D. Krebs

Ascorbic acid (vitamin C) improves the antineoplastic activity of doxorubicin, cisplatin, and paclitaxel in human breast carcinoma cells in vitro

Utilizing a microplate ATP bioluminescence assay, two human breast carcinoma cell lines, MCF-7 and MDA-MB-231, were tested against doxorubicin (DOX), cisplatin (DDP), and paclitaxel (Tx) alone and in combination with ascorbic acid (Vit C). In both cell lines, Vit C exhibited cytotoxic activity at high concentrations (i.e. 10(2)-10(3) microM). Both cell lines also were resistant to DOX. MCF-7 was found to be DDP-resistant, MDA-MB-231 was moderately sensitive to DDP. Both cell lines were strongly sensitive to Tx. Vit C both at non-cytotoxic (1 microM) and moderately cytotoxic concentrations (10(2) microM) improved the cytotoxicity of DOX, DDP, and Tx significantly. Combination effects between Vit C and DDP or Tx were partly synergistic and partly additive or subadditive whereas a consistent synergism was found between Vit C and DOX. The mechanisms by which Vit C potentiates the cytostatics studied are yet unclear and should be evaluated further.

Soluble HLA levels in early pregnancy after in vitro fertilization

Intact pregnancy can be interpreted as a state of maternal immunotolerance toward an haploidentical fetus. Soluble HLA (sHLA) molecules increase during episodes of allograft rejection and are discussed as candidates to modulate immune responses. We questioned whether after in vitro fertilization (IVF) the subsequent intact pregnancy, early abortion, or tubal pregnancy influence the courses sHLA serum levels. Therefore, serum samples of 65 IVF patients were assayed by ELISA for sHLA-I, sHLA-G, and sHLA-DR concentrations preovulatorily and after a positive HCG test weekly until the 9th gestational week (GW). In 20 patients experiencing an early abortion the preovulatory sHLA-G mean level of 25.9 +/- 3.9 SEM ng/ml and the share of 4.2 +/- 0.8 SEM % on total sHLA-I were significantly (p < 0.05) reduced compared to women with intact pregnancy. The same differences (p < 0.0001) were seen during the monitoring of sHLA-G and sHLA-I levels in intact pregnancy versus early abortion until 9th GW. Twin pregnancy revealed a drastically increase of sHLA-G levels from the 8th GW compared to singleton pregnancies. Further, individual sHLA-DR levels increased during intact pregnancy but decreased in the group of early abortion. With regard to sensitivity and specificity for pregnancy outcome sHLA quantitation reached similar weight as routine HCG determinations at GW 5. Especially women with preovulatory low sHLA-G levels appear to be on risk for early abortion after IVF.

Scintimammography with technetium-99m methoxyisobutylisonitrile: comparison with mammography and magnetic resonance imaging

The aim of the study was to compare the diagnostic accuracy of scintimammography with technetium-99m methoxyisobutylisonitrile (MIBI; SMM) in the detection of primary breast cancer with that of mammography (MM) and magnetic resonance imaging (MRI). Fifty-six patients with suspected lesions detected by palpation or MM were included in the study. Within the 4 weeks preceding excisional biopsy, MM and MRI were performed in all patients. Between 5 and 10 min after the injection of 740 MBq99mTc-MIBI, SMM in the prone position was performed. In the total group of 56 patients, 43 lesions were palpable, while 13 were non-palpable but were detected by MM. Breast cancer was confirmed by histopathology in 27 of the patients (22 palpable and 5 non-palpable carcinomas). The tumour size ranged from 6 to 80 mm in diameter. For non-palpable lesions, the sensitivity of SMM, MM and MRI was 60%, 60% and 100%, respectively, while the specificity was 75%, 25% and 50%, respectively. For palpable breast lesions, all methods showed high sensitivity (SMM 91%, MM 95%, MRI 91%) but SMM demonstrated significantly higher specificity (SMM 62%, MM 10%, MRI 15%). In two mammographically negative tumours (dense tissue), SMM showed a positive result. In comparison to MRI, one additional carcinoma could be diagnosed by SMM. It may be concluded that for palpable breast lesions, the diagnostic accuracy of SMM is superior to that of MM and MRI. Through the complementary use of SMM it is possible to increase the sensitivity for the detection of breast cancer and multicentric disease. In patients in whom the status of a palpable breast mass remains unclear, SMM may help to reduce the amount of unnecessary biopsies.

Transvaginal sonography of the endometrium during ovum pickup in stimulated cycles for in vitro fertilization.

The endometrium of a series of 190 in vitro fertilization patients was investigated by transvaginal ultrasound. The endometrial pattern was related to the likelihood of implantation. No such relationship was found for the thickness of the endometrium. There was not a strong correlation between the endometrium and hormonal values. However, three endometrial patterns were detected after hormonal stimulation. The predominant pattern consisted of an outer hyperechogenic layer and an inner hypoechogenic layer. This pattern correlated in a positive fashion with subsequent implantation. It is concluded that the texture of the endometrium at the time of ovum pickup has a prognostic value for the likelihood of implantation to occur.

Comparison of fluorine-18 fluorodeoxyglucose positron emission tomography and technetium-99m methoxyisobutylisonitrile scintimammography in the detection of breast tumours

The aim of this study was to compare, in breast cancer patients, the diagnostic accuracy of positron emission tomography (PET) using fluorine-18 fluorodeoxyglucose (FDG) and scintimammography (SMM) using technetium-99m methoxyisobutylisonitrile (MIBI). A total of 20 patients (40 breasts with 22 lesions) were evaluated serially with MIBI and, on the following day, with FDG. For SMM, planar and single-photon emission tomography imaging in the prone position was performed starting at 10 min following the injection of MIBI (740 MBq). For PET, scans were acquired 45–60 min after the injection of FDG (370 MBq) and attentuation correction was performed following transmission scans. Results from SMM and PET were subsequently compared with the histopathology results. True-positive results were obtained in 12/13 primary breast cancers (mean diameter=29 mm, range 8–53 mm) with both FDG and MIBI. False-negative results were obtained in two local recurrences (diameter <9 mm) with both FDG and MIBI. In benign disease, FDG and MIBI did not localize three fibrocystic lesions, two fibroadenomas and one inflammatory lesion (true-negative), but both localized one fibroadenoma (false-positive). Collectively, the results demonstrate a sensitivity of 92%, and a specificity of 86%, for primary breast cancer regardless of whether FDG or MIBI was used. In contrast to MIBI scintigraphy, FDG PET scored the axillae correctly as either positive (metastatic disease) or negative (no axillary disease) in all 12 patients. The tumour/non-tumour ratio for MIBI was 1.97 (range 1.43–3.1). The mean standard uptake value (SUV) for FDG uptake was 2.57 (range 0.3–6.2). The diagnostic accuracy of SMM was equivalent to that of FDG PET for the detection of primary breast cancer. For the detection of in situ lymph node metastases of the axilla, FDG seems to be more sensitive than99mTc-MIBI.

HLA-G Polymorphisms and Allele Frequencies in Caucasians

HLA-G, a nonclassical class I MHC molecule, is uniquely expressed on extravillous cytotrophoblasts of the maternal-fetal interface and is suggested to be essential for establishment of maternal-fetal immune tolerance. Although the level of polymorphism in HLA-G has originally been considered low, number, nature and site of polymorphisms seem to vary between different ethnic populations. We investigated HLA-G polymorphisms in a population of German and Croatian origin by SSCP-analysis and direct sequencing as well as RFLP analysis for presence of the 1597delC mutation. HLA-A alleles associated with the different HLA-G alleles were determined by SSP PCR-typing. In Caucasians, HLA-G exhibits a low degree of polymorphism on the amino-acid level and only slightly higher variability on the nucleotide level. In 264 independent chromosomes, 4 HLA-G alleles on the level of amino acid polymorphisms and an additional 6 variations of nucleotide sequences could be identified. The null-allele G*0105N was present at an allele frequency of 2.3%, which is higher than initially suggested for Caucasians but lower than in Hispanics and African-Americans. Furthermore, some HLA-G alleles exhibit strong linkage disequilibrium with HLA-A.

Exclusion of a major role for the PTEN tumour-suppressor gene in breast carcinomas

SummaryPTEN is a novel tumour-suppressor gene located on chromosomal band 10q23.3. This region displays frequent loss of heterozygosity (LOH) in a variety of human neoplasms including breast carcinomas. The detection of PTEN mutations in Cowden disease and in breast carcinoma cell lines suggests that PTEN may be involved in mammary carcinogenesis. We here report a mutational analysis of tumour specimens from 103 primary breast carcinomas and constitutive DNA from 25 breast cancer families. The entire coding region of PTEN was screened by single-strand conformation polymorphism (SSCP) analysis and direct sequencing using intron-based primers. No germline mutations could be identified in the breast cancer families and only one sporadic carcinoma carried a PTEN mutation at one allele. In addition, all sporadic tumours were analysed for homozygous deletions by differential polymerase chain reaction (PCR) and for allelic loss using the microsatellite markers D10S215, D10S564 and D10S573. No homozygous deletions were detected and only 10 out of 94 informative tumours showed allelic loss in the PTENregion. These results suggest that PTEN does not play a major role in breast cancer formation.

Polyspermy in in Vitro Fertilization of Human Oocytes: Frequency and Possible Causes

In an IVF program a total of 585 oocytes (180 patients) were examined for the presence of pronuclei 16 to 20 hours after the addition of spermatozoa. The overall fertilization rate was 71%, and in 58 (10%) of the fertilized oocytes, three or more pronuclei, indicating a failure of the block to polyspermy, could be observed. The frequency of polyspermy was related to the maturity of the oocyte, determined according to morphologic criteria. Immature oocytes showed a higher percentage of polyspermic fertilization (32%) compared to that of mature oocytes (6%). Preincubation of oocytes (for 0.5-1.5, 2-4, and 5-8 hours) prior to the addition of spermatozoa increased the fertilization rate (to 67%, 70%, and 83%, respectively). The polyspermy rate, however, was not significantly different between the various preincubation intervals (13%, 14%, and 19%, respectively). The polyspermy rate was affected by the number of spermatozoa used for in vitro fertilization. Insemination with 0.5-0.8, 1.0, or 1.5-2.0 X 10(6) spermatozoa/oocyte resulted in a polyspermy rate of 6%, 20%, and 32%, respectively. The appearance of polyspermic fertilization was not related to the age of the patient (which ranged from 20 to 45 years) nor to the method of ovarian stimulation (clomiphene, hMG, or clomiphene/hMG). Because of the high incidence of polyspermy under in vitro conditions it seems to be important to routinely examine the oocytes in the pronuclear stage. Reduction of the number of spermatozoa used for in vitro fertilization and the exact timing of insemination according to the maturity of the oocyte might reduce the occurrence of polyspermic fertilization.

Serum oxytocin concentration during embryo transfer procedure

OBJECTIVE To determine the effect of the embryo transfer (ET) procedure on serum concentration of oxytocin. STUDY DESIGN Prospective clinical study of 10 women undergoing in vitro fertilization (IVF) treatment with ET in the Section of Reproductive Medicine and Endocrinology at the Department of Obstetrics and Gynecology, University of Bonn, Germany. Serial blood samples were collected in time intervals of 20 s during embryo transfer procedure and serum oxytocin concentration was measured. RESULTS In the absence of tenaculum placement, none of the procedures associated with ET led to an increase in serum oxytocin concentration. When tenaculum placement was used, it was temporally (four out of five patients) associated with an elevation in oxytocin level, which remained elevated until of the end of ET procedure. CONCLUSION Application of a cervical tenaculum during ET or possibly also during intra uterine insemination (IUI) procedure can stimulate the release of oxytocin in some patients.

Homologous Intrauterine Insemination

Artificial intrauterine insemination using prepared husband’s spermatozoa (HI; formerly AIH) is a common procedure for the management of involuntary childlessness, even though in the literature there is no consensus on the effectiveness of such an approach. Especially in cases of male subfertility and persistent idiopathic fertility disorders, divergent views exist with regard to the therapeutic benefit of homologous insemination. Nevertheless, intrauterine insemination (IUI), both in spontaneous and, preferably, in ovulation induction cycles, is recommended as the first-choice option of assisted conception techniques, since — unlike gamete intrafallopian transfer (GIFT) or in vitro fertilization with embryo transfer (IVF/ET) — the procedure is noninvasive and also much more cost effective.