Female↗
PubMed1992

[Results of a prognosis study of in vitro fertilization after brief psychotherapy].

During the last four years, our research has been focussed on prediction criteria for IVF after a Short Course of psychotherapy. Our study can be divided in two phases: In an earlier phase (up to 1990), prediction criteria were derived from "expert" rating, whereas in a later phase (from 1990 onwards) they were derived by self-rating of the patients. The predictions refer to changes in the patients' behaviour, affective state, mood and attitude (acronym: BAMA) towards their infertility. Our work is based on the hypothesis, that a perceivable change in BAMA and in how the patients relate to their wanting a child, represents the solution of the problem, which facilitates the consequential events of either pregnancy or acceptance of childlessness or else adoption. As a rule, the absence of such a change entails none of the consequential events. - We report on and discuss the results of the earlier, as well as the later phase and finally compare them as to how well they correlate with prediction and consequences. A marked correlation could be found between changes in BAMA and the occurrence of pregnancy or acceptance of childlessness in both phases of our study. Comparing the two phases (expert- vs. self-rating) on the accuracy of prediction, we could find almost identical results. Because of the easier and more economical application of self-rating we decided to continue with this method and to refine it for further research.

Adaptation, Psychological↗
PubMed1992

[Steroid and protein hormone concentrations in serum and follicular fluid after stimulation for in vitro fertilization].

Sixty patients with tubal infertility were stimulated for IVF with a fixed schedule consisting of clomiphene and pure follicle stimulating hormone. They responded with an optimal estradiol increase and 2 to 4 embryos were transferred. Conception cycles (n = 17) could be differentiated from non-conception cycles (n = 43) by serum estradiol, serum progesterone, the serum progesterone/estradiol-ratio and serum LH concentrations. Testosterone, androstenedione and FSH in serum and follicular fluid showed no significant relation to a possible therapy outcome. We conclude from our findings, that, during the peri-implantation period, certain LH patterns followed by an optimal progesterone/estradiol-ratio in the serum support an embryo survival after transfer to the uterus.

Androstenedione↗
PubMed1992

Abbott IMx and serono MAIAclone assays compared for lutropin determinations in urine.

The urinary lutropin (luteinizing hormone; LH) profiles of 20 patients were determined in 10 consecutive urine samples (n = 200) with a manual technique (MAIAclone, Serono; y) and a fully automated (Abbott IMx; x) technique. For 132 single determinations within the range of detection, the regression line was y = 1.39 x - 0.07 (r = 0.85). Retrospective analysis of the LH profiles obtained by the IMx yielded the classification "not pregnant" (negative) in 14 cases, of which 13 were correctly classified; 6 profiles were classified as "pregnant" (positive), which correlated with the clinical findings in 4 cases. Nine of the LH profiles analyzed with the MAIAclone method were classified as negative, which was correct in seven cases. Retrospective analysis of 11 LH profiles established with the MAIAclone assay gave positive results, which agreed with the clinical outcome in three cases. The diagnostic sensitivity of the IMx and MAIAclone methods was 87% and 47%, respectively; diagnostic specificity was 80% and 60%, respectively. In smaller series (< or = 40 samples), the IMx method has considerable advantages over the MAIAclone method in cost and convenience, because standard curves can be stored and assays can be performed as single determinations.

Autoanalysis↗
PubMed1991

[Effect of incubation time in deep freezing human sperm].

For the protection of spermatozoa against deleterious influences during freezing and thawing, so-called "cryoprotective agents" are mixed with the ejaculate before freezing. Clinical experience indicated a decrease in the number of post-thaw motile sperms after increasing the incubation period of sperms with the added cryoprotective agents and prompted us to investigate this finding. In experiment 1 we used 21 ejaculates which had a mean motility rate of 63.3%. The ejaculates were divided into 3 portions (1 a, 1 b, 1 c), which were frozen after 10 (1 a), 30 (1 b), and 60 (1 c) minutes of equilibration respectively. After thawing, 18.4% (1 a), 12.8% (1 b), and 7.1% (1 c) of the sperms were motile. This decrease in sperm motility was highly significant (p = 0.0012). Cryosurvival was investigated more fully within the first 10 minutes of incubation in a second experiment with another 18 ejaculates. The ejaculates were divided into 2 portions; portion 2 a was mixed with the cryoprotectant and frozen immediately and portion 2 b was frozen after a 10 minute incubation time. There was no significant difference in post-thaw motility and in recovery rate between the groups 2 a and 2 b. Our results indicate that the cryoprotective agent has a toxic time-dependent negative influence on sperm quality during equilibration, which significantly increases after an incubation time of more than 10 minutes. We consider that the results of this study are applicable to other glycerol-containing cryoprotectants and we recommend that the incubation period is kept short.

Humans↗
PubMed1991

[Significance of a specific LH profile after follicle stimulation for producing a pregnancy with IVF/ET].

We report on a retrospective study of LH-profiles after follicular augmentation for in vitro fertilization in order to be able to predict more precisely the chance of success. In 30 out of 34 patients we were able to retrospectively characterize the patients as potentially pregnant or not pregnant. In a prospective application of this statistical method, it should be possible to decide on the basic of specific LH-profiles whether to continue with follicular aspiration or to cancel the cycle in order to increase the pregnancy rate.

Chorionic Gonadotropin↗
PubMed1991

[Effects of short-term psychotherapy on the success of an in vitro fertilization/embryo transfer program].

A pilot study investigates how the implementation of brief psychotherapy affects the results of an IVF/ET program. A main criterion in therapy is the reduction of the patients' psychic stress who are led to find new problem solutions building upon the abilities and faculties they already show. Emphasis is put on their gaining a cooperative relationship towards their body. Of the 15 patients in this study, 7 became pregnant by IVF after brief psychotherapy, which corresponds to a ratio of 47%. We assume that this is due to a change of attitude towards the desire for pregnancy as well as towards the possible ways of its realization.

Adaptation, Psychological↗
PubMed1988

[The significance of specific LH patterns before and following ovulation induction with hCG within the scope of in vitro fertilization and simplified diagnosis].

In 55 patients follicular stimulation for in vitro fertilisation (IVF) was carried out with a combination of clomiphene (Cl)/human follicle stimulating hormone (FSH) and human chorion gonadotropin (hCG) for ovulation induction. Two days before the anticipated hCG application LH measurements in 4-hourly urine samples were started and continued until follicular aspiration. A LH-IRMA (LH-MAIAclone, Serono) utilising monoclonal antibodies enabled the detection of LH also after hCG. In order to facilitate the organisation and to exclude sources of error the LH concentrations were correlated to the urinary creatinine concentrations and expressed as LH units per gram creatinine (LH U/g Cr). 6 characteristic LH profiles could be found: 31 patients (group a) had a baseline between 5-12 LH U/g Cr with an abrupt increase 10-12 hours after hCG, 2 patients (group b) had slightly elevated LH levels (10-15 LH U/g Cr), after hCG a decrease and 20-30 hours later a distinct increase. 7 patients (group c) had an endogenous LH surge before the hCG application. 3 patients (group d) had very low LH values (below 5 LH U/g Cr), 6 patients (group e) markedly increased concentrations (above 20 LH U/g Cr) with no change after hCG. 6 patients (group f) had significant LH fluctuations. Of the 15 pregnancies 12 were in group a, 2 in group b, 1 in group c, none in the groups d-f. The good correlation between the LH concentrations and creatinine concentrations in the urine proved the usefulness of this method and contributed well to organisational simplifications.(ABSTRACT TRUNCATED AT 250 WORDS)

Chorionic Gonadotropin↗