[Recurrent miscarriage or failed in-vitro fertilization: antibodies against annexin V, cardiolipin, beta-2-glycoprotein-1 and APC-resistance]. / Rezidivierende Aborte und frustrane In-vitro-Fertilisation. Antikörper gegen Annexin-V, Cardiolipin und beta-2-Glykoprotein-1, oder APC-Resistenz.

UNLABELLED: Antibodies against annexin-V, a potent anticoagulant abundant in placental tissues, were recently controversially reported to be associated with recurrent miscarriages or failures of in-vitro-fertilization (IVF) attempts. PATIENTS, MATERIAL, METHODS: We screened 56 women (34.7 +/- 4.3 years of age: mean +/- 1 SD) with recurrent IVF failures and/or early pregnancy losses for resistance against activated protein C, lupus anticoagulant and antibodies against annexin V, cardiolipin or beta(2)-glycoprotein-1. Among them the prevalence of APC-R (8/56, 14%) and elevated levels of IgG- or IgM-anti-cardiolipin antibodies (7/56, 12%) were more common than elevated levels of (IgG or IgM) antibodies against beta(2)-glycoprotein-1 (3/56, 5%) or annexin-V (1/56, 2%). 42 (75%) of the women had another IVF-attempt after this haemostaseological evaluation and received low molecular weight heparin and/ or acetylsalicylic acid in the case of positivity for APC-resistance, lupus anticoagulant or antibodies against annexin V, cardiolipin or beta(2)-glycoprotein-1. RESULTS: The outcome of these IVF-attempts were 19 pregnancies (34%): 4 early miscarriages (7%) and 15 so far uncomplicated pregnancies (27%). The only woman with an elevated anti-annexin V (IgG) level had had 7 IVF before and received 40 mg Enoxaparin (Lovenox) subcutaneously once daily during the 8(th) IVF, which resulted in a healthy pregnancy. DISCUSSION, CONCLUSION: Our findings suggest that among women with recurrent IVF failures anti-annexin V antibody positivity is less prevalent than APC-resistance, lupus anticoagulant (LA) or elevated levels of antibodies against cardiolipin, beta(2)-glycoprotein-1 and that the IVF-result of women with APC-R, LA or with elevated levels of antibodies against annexin V, cardiolipin or beta(2)-glycoprotein might be positively influenced by low molecular weight heparin.

The rare human fragile site 16B.

The rare human fragile site 16B (FRA16B) has been found to occur spontaneously. Its expression in lymphocyte cultures can also be induced or greatly enhanced by addition of chemicals which are known to bind to AT-rich DNA regions. Following optimal treatment with 150 microg/ml berenil 24 h prior to fixation, the heterozygote frequency of FRA16B is found to be about 5% in populations of European descent. Thus, FRA16B represents the most common of the rare fragile sites. Consistent with cytogenetic observations, the molecular characterization of FRA16B revealed that it is an amplified 33-base pair AT-rich minisatellite repeat. These interindividually variable, extremely large repeat expansions of 15-70 kb in size do not seem to interfere with the expression of genes essential for human development since heterozygotes and homozygotes for FRA16B are normal.

Comparative chromosome painting of chicken autosomal paints 1-9 in nine different bird species.

In a Zoo-FISH study chicken autosomal chromosome paints 1 to 9 (GGA1-GGA9) were hybridized to metaphase spreads of nine diverse birds belonging to primitive and modern orders. This comparative approach allows tracing of chromosomal rearrangements that occurred during bird evolution. Striking homologies in the chromosomes of the different species were noted, indicating a high degree of evolutionary conservation in avian karyotypes. In two species, the quail and the goose, all chicken paints specifically labeled their corresponding chromosomes. In three pheasant species as well as in the American rhea and blackbird, GGA4 hybridized to chromosome 4 and additionally to a single pair of microchromosomes. Furthermore, in the pheasants fission of the ancestral galliform chromosome 2 could be documented. Hybridization of various chicken probes to two different chromosomes or to only the short or long chromosome arm of one chromosome pair in the species representing the orders Passeriformes, Strigiformes, and Columbiformes revealed translocations and chromosome fissions during species radiation. Thus comparative analysis with chicken chromosome-specific painting probes proves to be a rapid and comprehensive approach to elucidate the chromosomal relationships of the extant birds.

X chromosomes of American marsupials contain minimal amounts of euchromatin.

The karyotypes of four South American didelphid marsupials, representing diploid numbers of 2n = 14 and 18, have been analyzed by a variety of banding techniques. The 2n = 14 karyotypes display a high degree of homoeology, but there also exist distinct similarities between the 2n = 14 and 2n = 18 karyotypes. The interspecific differences found are due to centric fissions, pericentric inversions, and variations in the amount and composition of the constitutive heterochromatin. Contrary to the evolutionary conservation of the banding patterns in all autosomal arms, there are multiple differences in the number and chromosomal location of the nucleolus organizer regions. In species with X-linked nucleolus organizers, the 18S + 28S ribosomal RNA genes escape inactivation in female cells. Measurements on the X chromosomes of Marmosa fuscata and Micoureus demerarae unexpectedly reveal the lowest quantities of euchromatin so far determined in the X chromosomes of mammals: 1.5% and 1.8%, respectively, of their haploid female genomes. This is significantly less than the amount of euchromatin in the basic X chromosomes of other marsupials (3%) or eutherians (5%).

Chromosome banding in Amphibia. XXVIII. Homomorphic XY sex chromosomes and a derived Y-autosome translocation in Eleutherodactylus riveroi (Anura, Leptodactylidae).

Extensive cytogenetic analyses on a population of the leptodactylid frog Eleutherodactylus riveroi in northern Venezuela revealed the existence of multiple XXAA male/XYAA female/XAA(Y) female sex chromosomes. The XAA(Y) karyotype originated by a centric (Robertsonian) fusion between the original, free Y chromosome and an autosome. 46.2% of the male individuals in this population are carriers of this Y-autosome fusion. In male meiosis the XAA(Y) sex chromosomes pair in the expected trivalent configuration. In the same population 53.8% of the male animals still possess the original, free XY sex chromosomes. E. riveroi is only the second vertebrate species discovered in which a derived Y-autosome fusion coexists with the ancestral free XY sex chromosomes. The free XY sex chromosomes, as well as the multiple XA(Y) sex chromosomes are still in a very primitive (homomorphic) stage of differentiation. With no banding technique applied it is possible to distinguish the Y from the X. Various banding techniques and in situ hybridizations have been carried out to characterize the karyotypes. DNA flow cytometric measurements show that the genome size of E. riveroi resembles that of other Eleutherodactylus species. The cytogenetic data obtained in E. riveroi are compared with those of the sole other vertebrate known to possess the extremely rare, multiple XXAA male/XYAA female/XAA(Y) female sex chromosomes. Surprisingly enough, this vertebrate again is a frog belonging to the genus Eleutherodactylus [E. ((maussi) biporcatus] which lives exactly in the same habitat in northern Venezuela as does E. riveroi.

Chromosome banding in Amphibia. XXVI. Coexistence of homomorphic XY sex chromosomes and a derived Y-autosome translocation in Eleutherodactylus maussi (Anura, Leptodactylidae).

A 15-year cytogenetic survey on one population of the leaf litter frog Eleutherodactylus maussi in northern Venezuela confirmed the existence of multiple XXAA male symbol /XAA(Y) female symbol sex chromosomes which originated by a centric (Robertsonian) fusion between the original Y chromosome and an autosome. 95% of the male individuals in this population are carriers of this Y-autosome fusion. In male meiosis the XAA(Y) sex chromosomes pair in the expected trivalent configuration. In the same population, 5% of the male animals still possess the original, free XY sex chromosomes. In a second population of E. maussi analyzed, all male specimens are characterized by these ancestral XY chromosomes which form normal bivalents in meiosis. E. maussi apparently represents the first vertebrate species discovered in which a derived Y-autosome fusion still coexists with the ancestral free XY sex chromosomes. The free XY sex chromosomes, as well as the multiple XA(Y) sex chromosomes are still in a very primitive (homomorphic) stage of differentiation. With no banding technique applied it is possible to distinguish the Y from the X. DNA flow cytometric measurements show that the genome of E. maussi is among the largest in the anuran family Leptodactylidae. The present study also supplies further data on differential chromosome banding and fluorescence in situ hybridization experiments in this amphibian species.

Terminal deletion of the long arm of chromosome 10: a new case with breakpoint in q25.3.

Since the first patient with partial deletion of the long arm of chromosome 10 was described in 1978, another 23 cases have been reported, with the breakpoint ranging from 10q23.3-26.2. To contribute further to the delineation of the monosomy 10qter syndrome, we describe a female child who, at age 3 6/12 years, was diagnosed with a de novo deletion of the long arm of chromosome 10, with a breakpoint in 10q25.3. The phenotypic manifestations in this child are compatible with those of previously reported cases. However, in contrast to most other patients, we found a moderate expression of the syndrome, with no genitourinary or cardiac malformations and with only mild retardation. Based on our observations and those of others, we conclude that a typical craniofacial appearance and varying degrees of psychomotor retardation are always found in patients with 10q- syndrome.

Follicle aspiration with interactive three-dimensional digital imaging (Voluson): a step toward real-time puncturing under three-dimensional ultrasound control.

OBJECTIVE: To evaluate the possibility of performing puncturing procedures under three-dimensional ultrasound (US) control in close to real time using a new commercially available system and to describe the technique. DESIGN: Descriptive case study. SETTING: Private outpatient infertility clinic. PATIENT(S): A volunteer undergoing IVF-ET treatment. INTERVENTION(S): Transvaginal needle-guided aspiration of 10 follicles using a newly developed US machine with a built-in rapid and powerful calculation software program for three-dimensional interactive volume and flow translation during operation. Interactive three-dimensional imaging was carried out during the aspiration of each follicle. MAIN OUTCOME MEASURE(S): The delays caused by volume acquisition and the search for the needle tip with and without "power Doppler" were estimated and compared. RESULT(S): Oocyte recovery was successful from all follicles. There was a mean (+/-SD) delay of 5.00 +/- 1.22 seconds from coasting to resuming real-time US scanning during interactive volume calculation and the search for the needle tip in the three-dimensional mode. This did not delay the procedure remarkably but enabled the precise localization of both the needle and its tip after each penetration. The integration of flow signals allowed an impressive color-coded demonstration of the needle within the tissue, but the delay was significantly longer for color-coded volume acquisition (18.40 +/- 4.56 seconds). CONCLUSION(S): Recent technology enables the performance of three-dimensional puncture procedures in close to real time; this technology is demonstrated for follicle punctures but will be of more importance in fields such as fetal medicine, oncology, and surgery.

Pregnancy following in vitro fertilization and embryo transfer using pure human serum as culture and transfer medium.

Pure heat-inactivated serum of patients adjusted to 280 mOsm was used for preincubation, insemination, and transfer of oocytes in an IVF-ET program. In patients where the oocyte recovery yielded more than two oocytes, two oocytes were chosen randomly for serum cultivation. The 15th patient of that series became pregnant after a transfer of two serum-cultured embryos. The follow-up weekly clinical investigation and ultrasound revealed a normal single pregnancy, now in the 20th week.

Successful clinical application of laser for micromanipulation in an in vitro fertilization program.

This is a report of our preliminary experience with assisted hatching by laser with a case of an ongoing twin pregnancy. We used an erbium laser to create a 20 to 30-micrometer opening in the zona pellucida of human embryos to facilitate the embryonic hatching after the embryo transfer. Our report proves that it is possible to obtain a pregnancy using a simple and quick laser method instead of mechanical or chemical procedures within the scope of micromanipulation.

Transvaginal sector scan sonography for needle guided transvaginal follicle aspiration and other applications in gynecologic routine and research.

A transvaginal sector scanner probe has been developed for precise needle-guided transvaginal follicle aspiration under sonographic control. The first results of transvaginal needle-guided follicle aspiration are presented. A high recovery rate was obtained (86% /follicle). The pregnancy rate after in vitro fertilization and embryo replacement was remarkably high (21.3%/cycle). Because exceptionally high-quality images of the organs in the pelvis are obtained, there are numerous other potential applications for the trans-vaginal sector scanner.

The use of two-component fibrin sealant for embryo transfer.

This is a report of our preliminary experience using fibrin sealant with a series of 38 patients undergoing IVF and ET. We used a two-component fibrin sealant to create a fibrin plug in the uterine cavity at the time of ET to decrease the possibility of embryo expulsion and also ectopic pregnancy. Our preliminary report proves that it is possible to obtain 26% pregnancies using this two-component biological glue instead of serum or culture medium for uterine embryo replacement. A prospective randomized study will be undertaken to evaluate whether the use of fibrin sealant could significantly improve IVF and ET results.

Results of laparoscopic recovery of preovulatory human oocytes from nonstimulated ovaries in an ongoing in vitro fertilization program.

The technique and results of 50 laparoscopies performed on sterility patients to obtain mature oocytes from Graafian follicles with a view to carrying out in vitro fertilization are described. In 32% of all laparoscopies and in 52% of all punctured follicles it proved possible to obtain a mature oocyte. Different diameters of puncture needles as well as different suction intensities are compared. As a result of increasing experience and improved technique, the exact time for the intervention was correctly determined in 92% of cases. The recovery rate for oocytes was also increased to 58% with respect to laparoscopies carried out.

Relationship of the individual uterine size and the endometrial thickness in stimulated cycles.

At present there is no agreement regarding the usefulness of monitoring the endometrial thickness in stimulated cycles. We tested prospectively the hypothesis of a linear relationship of the given individual uterine size and the endometrial thickness in stimulated cycles using a 240 degrees transvaginal sector scanner. There was a strong correlation of the uterine dimension with the endometrial thickness in the midfollicular and the early luteal phase. Our findings suggest that the endometrial thickness is determined by the individual uterine architecture and therefore not predictive of the likelihood of implantation.

Could aspiration of the Graafian follicle cause luteal phase deficiency?

Luteal phase quality was evaluated in 32 patients wih nonstimulated cycles after laparoscopic oocyte recovery for in vitro fertilization. A luteal phase deficiency occurred in two cases (6.2%), the mean duration of the luteal phase was 13.5 +/- 1.3 days in 30 patients, and two patients developed amenorrhea of 23 and 43 days respectively after laparoscopy in spite of normal progesterone values 7 and 9 days after oocyte recovery. Six embryo transfers were performed after fertilization and regular cleavage of the obtained oocytes. No pregnancy resulted from the embryo transfers, although the patients had apparently normal luteal phases. In one patient there was a transient beta-subunit human chorionic gonadotropin (beta-hCG) elevation in serum. Luteal phase deficiency should not be main cause of a nonsuccessful embryo transfer. However, a prophylactic luteal phase support after oocyte recovery and embryo transfer in nonstimulated cycles is proposed.

Vascular endothelial growth factor and its receptors in male fertility.

OBJECTIVE: To determine the concentration of vascular endothelial growth factor in the seminal fluid, the presence of vascular endothelial growth factor receptors Flt-1 and KDR in spermatozoa, and the predictive value of seminal vascular endothelial growth factor on fertilization and the chance of pregnancy in patients undergoing intracytoplasmic sperm injection (ICSI) or IVF. DESIGN: Retrospective analysis. SETTING: Private institute (semen collection, IVF/ICSI) and academic research environment (analysis of seminal fluid and spermatozoa). PATIENT(S): Eighty men whose spermatozoa were subsequently used for IVF or ICSI. INTERVENTION(S): Seminal vascular endothelial growth factor was measured by an EIA. Spermatozoa were analyzed by fluorescence-activated cell sorter analysis and by immunocytochemistry. MAIN OUTCOME MEASURE(S): Oocyte fertilization rate, pregnancy rate, and presence of vascular endothelial growth factor receptors on spermatozoa. RESULT(S): Patients with a seminal concentration of vascular endothelial growth factor of 2-100 ng/mL had a sixfold increased chance of pregnancy. Vascular endothelial growth factor concentration and patient's age remained the only independent prognostic factors for pregnancy. The concentration of vascular endothelial growth factor did not correlate with indices of male factor infertility or with the oocyte fertilization rate. Expression of vascular endothelial growth factor receptors (Flt-1, KDR) on spermatozoa was demonstrated. CONCLUSION(S): The seminal concentration of vascular endothelial growth factor correlates with the chance of pregnancy in patients undergoing IVF or ICSI. Vascular endothelial growth factor receptors Flt-1 and KDR were detected on spermatozoa for the first time.

Air in the uterine cavity after embryo transfer.

OBJECTIVE: To examine the effect on the pregnancy rate (PR) caused by air bubbles loaded into the transfer catheter to bracket the embryo-containing medium. DESIGN: Prospective, randomized study. SETTING: One hundred ninety-six consecutive ETs in the Institute of Sterility Treatment. INTERVENTIONS: Air bubbles were loaded into the transfer catheter concomitantly with the medium in group I (n = 98), whereas in group II (n = 98) no air bubbles were used. RESULTS: The PR of group I equals that of group II. CONCLUSIONS: Bracketing the embryo-containing medium by air bubbles offers several advantages, especially the possibility of tracking the air on the ultrasound monitor to localize the embryos after the ET. For these reasons we recommend using the proven method.

Successful treatment of a heterotopic pregnancy by sonographically guided instillation of hyperosmolar glucose.

OBJECTIVE: To describe a new conservative approach in the treatment of heterotopic pregnancy. DESIGN: Case report. SETTING: A university hospital and an outpatient IVF center. INTERVENTION(S): Transvaginal ultrasonography-guided puncture of the ectopic gestational sac and instillation of hyperosmolar glucose. RESULT(S): The ectopic pregnancy resolved without further intervention. The intrauterine pregnancy resulted in a full-term live birth. CONCLUSION(S): Transvaginal injection of hyperosmolar glucose may be an effective conservative treatment for unruptured ectopic pregnancies in cases of heterotopic pregnancy.