Expression of selected tumor suppressor and oncogenes in endometrium of women with endometriosis.

BACKGROUND: It is becoming increasingly evident that the eutopic endometrium of women with endometriosis shows certain genetic alterations which are not found in the endometrium of disease-free women. The aim of the study was to compare the expression level of mammalian target of rapamycin (mTOR) tumor suppressor and oncogene-related genes in the endometrium of women with and without endometriosis as well as in ovarian endometriosis. METHODS: A total of 81 regularly menstruating patients were recruited in the study. We applied the micro fluidic gene array to examine the expression of 15 human tumor suppressor and oncogenes in eutopic endometrium of 40 women with endometriosis and 41 controls without endometriosis. In 14 patients with endometriosis, gene expression was also studied in matched ovarian lesions. We studied the following genes: NF1, RHEB, mTOR, PTEN, TSC1, TSC2, KRAS, S6K1, TP53, EIF4E, LKB1, PIK3CA, BECN1, 4EBP1 and AKT1. Immunohistochemical studies were subsequently performed for selected proteins. RESULTS: Of the 15 studied genes, we found significantly higher levels of oncogene AKT1 (P = 0.006) and tumor suppressor gene 4EBP1 (P = 0.01) mRNAs in the eutopic endometrium of women with endometriosis compared with control patients. Immunohistochemistry showed that 4EBP1 and AKT1 proteins were expressed in eutopic endometrium. CONCLUSIONS: Our results suggest that up-regulation of AKT1 and 4EBP1 in eutopic endometrium may be associated with the pathogenesis of endometriosis, but their precise role remains to be established.

Profiling of Selected MicroRNAs in Proliferative Eutopic Endometrium of Women with Ovarian Endometriosis.

It has been well documented that aberrant expression of selected microRNAs (miRNAs) might contribute to the pathogenesis of disease. The aim of the present study is to compare miRNA expression by the most comprehensive locked-nucleic acid (LNA) miRNA microarray in eutopic endometrium of patients with endometriosis and control. In the study we recruited 21 patients with endometriosis and 25 were disease-free women. The miRNA expression profiles were determined using the LNA miRNA microarray and validated for selected molecules by real-time PCR. We identified 1198 human miRNAs significantly differentially altered in endometriosis versus control samples using false discovery rate of <5%. However only 136 miRNAs showed differential regulation by fold change of at least 1.3. By the use of selected statistical analysis we obtained 45 potential pathways that might play a role in the pathogenesis of endometriosis. We also found that natural killer cell mediated cytotoxicity pathway was found to be inhibited which is consistent with previous studies. There are several pathways that may be potentially dysregulated, due to abnormal miRNA expression, in eutopic endometrium of patients with endometriosis and in this way contribute to its pathogenesis.

The influence of lead ions on uterine activity in the rat.

Spontaneous uterine activity and reactivity to oxytocin, naproxen and PGF2 alpha were studied in vitro in 80 rats, which for 2-8 weeks had been exposed to different concentrations and combinations of Pb2+, Zn2+ and Cu2+ in their water supply or had been given clean water for control. In rats given only Pb2+ in concentrations of 1000 and 500 ppm for 6 weeks the uterine activity was significantly increased, whereas in groups given the other ions alone, or Pb2+ for 6 weeks followed by 2 weeks of clean water or Zn2+ or Cu2+ no change was observed. The responses to the oxytocin, naproxen and PGF2 alpha did not differ. These results suggest that contamination with lead ions might be one of the etiological factors involved in conditions with increased uterine activity.

Influence of high lead and cadmium soil content on human reproductive outcome.

The influence of lead and cadmium on human reproductive outcome was studied in areas of Eastern Poland with high specific soil contamination with these heavy metals. By personal interviews, information on socioeconomic background factors as well as obstetrical and gynecological histories were obtained from 136 women living in villages with high amounts of lead and cadmium and from 269 women of nearby villages with no increased content of these metals. Physical examination and estimations of blood concentrations of lead and cadmium were also performed in 89 and 175 women from these areas, respectively. In the contaminated areas, there were significantly fewer women with three or more pregnancies (39 vs. 52%) and deliveries at full term (35 vs. 44%) than in nearby villages with normal concentrations of these heavy metals, and the weight of preterm newborns was lower (1930 vs. 2225 g). Furthermore, hypertension was more common (15 vs. 22%) and of higher magnitude (mean blood pressure 140 vs. 129 mmHg) in the study group than in the control women. The mean blood concentrations of lead in the two groups. 675 and 621 micrograms/dl did not differ statistically, whereas the concentrations of cadmium of 29 and 25 micrograms/dl were significantly higher in the study than in the control group. There was a significant correlation between cadmium levels and number of preterm deliveries. These results suggest that cadmium and lead could be factors of importance in reproductive outcome.

[The role of adhesive molecules in pathogenesis of endometriosis. Laboratory findings and clinical aspects]. / Rola czynników adhezyjnych w patogenezie endometriozy. Badania doswiadczalne i implikacje kliniczne.

Adhesive molecules have played an important and well-established role in cell-to-cell communication for a long time. They reveal specific functions in an ongoing disturbed immunological reactions, including endometriosis. This review is to describe the role of topical factors involved in the pathogenesis of arising and course of endometriosis, taking adhesive molecules into special consideration. It does not only include the mechanisms and the effects of their action but also some advantages that can be gained from the knowledge of their function. The attention is also drawn to the putative role of certain proteolitical enzymes and their inhibitors which when more active can intensify or reduce the symptoms of the disease.

[The effectiveness of hysteroscopic intrauterine septum resection in terms of reproductive outcome]. / Skutecznosc hysteroskopowego usuniecia przegrody wewnatrzmacicznej w aspekcie uzyskania plodnosci.

Uterine septum is the major intrauterine disorder associated with infertility and recurrent abortions. The aim of the study was to find out what is the effectiveness of hysteroscopic intrauterine septum resection in terms of reproductive outcome and in comparison with conventional procedure. The mean follow up period was 18 +/- 8.3 months. The pregnancy wastage before operation in study group was 79% and decreased to 45% after hysteroscopic treatment. Hysteroscopic resection of intrauterine septum is the treatment of choice for patients suffering from infertility or recurrent pregnancy wastage.

[Pentoxifylline and verapamil influence on the phagocytosis of peritoneal macrophages from women with endometriosis]. / Wplyw pentoksyfiliny i werapamilu na aktywnosc fagocytarna makrofagów plynu otrzewnowego u kobiet z endometrioza.

OBJECTIVES: Disturbances in topical immunological response during endometriosis, cell and humoral, is of particular interest nowadays. Increase in the number and activity of peritoneal macrophages depends to a large extent on calcium concentration. Pentoxifylline and verapamil are both known for their ability to regulate calcium homeostasis. DESIGN: Evaluation of the pentoxifilline and verapamil influence on the phagocytic activity of the peritoneal macrophages from women with endometriosis. MATERIALS AND METHODS: Incubation of the peritoneal fluid with latex in four sets: 1) control with medium for 30 minutes; 2) with pentoxyfilline (3.8 mmol/l) for 30 minutes; 3) with verapamil 0.4 mmol/1; 4) with pentoxyfilline and verapamil together. Percentage of phagocyting cells, phagocytosis index and a score of phagocyting activity was calculated after 30 minutes incubation. Verapamil showed to increase all parameters of phagocytosis in comparison with control. CONCLUSIONS: Verapamil is effective reagent in activity of peritoneal fluid macrophages. Further studies on larger group of patients are necessary to see if the changes are statistically relevant.

[The comparison of efficiency of supplement of the second phase in the program IVF-ET by dydrogesterone and progesterone]. / Porównanie skutecznosci suplementacji drugiej fazy w programie IVF-ET dydrogesteronem i progesteronem.

The retrospective study was carried out to compare effectiveness of progesteron and dydrogesteron luetal phase support following in vitro fertilisation and embryo transfer. Two hundred and fifty eight patients undergoing IVF ET received progesteron in itramuscular injections. Two hundred sixty patients received dydrogesteron three times daily p.o. There were no differences in stimulation protocols, indications for IVF and IVF procedures. Pregnancy rate, implantation rate and spontaneous abortion rate were similar in both groups. The effectiveness of luteal phase support in patients undergoing IVF ET program by dydrogesteron and progesteron is similar with significantly lower costs for the last one.

[Monozygotic pregnancy after the treatment for infertility by transfer of frozen-thawed embryos]. / Ciaza mnoga monozygotyczna po leczeniu nieplodnosci metoda pozustrojowego zaplodnienia.

OBJECTIVES: It has been suggested that monozygotic pregnancies occur more frequently after in vitro fertilisation. This phenomenon is attributed to a number of factors including in vitro culture conditions, malformation of the zona pellucida due to manipulation on the oocytes and artificial opening of the zona pellucida. DESIGN: Retrospective analysis of monozygotic pregnancies in an IVF-ET procedures and obstetrical these pregnancies outcome. MATERIALS AND METHODS: A total of 2254 IVF-ET procedures were analysed. Three protocols were used for ovarian stimulation: short or long protocols with gonadotropins releasing hormone analogue or clomiphene citrate with hMG. In 811 cases male factor was diagnosed and intracitoplasmatic sperm injections were performed. RESULTS: 549 clinical pregnancies were achieved in analysed group. In six cases ultrasound examination 5 weeks after embryo transfer showed a greater number of foetus than the number of embryo transferred. In three of those cases the embryos were obtained after ICSI. Age and average thickness of zona pellucida were similar in group of patients with monozygotic pregnancies when compared with all pregnant patients after IVF treatment. CONCLUSIONS: The incidence of monozygotic pregnancy is increased in group pregnancies resulting from IVF ET. No single risk factor can explain this phenomena. This type of pregnancy needs special obstetrical attention.

[Seasonal changes in donor's sperm motility: CASA parameters]. / Wyniki komputerowej oceny seminologicznej w grupie dawców nasienia--zmiany sezonowe parametrów CASA.

Computer assisted semen analysis was employed to find out whether there are some changes in movement parameters between autumn-winter and spring-summer time. 473 sperm specimens obtained from 12 sperm donors were analyzed. Statistically significant percentage of motile spermatozoa was higher during spring-summer time.

[Pregnancy and delivery after the transfer of one blastocyst with enzymatically removed zona pellucida]. / Ciaza i poród po transferze blastocysty pozbawionej enzymatycznie oslonki przejrzystej.

OBJECTIVES: The embryonic development up to the blastocyst stage and hatching from zona pellucida are prerequisites for implantation and successful pregnancy. It is suggested that one of the possibilities limiting the implantation rate is impaired hatching. To overcome this problem an artificial alteration of the zona pellucida have been carried out in many laboratories. DESIGN: The report of the first pregnancy in Poland obtained after the transfer of enzymatically zona removed blastocyst. MATERIALS AND RESULTS: The patient was previously treated three times in in vitro fertilisation program without success. In the fourth program the embryos were cultured in co-culture of Vero cells to the blastocyst stadium. On the day 5 after insemination the zona of cavitating and expanding blastocyst was removed by pronase. The zona free blastocyst was transferred to the uterus. As a result of implanted blastocyst the ongoing pregnancy developed normally and the patient delivered healthy baby. CONCLUSION: Enzymatic zona pellucida hatching probably increases the rate of implantation. It is simple, safe and economic techniques.

[Enzymatic assisted hatching in the infertile couple after failed attempts of IVF ET]. / Enzymatyczne usuniecie oslonki przejrzystej blastocysty pronaza u pacjentek po kilkukrotnych niepowodzeniach w programie IVF ET.

The study was conducted on the patients who had attempted and failed previous IVF procedures. The aim of this study was to determinate effectiveness sequential culture of embryos to blastocyst and assisted hatching by zona pellucida digestation by pronase. In this group the pregnancy rate was 24.1% compared to control 17.8%. The implantation rate was higher in study group than control one. It suggests that the culture embryos to balstocyste stage and assisted hatching by digestation by pronase is safe and effective mode of treatment of infertile couple after failed attempts IVF ET.

[Intracytoplasmic sperm injection (ICSI): the Bialystok experience]. / Wyniki leczenia nieplodnosci metoda icsi w osrodku bialostockim.

OBJECTIVE: To present a complete results of Intracytoplasmic Sperm Injection Program (ICSI) performed at IVF Unit in Bialystok, Poland. DESIGN: Retrospective data analysis. MATERIALS AND METHODS: A total of 2593 cycles of ICSI treatment were analyzed. The results of controlled ovarian hyperstimulation (COH), parameters of fertilization, early embryo development and clinical pregnancy rates were compared depending on the type of COH used. RESULTS: Overall, for 16708 MII oocytes microinjected, the 2 pronuclear fertilization rate was 54%. Embryo transfer was performed in 94% of started cycles and 629 clinical pregnancies were recorded, giving a clinical pregnancy rate of 24% per cycle and 26% per transfer. No influence of COH on embryological and clinical results was noted. CONCLUSION: Intracytoplasmic sperm injection can be used successfully to treat couples with male factor infertility and those who have failed standard IVF. Precisely adapted protocol of ovarian hyperstimulation for every patient could diminish the costs of treatment without negative influence on ICSI results.

["Long protocol" for ovarian stimulation using D-Trp6 LHRH (Triptorelin Depot) and menopausal gonadotropin in the IVF-ET program]. / "Dlugi protokól"stymulacji jajeczkowania z uzyciem D-Trp6 LHRH (Triptorelina Depot) i gonadotropiny menopauzalnej w programie IVF-ET.

39 patients (age 35-40, anovulation, previous failures in the IVF-ET programme) were superovulated using GnRH analogue (Decapeptyl-Depot) in the long protocol and hMG 97 embryos were transferred and 109 were cryopreserved. The clinical pregnancy and implantation rates were 31.6% and 13.4%. The long protocol gives better results in the IVF-ET programme but increases the costs of treatment.

[Reinsemination of oocytes by intracytoplasmic sperm injection after the failure of conventional fertilization. Early embryonal development]. / Mikroiniekcja plemnika do cytoplazmy (ICSI) oocytów nie zaplodnionych w warunkach IVF. Wczesny rozwój embrionalny zarodkow.

OBJECTIVE: To analyze the influence of intracytoplasmic sperm injection (ICSI) on early embryo development used for reinsemination of unfertilized 1-day-old oocytes. DESIGN: Prospective observational study. MATERIALS AND METHODS: A total of 126 embryos resulted from reinsemination by ICSI were analyzed with regard for the time course of cleavage and the quality of embryos at 48 hours after injection. Results were compared with those obtained in 698 embryos obtained after routine ICSI program. RESULTS: It has been shown that embryos derived from reinsemination developed slower in comparison with those obtained after regular ICSI program. At 48 hours after microinjection 50% of embryos achieved 4 blastomeres stage and 37% remained on 2 cells stage. 71% of regular ICSI embryos at this same time showed 4 blastomere or more, only 16% remained on 2 blastomeres stage. The quality of embryos was similar in two compared groups, however those obtained following reinsemination at 4 blastomere stage were significantly poorer quality. CONCLUSION: Embryos derived from reinsemination of 1-day old oocytes unfertilized after conventional IVF developed slower and the quality of embryos at 4 blastomere stage was poorer comparing to regular ICSI embryos.

Profiling of selected angiogenesis-related genes in proliferative eutopic endometrium of women with endometriosis.

OBJECTIVE: To compare the expression level of the most relevant angiogenesis-related genes in the eutopic endometrium of women with and without endometriosis. STUDY DESIGN: 32 regularly menstruating patients (18 with endometriosis and 14 controls) underwent surgery in the proliferative phase of the cycle. Eutopic endometrium was collected by the use of aspirating biopsy prior to laparoscopy. Only patients with advanced (stage III and IV) histopathologically confirmed ovarian endometriosis were studied. Real-time PCR gene arrays were applied to examine the expression of 84 human angiogenesis-connected genes. Western-blot and enzyme-linked immunosorbent assays (ELISA) were used to confirm the expression of selected proteins. RESULTS: We found significantly higher levels of AKT1 (p=0.003), TYMP (p=0.02), JAG1 (p=0.007), LAMA5 (p=0.005) and TIMP-1 (p=0.03) in eutopic endometrium of patients with endometriosis as compared with controls. By the use of Western blot we found clearly positive expression of AKT1 whereas ELISA assays confirmed expression of AKT1, TYMP, JAG1, LAMA5 and TIMP1. CONCLUSION: Changes in the expression of selected genes might lead to or be a consequence of an early defect in the physiological activity of proliferative endometrium ultimately resulting in its overgrowth outside the uterine cavity.

Ghrelin in gestational diabetes: serum level and mRNA expression in fat and placental tissue.

OBJECTIVES: We studied the effect of an oral glucose load on circulating ghrelin, as well as ghrelin and ghrelin receptor (GHS-R1a) mRNA expression in subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT) and placental tissue from pregnant women with gestational diabetes (GDM) and normal glucose tolerance (NGT). METHODS: Plasma total ghrelin levels were measured in 58 patients with GDM and 61 women with NGT by radioimmunoassay. Ghrelin and GHS-R1a mRNA expression was studied in 16 subjects with GDM and 20 healthy pregnant women at term, using RT-PCR. RESULTS: Basal ghrelin concentrations and the maximal decrease in ghrelin levels after glucose load did not differ in the women with GDM and NGT (399.1 [299.6-563.3] pg/ml vs. 400.9 [302.3-475.8] pg/ml and 127.6 [23.1-213.1] pg/ml vs. 101.7 [44.0-217.6] pg/ml, respectively). Ghrelin mRNA expression in placental tissue was significantly higher in the subjects with GDM than in the healthy pregnant women (0.06 [0.03-0.07] AU vs. 0.02 [0.015-0.03 AU], p=0.02), whereas GHS-R1a mRNA expression in all three tissues studied did not differ between the two groups. Multiple regression analysis revealed that ghrelin mRNA expression in SAT was significantly predicted by serum insulin (beta=0.62, p=0.01), explaining 42% of its variability. CONCLUSIONS: Ghrelin mRNA expression in placental tissue was higher in the GDM than in NGT subjects, whereas no association between circulating ghrelin and GDM was observed.