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Biomedical subjects

Michael Ludwig

Publications and source records attributed to Michael Ludwig.

At least 55 records · Page 3Linked to original sources
PubMed2003

Are major defects in children conceived in vitro due to innate problems in patients or to induced genetic damage?

Birth anomalies recently detected in epidemiological studies indicate greater risks following assisted human reproduction than with natural conception. Some of these conclusions and assumptions are questioned in this paper, and the effects of specific causative factors unique to some infertile couples are analysed. Other recent studies have identified imprinting defects as causes of birth disorders following IVF or intracytoplasmic sperm injection. While few in numbers, they apparently involve unusual factors in conception such as aberrant responses among preimplantation embryos to culture medium or serum. Various genetic and developmental factors in infertile couples influencing the origin of such birth outcomes are assessed, and the significance of imprinting and its embryological roles are discussed.

Congenital Abnormalities↗
PubMed2003

Exclusion of WTAP and HOXA13 as candidate genes for isolated hypospadias.

OBJECTIVE: Different molecular factors have been identified as being associated with isolated or syndrome-associated forms of hypospadias. Nevertheless, the etiology of hypospadias is unknown in 70% of cases. As mutations in the homeobox gene A13 (HOXA13) were all found to be associated with hypospadias in affected males, some types of mutation may solely lead to the isolated form. Moreover, mutations in the Wilms' tumor suppressor gene WT1 have been found in patients with hypospadias without evidence of a Wilms' tumor and, therefore, its recently identified associated protein (WTAP) may be a further candidate gene for the genesis of hypospadias. MATERIAL AND METHODS: A total of 37 patients affected with different forms of isolated hypospadias were analyzed for mutations in their HOXA13 and WTAP genes. RESULTS: With the exception of two novel WTAP polymorphisms, no mutations could be observed. CONCLUSIONS: There seems to be no evidence that isolated hypospadias is commonly caused by mutations in HOXA13 or WTAP genes.

Carrier Proteins↗
PubMed2003

GnRH antagonists in the treatment of infertility.

GnRH analogues were introduced into clinical practice more than 20 years ago. The recent development of new GnRH antagonists (GnRHant) has revolutionized the treatment protocols of infertility. Ovarian stimulation has become easier, safer and more convenient for patients. This review presents data on the physiological background of GnRH and its analogues--agonists as well as antagonists--and highlights the different aspects of the clinical use of these substances. Two protocols, the single dose and multiple dose protocol are available and lead to comparable results. Data from prospective, randomized trials as well as meta-analyses suggest, that a more tailored, individualized approach concerning the day of administration of GnRHant may lead to more favorable results with regard to ovarian response, implantation and pregnancy rates. Possible negative effects of GnRHant, mainly based on in vitro models are discussed. The outcome of children born after GnRHant use has shown, that this medication is safe. On the whole, the use of GnRHant is advantageous as compared to the traditional way of ovarian stimulation, i.e., the GnRH agonist long protocol. However, especially the effectiveness of these compounds will have to be clarified in the near future by carefully designed prospective, randomized studies.

Female↗
PubMed2003

Pregnancy course and health of children born after ICSI depending on parameters of male factor infertility.

BACKGROUND: There is an ongoing debate as to whether the outcome of ICSI is affected by the origin of sperm used or by the severity of male factor infertility. METHODS: Data from a multicentric prospective, controlled cohort study in Germany were analysed to look for differences in pregnancy course and outcome following the use of either ejaculated, epididymal or testicular sperm. Pregnancies were recruited before the 16th week of gestation and included only when they were still ongoing in the 16th week of gestation. A total of 2809 pregnancies was originally included in the study. For 2545 pregnancies with 3199 fetuses/children, data of sperm count and origin were available. RESULTS: Regarding pregnancy course and complications, there was no influence of the origin of sperm. No higher risk of pre-eclampsia was associated with the use of surgically obtained sperm. The birth data of children were similar between the three groups (ejaculated, epididymal, testicular sperm). The risk of major malformation in abortions, stillbirths, livebirths and induced abortions was not significantly different between the three groups, but these results must be treated with caution because of the low numbers in some groups. Major malformation rates did not depend on the number of sperm in the ejaculate. CONCLUSIONS: The course of pregnancy as well as the outcome after ICSI is neither affected by the origin of sperm nor by the number of sperm in the ejaculate.

Child Welfare↗
PubMed2003

Four additional CLCN5 exons encode a widely expressed novel long CLC-5 isoform but fail to explain Dent's phenotype in patients without mutations in the short variant.

BACKGROUND: Dent's disease is caused by mutations in the CLCN5 gene coding for the chloride channel CLC-5. However, sequencing of CLCN5 exonic regions in some patients presenting with low-molecular-weight proteinuria and hypercalciuria - the hallmarks of Dent's disease - failed to identify causative mutations. AIM: Given the observation that some species harbour a CLCN5 mRNA encoding an extended CLC-5 aminoterminus compared with the so far known human form, we worked on the presumption that an orthologous (longer) CLCN5 transcript is also present in humans and that our patients may have mutations herein. METHODS: Extensive databank mining, reverse transcription polymerase chain reaction (RT-PCR) and automated sequencing were used in the search for novel CLCN5 transcripts. The human CLCN5 gene was investigated in 7 patients out of five families by direct automated sequencing of PCR-amplified DNA products. RESULTS: Two new human CLCN5 transcripts expressed in kidney and various other tissues could be identified. These arise from a novel site of transcription initiation, alternative splicing and the use of four additional CLCN5 exons. If being translated, both these mRNAs would lead to an enlarged CLC-5 protein consisting of 816 amino acids by adding 70 aminoterminal residues to the so far known 746-amino-acid-long isoform. Sequence analysis of the henceforward 17 CLCN5 exons revealed no mutation in the patients with a phenotype resembling Dent's disease. CONCLUSIONS: Despite the identification of further targets to explain Dent's disease, the molecular defect in our patients remains to be elucidated. Hence, their phenotype may be explained by mutations that affect so far unknown regulating elements of the CLCN5 gene or another gene(s), probably encoding CLC-5 accessory protein(s).

Adult↗
PubMed2003

Identification of a novel mutation in the human mineralocorticoid receptor gene in a german family with autosomal-dominant pseudohypoaldosteronism type 1: further evidence for marked interindividual clinical heterogeneity.

Pseudohypoaldosteronism (PHA) type 1 presents in infancy with potential life-threatening salt wasting and failure to thrive. Plasma renin activity and aldosterone levels are markedly elevated. PHA1 is inherited in either an autosomal recessive or autosomal dominant trait. The autosomal dominant form manifests with renal salt loss in infancy and a gradual improvement with advancing age. We report the case of a renal form of PHA1 in a German family. PCR and direct sequencing revealed a heterozygous point mutation, c488C>G (S163X), leading to a premature stop codon in the index patient. The segregation analysis revealed the identical mutation in the patient's father, who showed no symptoms of PHA at the time of investigation or before. The family was screened for amino acid polymorphisms in the amiloride-sensitive epithelial sodium channel, which could be a cause for phenotypic differences within the family. PCR and direct sequencing revealed identical epithelial sodium channel haplotypes in the patient and his father. These polymorphisms can, therefore, not be responsible for the difference in clinical presentation. It remains to be elucidated whether other defects or polymorphisms in genes coding for regulatory proteins participating in sodium homeostasis are a cause of the heterogeneity of the clinical manifestations in autosomal dominant PHA1.

Deoxyribonucleases, Type II Site-Specific↗
PubMed2003

Nonsupplemented luteal phase characteristics after the administration of recombinant human chorionic gonadotropin, recombinant luteinizing hormone, or gonadotropin-releasing hormone (GnRH) agonist to induce final oocyte maturation in in vitro fertilization patients after ovarian stimulation with recombinant follicle-stimulating hormone and GnRH antagonist cotreatment.

Replacing GnRH agonist cotreatment for the prevention of a premature rise in LH during ovarian stimulation for in vitro fertilization (IVF) by the late follicular phase administration of GnRH antagonist may render supplementation of the luteal phase redundant, because of the known rapid recovery of pituitary function after antagonist cessation. This randomized two-center study was performed to compare nonsupplemented luteal phase characteristics after three different strategies for inducing final oocyte maturation. Forty patients underwent ovarian stimulation using recombinant (r-)FSH (150 IU/d, fixed) combined with a GnRH antagonist (antide; 1 mg/d) during the late follicular phase. When at least one follicle above 18 mm was observed, patients were randomized to induce oocyte maturation by a single injection of either r-human (h)CG (250 microg) (n = 11), r-LH (1 mg) (n = 13), or GnRH agonist (triptorelin; 0.2 mg) (n = 15). Retrieved oocytes were fertilized by either IVF or intracytoplasmatic sperm injection, depending on sperm quality. Embryo transfer was performed 3-4 d after oocyte retrieval. No luteal support was provided. Serum concentrations of FSH, LH, estradiol (E(2)), progesterone (P), and hCG were assessed at fixed intervals during the follicular and luteal phase. The median duration of the luteal phase was 13, 10, and 9 d for the r-hCG, the r-LH, and the GnRH agonist group, respectively (P = 0.005). The median area under the curve per day (from 4 d post randomization until the onset of menses) for LH was 0.50, 2.34, and 1.07 for the r-hCG, the r-LH, and the GnRH agonist group, respectively (P = 0.001). The median area under the curve per day for P was 269 vs. 41 and 16 for the r-hCG, the r-LH, and the GnRH agonist group, respectively (P < 0.001). Low pregnancy rates (overall, 7.5%; range, 0-18% per started cycle) were observed in all groups. In conclusion, the nonsupplemented luteal phase was insufficient in all three groups. In the patients receiving r-hCG, the luteal phase was less disturbed, compared with both other groups, presumably because of prolonged clearance of hCG from the circulation and the resulting extended support of the corpus luteum. Despite high P and E(2) concentrations during the early luteal phase in all three groups, luteolysis started prematurely, presumably because of excessive negative steroid feedback resulting in suppressed pituitary LH release. Hence, support of corpus luteum function remains mandatory after ovarian stimulation for IVF with GnRH antagonist cotreatment.

Adult↗
PubMed2003

Does the addition of luteinizing hormone in ovarian stimulation protocols improve the outcome?

Ovarian stimulation is an integral part of assisted reproductive technologies (ART). Under physiologic conditions, both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) activity is necessary to guarantee follicle growth and maturation. This can be shown in patients with hypogonadotrophic hypogonadism, who have no endogenous FSH or LH activity. The use of FSH alone in these patients does not result in sufficient follicle growth and oocyte quality. Approximately 75IU of LH activity per day is necessary to guarantee optimal success. The use of gonadotropin-releasing hormone (GnRH) agonists in normogonadotrophic patients may result in suppression of LH levels below a certain threshold, resulting in suboptimal outcomes. The question under discussion in this article is the threshold level of LH below which exogenous LH activity should be added to provide optimal ovarian response. Different studies indicate that the endogenous LH level should be 0.5-1.5IU in long-term protocol situations. Patients treated in ultra-long GnRH agonist protocols, as well as older patients, patients with a low response to gonadotropin treatment, and patients treated with a GnRH antagonist protocol may benefit from exogenous LH activity. There are three ways of adding LH activity in ovarian stimulation cycles. Nowadays, lutropin alfa (recombinant LH) may be the optimal choice since it has no chorionic gonadotropin activity and allows individual dosage titration. Every menotropin preparation currently on the market contains some chorionic gonadotropin activity. However, more data are necessary before evidence-based recommendations regarding LH supplementation in ovarian stimulation protocols can be given.

Humans↗
PubMed2002

A novel stable polyalanine [poly(A)] expansion in the HOXA13 gene associated with hand-foot-genital syndrome: proper function of poly(A)-harbouring transcription factors depends on a critical repeat length?

Hand-foot-genital syndrome (HFGS) is a dominantly inherited congenital malformation affecting the distal limbs and genitourinary tract. Here, we describe the phenotype and its molecular basis in a family that presented with HFGS. Genetic analysis revealed that the condition is caused by an 18-bp in-frame duplication within a cryptic trinucleotide repeat sequence encoding an 18-residue polyalanine tract in the homeoboxgene ( HOX) A13. This mutation expands the stretch with six extra alanine residues. Similar types of mutation (plus eight alanines) have recently been found in another HFGS family and also in the human HOXD13 gene (plus seven up to plus 14 residues) where it leads to synpolydactyly (SPD), a further congenital limb malformation rarely associated with genital abnormalities. As observed in our family, all the expanded tracts encoding polyalanine, either reported for HOXA13 or HOXD13, are quite stable when transmitted within affected families. Unlike disorders with unstable expansions of perfect trinucleotide repeats the molecular mechanism underlying these polyalanine expansions should be unequal crossing-over rather than replication slippage. The alanine tract elongation may prevent protein-protein interactions of the mutant HOXA13, thereby inducing a localized heterochrony in the sequence of distal limb and genitourinary development.

Alleles↗
PubMed2002

Comparison of cryopreservation outcome with gonadotropin-releasing hormone agonists or antagonists in the collecting cycle.

OBJECTIVE: To compare the pregnancy rates of frozen-thawed 2-pronucleate (2PN) oocytes obtained either in a long protocol or in an antagonist protocol and ovarian stimulation with either human menopausal gonadotropin (hMG) or recombinant follicular stimulating hormone (recFSH). DESIGN: Retrospective data analysis. SETTING: Academic infertility center. PATIENT(S): Three hundred forty-two infertile couples who underwent a transfer of cryopreserved 2PN oocytes. INTERVENTION(S): hMG (n = 194) or recFSH (n = 92) in a long protocol or hMG (n = 16) or recFSH (n = 40) stimulation under pituitary suppression with the GnRH antagonist Cetrotide was used. The 2PN oocytes were transferred after endometrial preparation using E(2) valerate and vaginal progesterone (Crinone 8% vaginal gel). MAIN OUTCOME MEASURE(S): Implantation, pregnancy, and abortion rates. RESULT(S): Implantation rates in the freeze-thaw cycles were 5.6% (hMG) and 3.8% (recFSH) with 2PN oocytes from the long protocol and 7% from the antagonist cycles, irrespective of whether hMG or recFSH was used. Pregnancy rates were similar independent of whether they resulted from the long-protocol cycles with hMG (15.4%) and recFSH (13.1%) or from the antagonist protocol cycles with hMG (25.0%) and recFSH (17.5%). CONCLUSION(S): The potential to implant is independent of the gonadotropin-releasing hormone analogue and gonadotropin chosen for the collection cycle when previously cryopreserved 2PN oocytes were replaced after thawing in the cleavage stage.

Adult↗
PubMed2002

Ovarian stimulation: from basic science to clinical application.

Treatment for infertility, including ovarian stimulation, was first introduced almost 100 years ago. At this time, radiation therapy became an established treatment, and it was only some decades later that the problem of radiation-induced cancer emerged. Non-human gonadotrophins, such as pregnant mare serum gonadotrophin (PMSG), and human pituitary gonadotrophins (HPG), were commonly used for hormonal stimulation procedures. However, use of PMSG led to antibody formation, and it was therefore only useful for the first treatment cycle. HPG produced good results, but its use came to an end in the late 1980s when it was linked to the development of Creutzfeldt-Jakob disease. The first hormonal product from human menopausal urine to be used was human menopausal gonadotrophin (HMG), followed later by purified preparations of this product. All of these preparations contained a high percentage of unknown urinary proteins, which interfered with batch-to-batch consistency. This changed with the introduction of recombinant gonadotrophins, produced from an immortalized/standardized mammalian cell line (CHO). More recent developments include the introduction of long-acting gonadotrophin formulations. The development of gonadotrophin-releasing hormone (GnRH) analogues and more recently the use of GnRH antagonists has helped to improve ovarian stimulation protocols by optimizing their efficacy, and making them easier to administer.

Animals↗
PubMed2002

Characterization of the dehydroepiandrosterone (DHEA) metabolism via oxysterol 7alpha-hydroxylase and 17-ketosteroid reductase activity in the human brain.

Dehydroepiandrosterone and its sulphate are important factors for vitality, development and functions of the CNS. They were found to be subjects to a series of enzyme-mediated conversions within the rodent CNS. In the present study, we were able to demonstrate for the first time that membrane-associated dehydroepiandrosterone 7alpha-hydroxylase activity occurs within the human brain. The cytochrome P450 enzyme demonstrated a sharp pH optimum between 7.5 and 8.0 and a mean KM value of 5.4 micro m, corresponding with the presence of the oxysterol 7alpha-hydroxylase CYP7B1. Real-time RT-PCR analysis verified high levels of CYP7B1 mRNA expression in the human CNS. The additionally observed conversion of dehydroepiandrosterone via cytosolic 17beta-hydroxysteroid dehydrogenase activity could be ascribed to the activity of an enzyme with a broad pH optimum and an undetectably high KM value. Subsequent experiments with cerebral neocortex and subcortical white matter specimens revealed that 7alpha-hydroxylase activity is significantly higher in the cerebral neocortex than in the subcortical white matter (p < 0.0005), whereas in the subcortical white matter, 17beta-hydroxysteroid dehydrogenase activity is significantly higher than in the cerebral neocortex (p < 0.0005). No sex differences were observed. In conclusion, the high levels of CYP7B1 mRNA in brain tissue as well as in a variety of other tissues in combination with the ubiquitous presence of 7alpha-hydroxylase activity in the human temporal lobe led us to assume a neuroprotective function of the enzyme such as regulation of the immune response or counteracting the deleterious effects of neurotoxic glucocorticoids, rather than a distinct brain specific function such as neurostimulation or neuromodulation.

17-Hydroxysteroid Dehydrogenases↗
PubMed2002

The use of cryopreserved mature and immature testicular spermatozoa for intracytoplasmic sperm injection: risks and limitations.

Treatment of severe male subfertility has become available since the intracytoplasmic injection of a single sperm into an oocyte was successfully applied for the first time in 1992. Moreover, with the use of fresh and cryopreserved epididymal and testicular spermatozoa for this procedure, fertilization and pregnancies could be accomplished. This review addresses the development and performance of these techniques and discusses achievements and problems as well as future aspects of the feasibility of early spermatid injection. Furthermore, limitations of these procedures and concerns with regard to genetic and epigenetic risks of using immature gametes are stressed.

Cryopreservation↗
PubMed2002

Novel NCCT gene mutations as a cause of Gitelman's syndrome and a systematic review of mutant and polymorphic NCCT alleles.

BACKGROUND: Gitelman's syndrome (GS) is characterized by hypokalemic metabolic alkalosis, hypomagnesemia and hypocalciuria and these phenotypic features have been shown to be attributable to mutations in the gene encoding the thiazide-sensitive Na/Cl cotransporter (NCCT). Until now, 55 different mutations have been reported and most of the families affected with GS exhibit autosomal recessive inheritance. METHODS: All 26 exons of the human NCCT gene were investigated in 2 German NCCT-deficient patients and their families. Mutation detection was performed by either direct automated sequencing of polymerase chain reaction (PCR)-amplified DNA products or by sequence analysis of cloned PCR products. RESULTS: In a 47-year-old German GS female a novel non-conservative missense mutation (S314F) and a complex deletion/insertion in the NCCT gene were found to be associated with the disorder. A further novel non-conservative substitution (S402F) together with a frequently observed R209W exchange were found in a 19-year-old German GS female. CONCLUSIONS: The observation of a compound heterozygote state in both females affected and the absence of a GS phenotype in their relatives carrying a single mutant allele is consistent with an autosomal recessive pattern of inheritance.

Adult↗
PubMed2002

Gonadotropin-releasing hormone antagonists: pharmacology and clinical use in women.

Gonadotropin-releasing hormone (GnRH) antagonists are now widely used in protocols of patients with controlled ovarian hyperstimulation to treat infertility. By competitively binding to the pituitary GnRH receptor, they lead to a rapid suppression of gonadotropins and consecutively sex hormones. In the past, GnRH agonists have been exclusively used for these patients, with the disadvantage of an initial rise of gonadotropins--the flare-up effect. Several trials comparing the agonistic and antagonistic analogs of GnRH found no significant differences in oocyte quality, fertilization and pregnancy rates. Slightly lower implantation and pregnancy rates, and estradiol levels, in patients treated with GnRH antagonists has raised concern about eventual extrapituitary adverse effects. However, no convincing evidence has yet been found for any detrimental ovarian effects of GnRH antagonists. The lower rate of ovarian hyperstimulation syndrome, a potentially severe disadvantage of infertility treatment, is a positive feature of GnRH antagonists. The key point is that GnRH antagonists have been proven to be as effective and safe as GnRH agonists. This broadens the spectrum of indications for GnRH antagonists to sex hormone-dependent disorders like endometriosis, uterine fibroids, and gynecological cancers such as breast and ovarian cancer.

Female↗
PubMed2001

Impact of intracytoplasmic sperm injection on the activation and fertilization process of oocytes.

Intracytoplasmic sperm injection (ICSI) is used worldwide to treat preferentially severe cases of male factor infertility. In this review, data regarding the processes of oocyte activation and fertilization in non-assisted conception, conventional IVF and ICSI are discussed. The second messenger calcium shows a typical pattern after ICSI, which is different from that after subzonal insemination (SUZI), which is closer to the conditions of normal fertilization. The onset of calcium spikes is delayed. Sometimes a monophasic calcium pattern, in animals typical for parthenogenetic activation, is observed despite normal subsequent oocyte activation. Furthermore, the frequency of spikes is higher after SUZI, and only one phase instead of two is observed after ICSI for the second onset of calcium release. These alterations may be explained by the differences in oocyte activation after ICSI, since no oolemma-sperm contact is present. Sperm decondensation also follows another pattern after ICSI: as long as residuals of the acrosome are present on the sperm head, no sperm decondensation takes place at that site. Therefore, decondensation is delayed and pronucleus formation, especially that of the male pronucleus, takes longer after ICSI as compared with conventional IVF. Since studies have shown that gonosomes are located preferentially in the apical part of the sperm nucleus, this was proposed to be an explanation for a higher incidence of gonosomal aberrations in offspring after ICSI. However, other explanations, taking clinical data like the background risk of the parents into account, can also be offered for this phenomenon. These alternative theories are more likely to be associated with a slight instead of a frank increase in gonosomal aberrations. The inheritance of paternal mitochondrial DNA seems not to be a problem after ICSI, as shown by different studies. Mitochondrial DNA can be demonstrated in embryos after conventional IVF as after ICSI up to the blastocyst stage but not in children born after ICSI. Finally, lesion of the meiotic spindle by the ICSI procedure seems not to be a problem when data from different studies are taken into account. As assumed also at the beginning of the ICSI era, the meiotic spindle is almost always located in an area of < 90 degrees deviation from the polar body axis. Therefore, intrusion of the microinjection needle at the 90 degrees position might not endanger the spindle apparatus. To conclude, several studies using different approaches might show differences in the oocyte activation pattern, the 'choreography of fertilization' and pronucleus formation after ICSI. However, this different pattern does not necessarily mean that ICSI per se is a problem for embryonal development. The different pattern can be explained by the fact that ICSI uses another means of oocyte entry than the normal fertilization process. The clinical data of a high fertilization, cleavage and implantation rate, and especially the data from newborn babies, show that ICSI is a reliable procedure.

Journal Article↗
PubMed2001

Fertilization rate and preimplantation development after intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is the treatment of choice in cases of male factor infertility. In the present review, all data from the literature regarding early fertlilization and preimplantation embryo development are analysed. ICSI seems to offer better fertilization rates than conventional IVF, even in the absence of a male factor and in prospective, randomized studies on sibling oocytes. Blastocyst formation is lower in ICSI than in IVF. This may be due to sublethal disturbances in oocyte architecture and function. Implantation and pregnancy rates, however, are similiar in ICSI and conventional IVF. Therefore, it can be concluded that ICSI offers a safe approach, as far as is known to date, for the treatment of male factor infertility. Embryo selection in cases of sublethal damage to oocytes seems to take place in the early preimplantation stages. Nor does the risk of monozygotic twinning, which was also proposed to be higher in ICSI compared with conventional IVF, seem to increase with this procedure.

Journal Article↗