Hans Wilhelm Michelmann

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

Abstract 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 (χ2 = 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.

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 Klinefelters syndrome. DESIGN Case report. SETTING University womens 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 Klinefelters syndrome, when used for ICSI, can lead to a pregnancy.

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

Abstract 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 μm 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 μm 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 μm in diameter until the follicles had reached 150 μm 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.

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

Abstract: 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.

Text Mining and Natural Language Processing Approaches for Automatic Categorization of Lay Requests to Web-Based Expert Forums

Background Both healthy and sick people increasingly use electronic media to obtain medical information and advice. For example, Internet users may send requests to Web-based expert forums, or so-called “ask the doctor” services. Objective To automatically classify lay requests to an Internet medical expert forum using a combination of different text-mining strategies. Methods We first manually classified a sample of 988 requests directed to a involuntary childlessness forum on the German website “Rund ums Baby” (“Everything about Babies”) into one or more of 38 categories belonging to two dimensions (“subject matter” and “expectations”). After creating start and synonym lists, we calculated the average Cramer’s V statistic for the association of each word with each category. We also used principle component analysis and singular value decomposition as further text-mining strategies. With these measures we trained regression models and determined, on the basis of best regression models, for any request the probability of belonging to each of the 38 different categories, with a cutoff of 50%. Recall and precision of a test sample were calculated as a measure of quality for the automatic classification. Results According to the manual classification of 988 documents, 102 (10%) documents fell into the category “in vitro fertilization (IVF),” 81 (8%) into the category “ovulation,” 79 (8%) into “cycle,” and 57 (6%) into “semen analysis.” These were the four most frequent categories in the subject matter dimension (consisting of 32 categories). The expectation dimension comprised six categories; we classified 533 documents (54%) as “general information” and 351 (36%) as a wish for “treatment recommendations.” The generation of indicator variables based on the chi-square analysis and Cramer’s V proved to be the best approach for automatic classification in about half of the categories. In combination with the two other approaches, 100% precision and 100% recall were realized in 18 (47%) out of the 38 categories in the test sample. For 35 (92%) categories, precision and recall were better than 80%. For some categories, the input variables (ie, “words”) also included variables from other categories, most often with a negative sign. For example, absence of words predictive for “menstruation” was a strong indicator for the category “pregnancy test.” Conclusions Our approach suggests a way of automatically classifying and analyzing unstructured information in Internet expert forums. The technique can perform a preliminary categorization of new requests and help Internet medical experts to better handle the mass of information and to give professional feedback.

PSCDGs of mouse multipotent adult germline stem cells can enter and progress through meiosis to form haploid male germ cells in vitro

Spermatogonial stem cells (SSCs) provide the basis for spermatogenesis throughout adult life by undergoing self-renewal and differentiation into sperm. SSC-derived cell lines called multipotent adult germline stem cells (maGSCs) were recently shown to be pluripotent and to have the same potential as embryonic stem cells (ESCs). In a differentiation protocol using retinoic acid (RA) and based on a double selection strategy, we have shown that ESCs are able to undergo meiosis and produce haploid male germ cells in vitro. Using this differentiation protocol we have now succeeded to generate haploid male germ cells from maGSCs in vitro. maGSCs derived from a Stra8-EGFP transgenic mouse line were differentiated into stable spermatogonial stages and further cultured. These cells were transfected with a postmeiotic specific promoter construct Prm1-DsRed to monitor retinoic acid (RA) induced differentiation into haploid male gametes. Our protocol is another approach for the production of pluripotent stem cell derived gametes (PSCDGs) and is an alternative for the investigation of mammalian spermatogenesis, germ line gene modification and epigenetic reprogramming. If reproducible with pluripotent cell lines derived from human SSCs, it could also be used as a therapeutic approach for the treatment of male infertility.

Epidermal growth factor effects on marmoset monkey (Callithrix jacchus) oocyte in vitro maturation, IVF and embryo development are altered by gonadotrophin concentration during oocyte maturation

BACKGROUND This is the first study of the effect of epidermal growth factor (EGF) on marmoset monkey oocytes matured in vitro. METHODS We have evaluated the effects of 10 ng/ml EGF in combination with 1 or 10 IU/ml of gonadotrophins (FSH/hCG 1:1 ratio) during in vitro maturation (IVM) of marmoset oocytes. Immature cumulus-oocyte complexes (COCs) were retrieved from ovarian antral follicles of unprimed monkeys. COCs from six animals (n= 268) used in this study were randomly distributed among four experimental groups: (A) 1 FSH +1 hCG; (B) 10 FSH +10 hCG; (C) 1 FSH +1 hCG + EGF; and (D) 10 FSH +10 hCG + EGF (where 1 and 10 are concentrations, IU/ml). After IVM, oocytes were fertilized in vitro and embryos were allowed to progress up to 87-88 h. RESULTS the highest rate of total and radial cumulus expansion was observed in Group A, with the lowest in Group B (P < 0.05). Neither maturation nor fertilization rate were affected by gonadotrophin concentration or presence of EGF. Addition of EGF increased degeneration and decreased first cleavage rate, which was significantly lower in Group C than Group A (P < 0.005). Interestingly, in the EGF groups some embryos cleaved faster than without EGF. CONCLUSIONS The effects of EGF are highly dependent on concentration of gonadotrophins present in IVM medium. EGF has a negative effect on oocytes in the presence of low gonadotrophins, but contrastingly partially protects oocytes from the negative effects of high gonadotrophins. We propose that these observed negative effects of EGF may suggest use of an inappropriate dose of growth factor.

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

Summary. 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 (100000 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.

Prognostic value of an automated sperm analysis in IVF or insemination therapy

Summary. During the course of sterility treatment semenograms of 271 IVF and 316 insemination patients were carried out by two automated semen analysers. The parameters of these analyses were correlated to pregnancies resulting from the treatment. Semen samples were analysed in the ejaculate and after swim‐up preparation. In addition, the swim‐up suspension of IVF patients was measured again 18 h after sperm preparation. Patients were divided into three groups: (1) couples who achieved a pregnancy, (2) couples who did not achieve a pregnancy, and (3) IVF patients with no fertilization of the oocytes. Because of large standard deviations in the quality of ejaculates, the results in the IVF group show no significant differences in the semen parameters of husbands of pregnant and non‐pregnant women. In contrast husbands of women with no fertilization have a significantly lower sperm motility. After swim‐up preparation these differences no longer occur. A further measurement, taken 18 h later, reveals that there are no differences in the sperm parameters between the pregnant and non‐pregnant group. However, the semen quality in the group with no fertilization is significantly reduced. The results of the insemination patients are similar to those of the IVF group. Thus, the results from automated sperm analysers cannot replace either the microscopic or biochemical analysis of an ejaculate and, moreover, cannot be used as prognosis for the fertilization capacity of sperms or a following pregnancy.

A case report of spontaneous opening of congenitally fused labia in a female common marmoset (Callithrix jacchus) followed by pregnancy and birth of twins

A first case of spontaneous opening of congenitally fused labia (CFL phenotype) in a captive common marmoset followed by pregnancy and birth is presented here. The occurrence of this phenotype has been previously published in captive marmosets, but so far the etiology is unknown.