Background/Aims: Catheter injection speed affects depth and placement of the embryo into the uterine cavity and is shown to be highly variable in, and between, subjects in a manually performed embryo transfer. In an effort to standardize the injection speed during embryo transfer, we developed an automated transfer pump: the pump-regulated embryo transfer (PRET) device. In this randomized controlled trial, we aimed to investigate if standardization of the injection speed and pressure with this PRET results in a better controlled positioning of the transferred embryo(s). Methods: Five hundred ninety-nine in-vitro fertilization/intracytoplasmic sperm injection/frozen-thawed embryo transfer cycles were randomly assigned to the PRET or manual transfer. Positioning of the embryo(s) into the uterine cavity was measured with ultrasound. Results: The PRET device generates a significantly smaller variance of the positioning of the embryo(s) into the uterine cavity. This resulted in an ongoing pregnancy rate of 21% in the PRET versus 17% in the manual (p = 0.22) transfer group; frozen-thawed embryo transfers resulted in 17.5 versus 10.9% (p = 0.097), respectively. Conclusion: The PRET results in better controlled positioning of the embryo(s), and it also gives the opportunity to standardize embryo transfer. Whether the PRET may positively influence pregnancy rates, needs to be investigated in a multicenter trial.

Background: Breast cancer risk is temporarily increased after a full-term pregnancy and declines thereafter, possibly due to increased levels of gonadal and placental hormones during pregnancy. Inconsistent results, however, have been reported after twin pregnancies with higher hormone levels. Among women treated with in vitro fertilisation (IVF), for whom the number of embryos available for implantation is known, we recently observed that a multiple birth after implantation of all transferred embryos is associated with higher levels of vascular endothelial growth factor (VEGF). As VEGF is involved in breast cancer progression, we studied the effects of embryo implantation and a multiple birth on breast cancer risk in a nationwide Dutch cohort of IVF-treated women.Methods: We performed a cohort analysis among 12,589 women who had been treated with IVF between 1983 and 1995 and completed a risk factor questionnaire between 1997 and 1999. Data on IVF treatment were obtained from medical records. Breast cancer cases were ascertained through linkage with the population-based Netherlands Cancer Registry. Breast cancer risks associated with singleton and multiple births were estimated with Cox regression.Findings: There were 1688 women (13.4%) with multiples, 6027 (47.9%) with singletons and 4874 (38.7%) nulliparous women. Breast cancer occurred in 317 women of whom 57 had multiples. Breast cancer risk was 1.44 times higher in mothers of multiples than in mothers of singletons (95% confidence interval (CI) 1.06-1.97). Risk was highest in women who gave birth to multiples from all embryos transferred (adjusted hazard ratio (HR) 1.86, 95% CI 1.01-3.43), and lower for those with multiples after incomplete embryo implantation (adjusted HR 1.31, 95% CI 0.76-2.25).Interpretation: A woman's potential to implant all transferred embryos may be associated with breast cancer risk. Further research is needed to confirm our results and to identify the underlying biological mechanisms. (C) 2014 Elsevier Ltd. All rights reserved.

STUDY QUESTION:Does preconceptionally started low-dose aspirin prevent hypertensive pregnancy complications and preterm delivery in IVF patients?SUMMARY ANSWER:The current data do not support the use of preconceptionally started low-dose aspirin treatment for the prevention of hypertensive pregnancy complications and preterm delivery in IVF women.WHAT IS KNOWN ALREADY:Studies starting low-dose aspirin treatment as prevention in the second trimester of pregnancy found no or only moderate reductions in the relative risk of developing pre-eclampsia. Low-dose aspirin was possibly started too late, that is after the first episode of trophoblast invasion.STUDY DESIGN, SIZE, DURATION:We performed a meta-analysis with individual patient data (IPD), in which four authors could provide IPD on a total of 268 pregnancies (n = 131 treated with aspirin, n = 137 placebo). Data on hypertensive pregnancy complications and preterm delivery were collected.PARTICIPANTS/MATERIALS, SETTING, METHODS:All separate databases were merged into a summary database. Treatment effect of aspirin on the incidence of hypertensive pregnancy complications (n = 187) and preterm delivery (n = 180) were estimated with odds ratios (OR) and 95% confidence intervals (95% CI) using multivariable logistic regression.MAIN RESULTS AND THE ROLE OF CHANCE:There were significantly fewer twin pregnancies in the aspirin group (OR 0.55 95% CI 0.30-0.98), but no significant differences for hypertensive pregnancy complications and preterm delivery: for singletons OR 0.62 (95% CI 0.22-1.7) and OR 0.52 (95% CI 0.16-1.7), respectively, as well as for twin pregnancies OR 1.2 (95% CI 0.35-4.4) and OR 1.6 (95% CI 0.51-5.0), respectively.LIMITATIONS, REASONS FOR CAUTION:We have to bear in mind that the included studies showed clinical heterogeneity; there was variation in the duration of low-dose aspirin therapy and degree of hypertension between the different studies. Although we combined IPD from four studies, we have to realize that the studies were not powered for the outcome of the current IPD meta-analysis.WIDER IMPLICATIONS OF THE FINDINGS:Based on the current meta-analysis with IPD we found no confirmation for the hypothesis that preconceptionally started low-dose aspirin reduces the incidence of hypertensive pregnancy complications or preterm delivery in IVF women. Larger studies are warranted.

BACKGROUND:Dizygotic twin pregnancies after IVF treatment are the result of multiple embryos transferred into the uterine cavity, followed by successful double implantation. Factors that increase the chance of multiple implantation after IVF are relatively unknown. The present study aimed to investigate whether features of body composition, such as maternal height, weight and body mass index (BMI) are associated with an increased chance of dizygotic twinning after IVF with double embryo transfer (DET).METHODS:This study was conducted using data from a large Dutch nationwide cohort that comprised 19 861 women who had IVF or ICSI treatment between 1983 and 1995 (OMEGA study). First 'fresh' IVF and ICSI cycles with DET resulting in a delivery of a singleton or twin (living as well as stillborn) were selected. A multivariable logistic regression analysis was performed, with the delivery of a singleton or twin as the dependent variable and height, weight, BMI, maternal age, number of retrieved oocytes, use of alcohol, smoking, highest level of education and parity as independent variables.RESULTS:Of the 6598 women who completed their first IVF or ICSI cycle, 2375 had DET, resulting in 496 deliveries of 371 singletons and 125 twins. Multivariable regression analysis revealed that tall women (>1.74 cm) and women with a high number of retrieved oocytes (>8) had an increased chance of dizygotic twinning [OR: 1.8 (95% CI: 1.0-3.4) and OR: 2.2 (95% CI: 1.3-3.8), respectively].CONCLUSIONS:Our data demonstrate that tall stature and increased number of retrieved oocytes independently increase the chance of dizygotic twinning after IVF with DET.

The position of transfer air bubbles after embryo transfer is related to the pregnancy rate. With the conventional manual embryo-transfer technique it is not possible to predict the final position of the air bubbles. This position mainly depends on the catheter load speed at transfer (injection speed), a parameter that remains uncontrollable with the conventional technique even after standardization of the protocol. Therefore, the development of an automated device that generates a standardized injection speed is desirable. This study aimed to examine the variation in injection speeds in manual embryo transfer and pump-regulated embryo transfer (PRET). Seven laboratory technicians were asked to perform simulated transfers using the conventional embryo-transfer technique. Their injection speeds were compared with that of a PRET device. The results indicate that in manually performed transfers, even after standardization of the protocol, there is still a large variation in injection speed, while a PRET device generates a reliable and reproducible injection speed and therefore brings new possibilities for further standardization of the embryo-transfer procedure. Future research should reveal whether these experiments mimic real clinical circumstances and if a standardized injection speed results in more exact positioning of the transferred embryos and therefore higher pregnancy rates.

BACKGROUNDAspirin is believed to improve the outcome of IVF, but previous conventional meta-analyses on the subject are conflicting. Therefore, we performed a meta-analysis with individual patient data (IPD MA) of randomized clinical trials (RCTs) on the subject.METHODSA systematic literature search was conducted to identify RCTs assessing the effectiveness of aspirin in IVF. Authors were asked to share their original data. In a one step meta-analytic approach, the treatment effect of aspirin was estimated with odds ratios (ORs) and 95% confidence intervals (CIs) using logistic regression, based on the intention to treat principle.RESULTSTen studies fulfilled the inclusion criteria. Authors of six studies provided IPD, including 1119 patients (562 placebo and 557 aspirin). There were 160 clinical pregnancies in the aspirin (28.8%) and 179 (31.9%) in the placebo group [OR 0.86, 95% CI (0.69-1.1)]. There were 129 ongoing pregnancies in the aspirin (23.6%) and 147 in the placebo group (26.7%) [OR 0.85, 95% CI (0.65-1.1)]. Whereas the conventional meta-analysis limited to studies that could provide IPD showed an OR of 0.89 (95% CI 0.69-1.2), the conventional meta-analysis limited to the eight studies of which method of randomization could be confirmed showed an OR of 0.94 (95% CI 0.76-1.17) and the conventional meta-analysis including all 10 eligible RCTs identified with our search changed the OR to 1.07 (95% CI 0.81-1.41). This difference in direction of effect, derived from the studies not able to share IPD of which quality of randomization could not be confirmed.CONCLUSIONSAspirin does not improve pregnancy rates after IVF.

Objective: In assisted reproductive techniques it is important to find a balance between high pregnancy and acceptable multiple pregnancy rates. In IVF treatment, stimulation with highly purified human menopausal gonadotropin (hMG) results in comparable or even higher pregnancy rates at lower oocyte yields compared to recombinant FSH. Since highly purified hMG contains LH activity, a number of the advantages of highly purified hMG may be attributed to this LH activity. In IUI treatment the effectiveness of highly purified hMG has been barely investigated. The aim of this study was to examine the effectiveness of highly purified hMG in IUI patients treated with a mild stimulation protocol.Study design: In this retrospective study 378 patients were included, receiving 1400 IUI cycles between January 2006 and December 2007. Patients were first treated with three subsequent natural cycles without controlled ovarian hyperstimulation, followed by three subsequent cycles stimulated with highly purified hMG. Primary outcomes were ongoing pregnancy rate and multifollicular growth. Secondary outcomes were multiple pregnancy and miscarriage rates. Primary and secondary outcomes were expressed in percentages with associated 95% confidence intervals (95%CI). Differences in the outcomes between natural and stimulated cycles were calculated using chi(2) tests. Statistical differences were determined at P < 0.05.Results: Ongoing pregnancy rates increased from 6% (95%CI 4.7-7.7) per natural cycle to 7.4% (95%CI 5.2-10.3) per highly purified hMG stimulated cycle (p = 0.34). The highest ongoing pregnancy rate was observed in the fifth treatment cycle (10.8% (95%CI 6.6-17)), which is significantly higher than the ongoing pregnancy rate in the unstimulated group (p = 0.03). In the highly purified hMG group three (9.7% (95%CI 3.3-24.9)) of the ongoing pregnancies were twin pregnancies, in the unstimulated group there was one (1.7% (95%CI 0.3-9.0)) twin pregnancy (p = 0.08).Conclusion: Our results indicate that mild stimulation with highly purified hMG in IUI treatment results in an acceptable balance between ongoing and multiple pregnancy rates. Future prospective trials should compare mild stimulation protocols to protocols directly starting with controlled ovarian hyperstimulation. Furthermore, these trials should compare other types and dosages of gonadotropins. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

This study aimed to compare longitudinal serum concentrations of angiogenic implantation factors between ongoing singleton and twin pregnancies after double-embryo transfer and to investigate whether these are involved in sustained double implantation. Sixteen patients with an ongoing singleton and nine patients with an ongoing twin pregnancy after double-embryo transfer were included in this prospective observational study. Main outcome measures were concentrations of vascular endothelial growth factor-A (VEGF-A), inhibin A, glycodelin A, insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), insulin-like growth factor binding protein-1 (IGFBP-1) and insulin-like growth factor binding protein-3 (IGFBP-3) at baseline, during the IVF treatment and in early pregnancy. It appeared that VEGF-A concentrations prior to any treatment and at early implantation as well as body mass index (BMI) were higher in women who conceived a twin pregnancy (P = 0.04). Soon after implantation, inhibin A concentrations were higher in twin pregnancies (P=0.02). Secretion profiles of glycodelin A and members of the IGF family did not differ between singleton and twin pregnancies. VEGF-A appears to play a role in sustained double implantation. Furthermore a high BMI is associated with ongoing double implantation. Future studies should investigate the predictive value of VEGF-A for having an ongoing singleton or twin pregnancy.

OBJECTIVE: Aspirin, a simple, safe and inexpensive drug, might improve the effectiveness of IVF. Previous systematic reviews for aspirin in IVF have shown conflicting results. Performing a meta-analysis on individual patient data from each trial permits us to adjust for important variables, such as female age, and allows an analysis of differential treatment effects in patient subgroups. The aim of this IPD MA was to compare the effectiveness of aspirin versus placebo in IVF. DESIGN: Individual patient data meta-analysis. MATERIALS AND METHODS: A systematic search of the literature was conducted to identify randomised controlled trials examining aspirin in IVF. Authors of these trials were requested to send their original data. Treatment effect of aspirin was estimated with odds ratios (OR) and 95% confidence intervals (CI) using logistic regression, based on the intention to treat principle. The analysis was stratified for centre. We controlled for female age as well as interaction between age and treatment effect. RESULTS: Ten studies fulfilled the inclusion criteria. Three authors were not able to share data. We are currently waiting for two datasets. Therefore, five trials comparing aspirin with placebo in a total of 1060 patients could be analysed. Data on outcome or allocation were missing in 54 patients, leaving data of 1006 patients for analysis. Mean female ages in the aspirin and placebo groups were 31.1 years and 31.9 years, respectively. The number of clinical pregnancies was 174 (34.5%) versus 168 (33.5%) in the placebo group (OR 1.04, 95% CI (0.80 to 1.4). Addition of female age to the model did not alter treatment effect (OR 1.03, 95% CI (0.79 to 1.3). This point estimate of 1.03 was lower than the 1.09 reported in conventional meta-analysis published in Cochrane. CONCLUSIONS: Previous conventional meta-analyses have overestimated the treatment effect of aspirin. According to our meta-analysis, low dose aspirin does not improve pregnancy rates after IVF.

Introduction: HLA-G is thought to play an important role in the immuological recognition of pregnancy since it is the main HLA molecule expressed on the trophoblast and it seems to display immunosuppressive properties during normal implantation and pregnancy.A 14 basepair (bp) long insertion in exon 8 of the HLA-G gene seems to decrease stability of the HLA-G transcript.We have previously found that plasma concentrations of soluble HLA-G are lower in homozygous carriers of this HLA-G14bp insertion.In theory, homozygous carriers of the HLA-G14bp insertion are at an increased risk of impaired trophoblast invasion and growth since immune protection is defect.Material and Methods: We investigated the HLA-G14bp insertion/deletion polymorphism in 301 Caucasian patients with three or more consecutive miscarriages that remained unexplained after routine anatomical, chromosomal, endocrine and autoimmune screening and in 168 Caucasian women with two or more children and no miscarriages.The HLA-G14bp polymorphisms were investigated after amplification of the HLA-G exon 8 using genomic DNA and primers GE14HLAG and RHG4.Results: In all recurrent miscarriage patients 56 (18.6 %) were homozygous for the HLA-G14bp insertion compared with 19 (11.3%) of the controls (p < 0.05).HLA-G14bp insertion homozygocity was found in 19.2% of patients with secondary recurrent miscarriage (p < 0.05 compared with controls) and 17.7% of patients with primary recurrent miscarriage (not significant compared with controls).Among the children born prior to a secondary recurrent miscarriage diagnosis, the mean birth weight was 2766.3 g in 35 children born to HLA-G14bp homozygous women, which was significantly lower than the mean birth weight of 3082.7g in the 119 children born to women who were HLA-G14bp insertion/ deletion heterozygous or HLA-G14bp deletion homozygous (p < 0.01).Conclusions: Homozygocity for the HLA-G14bp insertion seems to be associated with recurrent miscarriage and especially secondary recurrent miscarriage.In first pregnancies of patients with secondary recurrent miscarriage, maternal carriage of this HLA-G14bp genotype seems to predispose to birth of children with significantly reduced birth weight compared with children born to similar patients with other HLA-G14 bp genotypes.HLA-G14bp insertion homozygocity may affect maternal immune tolerance to the trophoblast thereby causing miscarriage or intrauterine growth restriction.

BACKGROUND:The use of aspirin during in vitro fertilization (IVF) has been investigated for its effect on pregnancy rates after IVF. In most of these studies, aspirin administration was then prolonged throughout the first trimester of pregnancy. By inhibiting vasoconstriction, the use of low-dose aspirin in the first trimester could influence placentation and therefore prevent or delay development of hypertensive pregnancy complications, such as pregnancy-induced hypertension (PIH) and pre-eclampsia (PE).METHODS:This study involved the follow-up by questionnaires and hospital records of patients with an ongoing pregnancy in a prospective, randomized, double-blind, placebo-controlled trial on the effect of low-dose aspirin during IVF. Aspirin treatment was continued throughout the first trimester of pregnancy. The primary end-point of this follow-up study was the incidence of pregnancy complications. The original trial is registered with the Dutch Trial Register and as an International Standard Randomized Clinical Trial, No. ISRNCTM97507474.RESULTS:There were 54 patients who had ongoing pregnancies in the original trial; 90.7% returned the questionnaire and all Dutch hospital records were retrieved. A significant difference was found in the incidence of hypertensive pregnancy complications: 3.6% in the aspirin group and 26.9% in the placebo group (P < 0.05), resulting in numbers-needed-to-treat (NNT) of 10.3 to prevent hypertensive complications in one pregnancy after IVF treatment.CONCLUSIONS:The incidence of hypertensive complications was significantly lower in the group of women treated with low-dose aspirin throughout IVF treatment and first trimester of pregnancy. These results suggest a potential benefit of low-dose aspirin during IVF and first trimester to prevent hypertensive pregnancy complications. The findings justify further investigation in placebo-controlled randomized trials.

OBJECTIVE: The basis of (late) vascular complications in pregnancy, such as high blood pressure and pre-eclampsia, is known to be founded in early stages of pregnancy. The process of placentation plays a role in development of high blood pressure or pre-eclampsia later in pregnancy. As an inhibitor of the cyclo-oxygenase (COX) enzyme aspirin inhibits thromboxane A2 and reduces vasoconstriction. The use of low-dose aspirin early in pregnancy may therefore influence the process of placentation and potentially the chance of developing complications later in pregnancy.DESIGN: Follow-up in a prospective randomised controlled trial.MATERIALS AND METHODS: Fifty-four patients had an ongoing pregnancy in a prospective randomised double blind placebo controlled trial investigating the effect of low-dose aspirin on pregnancy rates among IVF/ICSI patients with previous implantation failure. None of these patients had a history of high blood pressure. All patients had used 100 mg of aspirin or 100 mg placebo daily during a long GnRH-agonist protocol and had continued the study medication until 12 weeks of pregnancy. After delivery all patients received a questionnaire inquiring after complications during pregnancy, delivery and after the health their babies. These data were linked with the data of their hospital records, that where checked for complications during pregnancy, delivery and the health the baby.RESULTS: All 54 patients received a questionnaire, 28 patients were treated with aspirin (group A) and 26 patients with placebo (group B). Four women in group A and nine women in group B had a twin pregnancy. The overall response rate was 85%. In group A twelve women had no complications during pregnancy, one woman had a high blood pressure during pregnancy and 4 women reported blood loss during pregnancy. In group B six women had no complications during pregnancy, 5 women had a high blood pressure, 2 women had a high blood pressure with pre-eclampsia, of which one developed a HELLP-syndrome, one woman had a pulmonary embolism and 4 women reported blood loss during pregnancy. The incidence of vascular complications during pregnancy was significantly higher in group B: 3.6% vs 26.9%, p<0.05. Other outcome was not different between the groups.CONCLUSIONS: Women treated with low dose aspirin during IVF treatment and the first 12 weeks of their pregnancy had significantly lower incidence of blood pressure related complications during pregnancy. OBJECTIVE: The basis of (late) vascular complications in pregnancy, such as high blood pressure and pre-eclampsia, is known to be founded in early stages of pregnancy. The process of placentation plays a role in development of high blood pressure or pre-eclampsia later in pregnancy. As an inhibitor of the cyclo-oxygenase (COX) enzyme aspirin inhibits thromboxane A2 and reduces vasoconstriction. The use of low-dose aspirin early in pregnancy may therefore influence the process of placentation and potentially the chance of developing complications later in pregnancy. DESIGN: Follow-up in a prospective randomised controlled trial. MATERIALS AND METHODS: Fifty-four patients had an ongoing pregnancy in a prospective randomised double blind placebo controlled trial investigating the effect of low-dose aspirin on pregnancy rates among IVF/ICSI patients with previous implantation failure. None of these patients had a history of high blood pressure. All patients had used 100 mg of aspirin or 100 mg placebo daily during a long GnRH-agonist protocol and had continued the study medication until 12 weeks of pregnancy. After delivery all patients received a questionnaire inquiring after complications during pregnancy, delivery and after the health their babies. These data were linked with the data of their hospital records, that where checked for complications during pregnancy, delivery and the health the baby. RESULTS: All 54 patients received a questionnaire, 28 patients were treated with aspirin (group A) and 26 patients with placebo (group B). Four women in group A and nine women in group B had a twin pregnancy. The overall response rate was 85%. In group A twelve women had no complications during pregnancy, one woman had a high blood pressure during pregnancy and 4 women reported blood loss during pregnancy. In group B six women had no complications during pregnancy, 5 women had a high blood pressure, 2 women had a high blood pressure with pre-eclampsia, of which one developed a HELLP-syndrome, one woman had a pulmonary embolism and 4 women reported blood loss during pregnancy. The incidence of vascular complications during pregnancy was significantly higher in group B: 3.6% vs 26.9%, p<0.05. Other outcome was not different between the groups. CONCLUSIONS: Women treated with low dose aspirin during IVF treatment and the first 12 weeks of their pregnancy had significantly lower incidence of blood pressure related complications during pregnancy.

The beta1D protein is a recently characterized isoform of the integrin beta1 subunit that is present in cardiac and skeletal muscles. In this study, we have examined the expression of beta1D in different types of skeletal muscle and in cardiac muscle and studied its distribution during mouse development, using new monoclonal antibodies specific for beta1D. Immunoprecipitation studies revealed that, while beta1A is strongly expressed in proliferating C2C12 myoblasts, beta1D is only expressed after their differentiation to myotubes. In these myotubes, beta1D is associated with different alpha subunits, namely alpha3A, alpha5, alpha7A, or alpha7B. Initially, during embryogenesis, the alpha1A subunit is the only beta1 variant expressed in skeletal and cardiac muscle. The beta1D subunit is first detected in skeletal muscle at E17.5, whereas in cardiac muscle its expression begins around the time of birth. Later the expression of beta1A in skeletal and cardiac muscle becomes restricted to capillary cells, whereas beta1D eventually becomes the only variant expressed in adult cardiac and skeletal muscle cells. The switch from the beta1A to the beta1D subunit in cardiac muscle cells coincides with the expression of alpha7. In adults there is a distinct concentration of beta1D at the myotendinous junctions of muscle fibers and at costameres in both cardiac and skeletal muscle. In addition, beta1D is present at intercalated discs in cardiac muscle and at neuromuscular junctions in skeletal muscle cells. The amount of beta1D in different types of skeletal muscle (fast, slow, and mixed-type) was similar, but cardiac muscle expressed almost five times as much of this protein. We suggest that beta1D plays a role in the maintenance of the cytoarchitecture of mature muscle and in the functional integrity of the muscle cells.

Immunohistochemical and biochemical procedures were used to study the influence of retinoic acid (RA) on cellular expression and distribution of cytokeratins (CKs) in feline mammary carcinoma cells. These cells were grown in vitro as established cell lines (K248C and K266) and in vivo as xenografts in athymic mice. The results were compared with the distribution of CKs in normal feline mammary gland and in a series of invasive mammary carcinomas previously probed with a panel of monoclonal antibodies specific for individual CKs. Coexpression of CKs of both major mammary gland cell types (myoepithelial cells, MECs, CKs 5/14 positive, and luminal epithelial cells, LECS, CKs8/18 positive) by K248C and K266 cells, suggested a stem cell-like character of both cell lines. RA increased CK19 expression in both cell lines and CK19 was also present in tumors developed in nude mice from both RA untreated (CK19 negative) and RA-treated (CK19 positive) K248C and K266 cells. In addition, RA had cell line specific effects as well. RA treatment induced differentiation of K248C cells to more mature LEC-like cells and this change was accompanied by the loss of the MEC keratins CKs 5/14. Under the same culture conditions however, RA treatment did not induce morphological changes in the K266 cell line and the expression of CKs 5/14 was not significantly reduced. These findings suggest that the modulation of CK19 and CKs 5/14 expression observed in mammary carcinoma cells upon RA treatment might be regulated through different pathways.

Expression of keratins (cytokeratins, CK) known to be suitable markers for different types of epithelial differentiation was analyzed in specimens of feline mammary tissue. A panel of specific anti-CK monoclonal antibodies (MAb) was used to determine CK distribution pattern in normal feline tissues (n = 3), and in benign (n = 18) and malignant (n = 20) feline mammary tumors. In selected tumors, the CK distribution pattern was also determined by biochemical methods. A MAb specific for alpha-smooth muscle actin was used to discriminate between myoepithelial cells and luminal epithelial cells. In normal mammary gland tissues, 6 MAb reacted exclusively, either with myoepithelial cells or with luminal epithelial cells. Luminal epithelial cells reacted with MAb specific for CK typical of simple epithelia, whereas myoepithelial cells reacted with MAb specific for CK in basal cells of stratified epithelia. A similar distribution of CK was detected in specimens from benign tumors, except that CK4 was not detected in normal mammary gland tissues and was detected in some ducts in specimens with adenosis. Almost all tumor cells in specimens from malignant tumors reacted with MAb specific for CK typical of simple epithelia. Concomitant expression of CK typical of stratified epithelia was detected in small or large subpopulations of tumor cells in 70% of carcinomas. Cytokeratins typical of basal cell layers and typical for suprabasal layers of inner stratified epithelia were detected. Cytokeratins typical of stratified epithelia were always found in areas of squamous metaplasia, but also were found in adenocarcinomal cells surrounding these areas.(ABSTRACT TRUNCATED AT 250 WORDS)