Biomedical subjects

H W Michelmann

Publications and source records attributed to H W Michelmann.

At least 19 recordsLinked to original sources

PubMed2006

Cryopreservation of human embryos.

Cryopreservation of human embryos from the 2-cell stage up to the morula stage is a safe procedure which has been carried out for the last 25 years. Experience with blastocyst cryopreservation is still limited and pregnancy rates after the use of frozen, thawed blastocysts vary extremely. Vitrification has improved the success of embryo cryopreservation. However, this technique cannot yet be considered as a routine procedure. Despite all of the advantages for infertile couples, cryopreservation of human embryos creates severe ethical problems, because of surplus frozen embryos which either have to be destroyed or perhaps used for research. Embryo adoption may provide a solution to solve imminent medical, ethical and social problems.

Blastocyst↗

PubMed2006

Failure of phospholipid hydroperoxide glutathione peroxidase expression in oligoasthenozoospermia and mutations in the PHGPx gene.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein belonging to the family of glutathione peroxidases. PHGPx has long been considered a major antioxidant that, in cooperation with vitamin E, protects biomembranes. To determine the expression pattern of PHGPx mRNA in human, quantitative RT-polymerase chain reaction (PCR) analyses using RNA from different embryonal and adult tissues were performed. A predominant expression was found in testes. In spermatozoa, PHGPx was found to be localized in the mid-piece of spermatozoa. We studied the relationship between spermatozoa PHGPx expression, mutations in PHGPx gene and human oligoasthenozoospermia, a defect in which both the number and the motility of spermatozoa are significantly below normal. Spermatozoa specimens from 45 infertile males were analysed for fertility-related parameters according to World Health Organisation and were classified as suffering from oligoasthenozoospermia. Two patients (4.44%) showed no expression of PHGPx and in nine patients (20.00%), a reduced expression of the enzyme was observed. DNA sequences of various regions of the PHGPx gene (coding, 5'flanking region and intron 1) from these patients and 58 fertile volunteers were analysed for mutations by PCR amplification and direct sequencing. Sequence data revealed no cause/effect relationship for any of the variants. From these data it can be concluded that oligoasthenozoospermia is associated with a decrease in the level of expression of PHGPx in the spermatozoa of some infertile men (24.44%), but is not linked to mutations in PHGPx gene.

Female↗

PubMed2006

Structural, biochemical and functional aspects of sperm-oocyte interactions in pigs.

Polyspermic fertilization is still a major issue in porcine IVF systems. New information is available to characterize the zona pellucida (ZP) at different developmental stages by scanning electron microscopy (SEM) and by confocal microscopy to show the distribution of ZP glycoproteins. SEM images indicated no differences between in vivo and in vitro matured oocytes; however a change in the surface structure between immature and matured oocytes, as well as between mature oocytes and preimplantation embryos was obvious. In addition, spermatozoa were more tightly fixed in the ZP of in vivo produced compared to the ZP of in vitro produced embryos. The ZP undergoes biochemical changes during maturation prior to fertilization. The acidity of the ZP increases during maturation as indicated by a shift of 1.3 pl units for ZPB/ZPC and 0.8 pl units for ZPA in 2D gel electrophoresis, which is based on increasing sulfation of the oligosaccharides during maturation. Mass spectrometry in combination with in-gel deglycosylation allowed the mapping of new glycosylation sites. Functionality of the ZP also depends on its maturation status. Induction of the acrosome reaction was delayed when capacitated spermatozoa were exposed to immature oocytes.

Animals↗

PubMed2003

Oocyte-sperm interaction in the course of IVF: a scanning electron microscopy analysis.

Interaction between the spermatozoon and the zona pellucida during the first steps of fertilization was analysed on approximately 500 polyploid and unfertilized IVF oocytes by scanning electron microscopy (SEM). Oocytes demonstrate high inter- and intra-individual variations in size and morphology which do not correlate either with the maturity of the oocytes or with the age of the women. During gamete interaction, corona radiata cells are widely dispersed around the zona but are still in contact with it through cytoplasmic filaments. Channels between granulosa cells guide spermatozoa towards the zona. In the course of fertilization, different types of attachment of the spermatozoon to the oocyte occur. Most commonly, a flat, tangential attachment of the sperm head to the surface of the zona appears, which is then followed by intrusion into the zona in precisely this horizontal position. However, vertical binding with penetration by the tip of the head first also occurs. In oocytes where large, cluster-like numbers of bound spermatozoa are visible, vertical binding and penetration is the most usual position. In the process of gamete interaction, both spermatozoa and zona pellucida are actively involved. Spermatozoa, including their tails, which are attached to the zona, are overgrown by filaments of zona material. These filaments of the zona are made of granules, which are the basic components of zona material. After the removal of the zona pellucida by laser, the oolemma becomes visible. It is covered by microvilli of highly variable numbers. Between these microvilli, cortical granules are evident, and appear even before sperm penetration.

Female↗

PubMed2003

Influence of autogenous leucocytes and Escherichia coli on sperm motility parameters in vitro.

Urogenital infections are considered important factors in male infertility. In this in vitro study we have evaluated the impact of leucocytes in association with an artificial infection with Escherichia coli on the motility of human spermatozoa. Ejaculates and blood samples were obtained from healthy donors with normal semen parameters. Ejaculates were prepared by swim-up technique and five fractions were isolated for incubation. Leucocyte subtypes were separated from blood samples by gradient centrifugation. Purified sperm suspensions were adjusted to a concentration of 20 x 106 ml-1 and incubated with lymphocytes/ monocytes, polymorphonuclear granulocytes (PMN), and E. coli. Samples were incubated for up to 6 h at 37 degrees C. Motility analysis was performed using a computer-assisted sperm analyzer (CASA). Spermatozoa incubated with 3 x 106 PMN ml-1 revealed a significant (P=0.003) decrease in progressive motility after 2 h. This decrease remained weakly significant (P=0.024) after 4 and 6 h. Lymphocytes and monocytes had no effect on sperm motility. Spermatozoa incubated with granulocytes and E. coli demonstrated highly significant alterations in motility after 4 and 6 h of incubation (P < 0.001). The PMN indicate an effect on motility of spermatozoa under experimental conditions. However, the results suggest that bacteria are the primary agents that interfere with sperm motility.

Ejaculation↗

PubMed2003

In vitro development of marmoset monkey oocytes by pre-antral follicle culture.

A technique for in vitro maturation of oocytes from small ovarian follicles of marmoset monkeys (Callithrix jacchus) has been developed. We employed a two-step culture system for primary follicles (45-85 microm) and a one-step culture technique for secondary follicles (>85 microm). The two-step technique started with the culture of stromal tissue fragments for 2 days. Thereafter, mechanically isolated follicles were transferred to a culture system where they attached to the culture surface and grew for up to a further 12 days. Significant growth of the small follicles and their oocytes was only achieved with gonadotrophins in the medium. Oocytes with a mean diameter of 39 microm from follicles <85 microm reached a mean diameter of 90 microm by the end of the two-step culture. After in vitro maturation, 19% of oocytes from these follicles had progressed to the germinal vesicle breakdown (GVBD). Follicles between 85 and 170 microm in diameter were isolated from the stroma and placed directly in the culture. Oocytes from these follicles had a mean diameter of 64 microm. The maximum size the oocytes reached in culture was related to the age of the females (pre-pubertal females: 102 +/- 1.3 microm; adults: 96 +/- 1.4 microm). Twenty-seven per cent of oocytes from pre-pubertal ovaries achieved GVBD and nearly two-thirds of these progressed to polar body stage. From adult ovaries, only 12% progressed to GVBD and one-third of these to polar body stage. It is possible to develop mature oocytes in vitro from marmoset secondary pre-antral follicles (>85 microm). From primary follicles, although near full size oocytes were developed, maturation capacity was incomplete.

Age Factors↗

PubMed2002

Congenitally caused fused labia in the common marmoset (Callithrix jacchus).

In this paper, the occurrence of an external genital abnormality in female marmoset monkeys (fused labia) is discussed. This malformation was detected, for the first time, in a group of animals at the German Primate Center (GPC), Goettingen. The malformed vulva was completely sealed except for an opening of 1.5-2.5 mm around the urethra sufficient for urination. Because of this defect the animals were not able to copulate. As a consequence, the affected females were functionally infertile although they had a normal genital tract and a regular cycle. This vulvar abnormality was found in 12 females, offspring of 10 pairs in which either one or both came to the German Primate Center from two genetically related colonies in Munich, Germany, and one colony in Basel, Switzerland. The abnormality appeared to be recessive and inheritable from either parent. In pairs in which both animals were from one of the mentioned colonies, 45% of the female offspring were affected. In pairs where only one partner came from these colonies, 26% of female offspring had the malformation. These results indicate that avoidance of inbreeding, which is frequently performed in primate colonies, may reduce, but not eliminate the expression of abnormalities of genetic origin. Therefore selective breeding is required, and, in colonies where these recessive mutations are widespread, the development of genetic screening tests would be advantageous.

Animals↗

PubMed2001

Lipolysis and ultrastructural changes of intracellular lipid vesicles after cooling of bovine and porcine GV-oocytes.

The aim of our investigation was to compare the ultrastructure of lipid droplets, and the effect of cooling on intracellular lipid vesicles of bovine and porcine GV oocytes. The lipid droplets in bovine GV oocytes have a homogeneous structure. The utilization of lipids takes place directly from these vesicles without formation of interim lipid compounds. In contrast, there are two kinds of lipid droplets in porcine GV oocytes: 'dark', homogeneous vesicles next to 'grey' vesicles with electron-lucent streaks. Vesicles of each specific group are connected to each other. After a 12-h culture, the formation of the cisternal smooth endoplasmic reticulum layer was always associated with 'grey' lipid vesicles. This is evidence that during oogenesis lipolysis takes place only in 'grey' vesicles. It is supposed that cytoplasmic lipolysis has two stages: 'dark' vesicles change into a 'grey' form followed by a utilization of these 'grey' lipids. Furthermore, both types of lipid droplets in porcine oocytes changed morphologically during cooling: they changed into a spherical form with lucent streaks. Lipid droplets in bovine GV oocytes revealed no visible morphological changes after cooling.

Animals↗

PubMed2001

Use of microbeads for the detection of binding sites on the human zona pellucida: a scanning electron microscopy (SEM) assay.

One prerequisite for fertilization is the specific binding of spermatozoa to the zona pellucida. However, the factors and mechanisms involved in this gamete contact are not well understood. Gamete recognition and binding are species-specific and are controlled by oligosaccharides of the zona and their corresponding carbohydrates on the spermatozoon. By using a specific lectin we developed a technique to detect those oligosaccharides on the human zona pellucida that might be involved in the binding process. Microbeads (Ø = 2.8 microm), used as artificial spermatozoa, were coated with lectin Con A and cultured together with 75 unfertilized oocytes (group A) remaining after intracytoplasmic sperm injection. Con A binds specifically to alpha-d-mannose and alpha-d-glucose. As a control, 75 unfertilized oocytes after intracytoplasmic sperm injection (group B) were also cultured together with Con A-covered microbeads, but in a medium containing a binding inhibiting sugar (alpha-methyl-mannopyrasosid). The number and distribution of the microbeads on human oocytes of both groups were analysed on scanning electron microscopy images. Beads on oocytes of group A had binding patterns similar to those of spermatozoa. They were distributed in an extremely heterogeneous way with various numbers of bound beads both on individual and different oocytes. Most of the group A oocytes (85%) had more than 50 beads bound to the zona, in contrast to the control oocytes of group B, where 68% had less than 10 bound beads. The use of an inhibiting sugar abolished the binding capacity of the microbeads nearly completely. This technique is a powerful tool for the detection of binding sites on the zona pellucida, i.e. those sugars that are responsible for contact between spermatozoa and the zona pellucida.

Binding Sites↗

PubMed2001

Ultrastructure of centrifuged bovine oocyte-cumulus complexes after pre-treatment with cytoskeletal relaxant.

The objective of this study was the electron microscope examination of the localization of lipid droplets, mitochondria and other intracellular organelles in bovine oocytes and cumulus cells after Cytochalasin B pre-treatment and ultra-centrifugation. Bovine (n = 180) oocyte cumulus complexes on a germinal vesicle stage were treated with 5 micrograms/ml Cytochalasin B at 38.5 degrees C for 10-15 min. They were then centrifuged at 15,800 g and fixed at 39 degrees C immediately after centrifugation in 3% glutaraldehyde with 0.5% formaldehyde for the microscopic examinations. The centrifugal pole of the oocytes was filled with mitochondria. The centripetal part contained lipid granules and vesicles. Cytoplasm of low density was located in the equatorial region. Hyaloplasm with spontaneously formed membrane and non-membrane vacuoles was located in a supra-equatorial zone of the oocytes. In the cumulus cells the lipid vesicles formed one dark mass in the centripetal pole. The nuclei of these cells were deformed and vacuolization of the cytoplasm was noted.

Animals↗

PubMed2000

Escherichia coli-induced alterations of human spermatozoa. An electron microscopy analysis.

This study evaluated if the negative influence of Escherichia coli on the motility of human spermatozoa is a consequence of E. coli-induced ultrastructural alterations. Suspensions of spermatozoa were artificially infected with E. coli from a serotyped, pathogenic strain and incubated at 37 degrees C for 6 h. After incubation, spermatozoa were fixed in glutaraldehyde, stained with osmium tetroxide and ruthenium red and embedded in Spurr(R)-resin followed by ultramicrotomy. The sections were analysed subsequently by use of transmission electron microscopy. Uninfected suspensions of spermatozoa in medium and bacterial suspensions served as controls. Negative contrast technique was performed to facilitate visualization of ultrastructural details of the bacterial capsule after experimental exposure to spermatozoa. Electron microscopic evaluation revealed multiple and profound alterations in the ultrastructure of spermatozoa such as membrane defects and cytoplasmic vacuoles exclusively in spermatozoa of infected samples (> 90%). Morphological alterations involved all superficial structures of spermatozoa, in particular the plasma membrane of the mid-piece and neck as well as the inner and outer acrosomal membrane of the acrosome, indicating that morphological defects account for the immobilization of spermatozoa by E. coli. The results suggest that E. coli infection of ejaculates results in immobilization and impaired acrosomal function in human spermatozoa, findings that support the indication for antimicrobial chemotherapy in symptomatic and silent infections that affect the ejaculate.

Escherichia coli↗

PubMed2000

Luteinizing hormone has a stage-limited effect on preantral follicle development in vitro.

Although it is known that LH receptors are present from the time of thecal differentiation, the role of LH during early follicle development is not yet clear. The effect of LH on preantral follicle development has therefore been investigated in vitro using a culture system that supports the development of intact follicles. We have previously shown that although preantral follicles 150 micrometer in diameter (2-3 granulosa cell layers) do not require LH to proceed through antral development, smaller follicles (1-2 granulosa cell layers, 85-110 micrometer in diameter) do not develop beyond the large preantral stage in the presence of only FSH and 5% mouse serum. Follicles of this size were therefore used to determine the effects of LH and serum on their development in vitro. The results showed that although FSH must be continuously present, a low concentration of LH together with a slight increase in serum concentration was necessary, specifically during the primary stage of follicle development (from 85 micrometer in diameter until the follicles had reached 150 micrometer in diameter) to induce the capacity for subsequent LH-independent rapid growth and antral development. The in vitro development of maturable oocytes with normal spindle and chromatin morphology was also supported. These results indicate that LH probably induces changes in the early differentiating thecal cells, which are critical for the completion of subsequent follicular and oocyte development.

Animals↗

PubMed2000

X-Y sperm selection: fact or fiction?

Selecting the gender of offspring has given rise to various and sometimes amusing stories. But regardless of which prefertilisation technique is used to influence the sex ratio of offspring it must fulfill certain criteria. First of all it must achieve a complete separation of the X and Y bearing sperm in sufficient quantities. Secondly sperm must be viable after separation and capable of fertilising. Sex preselection methods can be divided into two general groups which either separate spermatozoa on the basis of subtle physical or kinetic features or those which rely on distinctive nuclear characteristics unique either to X or Y chromosome bearing sperm. These, in turn, can be divided into in vivo methods designed to produce optimal conditions for fertilisation by either the X or Y bearing sperm, or in vitro sperm separation methods designed to separate X or Y bearing sperm. According to all published data, the different separation techniques have been shown not to be very effective. Only sex selection of spermatozoa by chromatin differences (cell sorting by flow cytometry) has demonstrated a significant enrichment of the X bearing sperm.

Chromosomes, Human, X↗

PubMed1999

Scanning electron microscopy analysis of the human zona pellucida: influence of maturity and fertilization on morphology and sperm binding pattern.

Human oocytes from the same as well as from different patients have an extremely heterogeneous morphology of the zona pellucida surface as shown by scanning electron microscopy. For years it has been believed that this heterogeneous morphology plays an important part in the sperm-oocyte interaction. It was the aim of this investigation to analyse the morphology and the sperm binding patterns of the human zona pellucida. Oocytes were divided into four categories: mature, immature, fertilized and unfertilized. Four different types of zona morphology were detectable. They ranged from a porous, net-like structure to a nearly smooth and compact surface. No correlation could be established between zona type and oocyte maturity or zona type and achieved fertilization. However, fertilized (polyploid) oocytes had a more compact and smooth zona surface than unfertilized ones. The analysis of the number and distribution patterns of bound spermatozoa on the zona pellucida revealed extremely variable patterns regardless of the zona morphology. Significant differences between mature and immature oocytes did not appear. In both groups there were oocytes with either no or numerous bound spermatozoa on the zona pellucida. Oocytes overloaded with spermatozoa could only be found in the mature group. Unfertilized oocytes had fewer bound spermatozoa on average than polyploid zygotes.

Cell Differentiation↗

PubMed1998

Influence of bacteria and leukocytes on the outcome of in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).

The influence of bacteria and/or leukocytes on the outcome of IVF or ICSI is influenced by three factors which have little in common with in vivo conditions: 1) The process of ejaculate preparation (swim-up, Percoll) with antibiotic buffered media; 2) The small amount of inseminated spermatozoa (100,000 per culture); 3) The short cultivation time. From the very beginning, these factors limit whatever the influence of bacteria and leukocytes on fertilization and embryonic development in vivo may be. Despite the contradictory results published so far, the influence of bacteria and/or leukocytes on the functional integrity of spermatozoa during the process of IVF or ICSI can be ignored. Furthermore, during IVF or ICSI the spermatozoon does not act as a vector for the transportation of bacteria into the ooplasm.

Bacteria↗

PubMed1997

Segregation of sex chromosomes into sperm nuclei in a man with 47,XXY Klinefelter's karyotype: a FISH analysis.

Meiotic segregation of the sex chromosomes was analysed in sperm nuclei from a man with Klinefelter's karyotype by three-colour FISH. The X- and Y-specific DNA probes were co-hybridized with a probe specific for chromosome 1, thus allowing diploid and hyperhaploid spermatozoa to be distinguished. A total of 2206 sperm nuclei was examined; 958 cells contained an X chromosome, 1077 a Y chromosome. The ratio of X:Y bearing sperm differed significantly from the expected 1:1 ratio (chi2 = 6.96; 0.001 < P < 0.01). Sex-chromosomal hyperhaploidy was detected in 2.67% of the cells (1.22% XX, 1.36% XY, 0.09% YY) and a diploid constitution in 0.23%. Although the frequency of 24,YY sperm was similar to that detected in fertile males, the frequencies of 24,XX, 24,XY and diploid cells were significantly increased. A sex-chromosomal signal was missing in 4.26% of the spermatozoa. This percentage appeared to be too high to be attributed merely to nullisomy for the sex chromosomes and was considered, at least partially, to be the result of superposition of sex-chromosomal hybridization signals by autosomal signals in a number of sperm nuclei. The results contribute additional evidence that 47,XXY cells are able to complete meiosis and produce mature sperm nuclei.

Adult↗

PubMed1997

Pregnancy after intracytoplasmic sperm injection with sperm from a man with a 47,XXY Klinefelter's karyotype.

OBJECTIVE: To report the initiation of a pregnancy that was achieved by intracytoplasmic sperm injection (ICSI) with sperm from a patient with Klinefelter's syndrome. DESIGN: Case report. SETTING: University women's hospital IVF center. PATIENT(S): A couple with primary infertility and nonmosaic 47,XXY karyotype of the male partner. INTERVENTION(S): Intracytoplasmic sperm injection after ovarian stimulation and transvaginal ultrasound-guided oocyte pick-up with sperm from a hypergonadotropic man with a nonmosaic 47,XXY karyotype. MAIN OUTCOME MEASURE(S): Clinical pregnancy. RESULT(S): Despite a 47,XXY karyotype in all 50 analyzed lymphocyte metaphases, the sperm of the patient led to a clinical pregnancy with the first attempt of ICSI and intrauterine transfer of three embryos. The pregnancy stopped developing in the ninth week. Cytogenetic investigation of the abortion material revealed a numerical normal 46,XXY karyotype. CONCLUSION(S): Sperm from a patient with hypergonadotropic nonmosaic Klinefelter's syndrome, when used for ICSI, can lead to a pregnancy.

Abortion, Induced↗

PubMed1997

Incidence of diploid and disomic sperm nuclei in 45 infertile men.

Sperm cells from 45 infertile patients were investigated for disomy rates of chromosomes 1, 7, 10, 17, X and Y as well as for diploidy by single- and double-target in-situ hybridization. The patients who attended the infertility clinic were aged 23-46 years. Semenograms showed that the patients had oligo-, astheno-, oligoastheno-, oligoterato-, oligoasthenoterato-, or asthenoteratozoospermia. The average disomy rates determined in the patients were similar for all chromosomes, ranging from 0.10% (chromosome Y) to 0.14% (chromosomes 10 and X). Diploidy was detected with a mean incidence of 0.1%. With the exception of two patients who exhibited significantly increased diploidy rates of 0.35 and 1.6%, neither disomy nor diploidy was increased in the group of infertile patients as compared to healthy, fertile males.

Adult↗