Department of Genetics, Wroclaw Medical University, Poland
Immunolocalization of estrogens and progesterone receptors within the ovary of the lizard Uromastyx acanthinura from vitellogenesis to rest season
Hammouche Sadjia, Therese Gernigon-Spychalowicz, Jean Marie Exbrayat
Laboratoire de Recherche en Zones Arides, Faculte des Sciences Biologiques, Universite des Sciences et de la Technologie Houari Boumediene, Algeria
The sites of action and the physiological role of estrogens and progesterone in the ovary are poorly understood in Reptiles. We have undertaken a systematic study of the immunoexpression of classical oestrogen receptor (ER or ERα) and progesterone receptor (PR) in the female lizard during the reproductive cycle. During vitellogenesis, ER was not expressed in vitellogenic follicles whereas PR was weakly detected in the nucleus of some follicular cells and well expressed in the internal theca cells. The follicular and theca cells were immunopositive for ER in the previtellogenic follicles, the signal in both was cytosolic. PR was strongly expressed in the follicular cells, the signal was localised in the nucleus. In the post-reproductive period, ER was detected in the previtellogenic follicles in the same manner as in the breeding period. The staining for PR was expressed in both the nucleus and cytoplasm of follicular cells and theca cells. In the sexual rest, the previtellogenic follicles were all negative for ER and PR immunoexpression. These findings suggest that the main action of estrogens in the ovary is not mediated by ER. The expression of cytosolic PR only in the post-reproduction period, at the same time at the progesterone synthesis, supports the hypothesis which stipulates an exclusive nuclear localization in the absence of progesterone.
The influence of short-time period of an adaptation to decreased ambient temperature on interleukin-6 and corticosterone levels in female Wistar strain rats in the proestrous phase of the reproductive cycle
Arkadiusz Baran, Grzegorz Jakiel, Grazyna Wojcik
Department of Reproduction and Andrology, Medical University of Lublin, Poland
To date, there has been little research examining whether short-time changes of external environmental conditions exert any effects on immune responses. The activation of metabolic changes, release of hormones responsive for immunomodulation and the action of interleukins play an important role in interaction with hormones of an anterior pituitary gland in the proestrous phase of the reproductive cycle. The aim of our study was to determine the effects of a short-time change of ambient temperature (30 minutes) on interleukin-6 (IL-6) and corticosterone plasma concentration of female rats in the proestrous phase of the reproductive cycle. The climatic chamber with automatically adjustable and monitored internal environmental parameters (temperature, oxygenation, humidity) was used during the experiment. The estimation of the vaginal lavage using a microscope was done to determine the estrous cycle. On the day of the experiment, animals were divided into 2 groups: the control group (ambient temperature 21oC ± 1oC; normoxia 21% O2) and the test group (ambient temperature 10oC ± 1oC; normoxia 21% O2) stayed in the climatic chamber for 30 minutes. The blood samples were collected before the experiment and after 30, 60, 90, 150 and 210 minutes from the beginning of the experiment. The concentrations of IL-6 and corticosterone were measured in blood plasma samples using ELISA method. There was a significant elevation of IL-6 levels after staying in 10oC during the first 150 minutes from the beginning of the experiment, with the highest value occuring after 60 minutes (426.6 pg/ml; SE - 146.1) with comparison to the value at first sampling (108.5 pg/ml; SE - 29.5; p<0.05) and with comparison to the control group at the same time from the beginning of the experiment (87.6 pg/ml; SE - 2.3; p<0.05). The changed level of corticosterone in the test group in comparison to control group was observed but the differences were insignificant. Our observations confirm the proposition, that even short-time changes of ambient conditions can activate adaptation mechanisms in the organism, which in part, is the activation of the immune system.
Influence of Echinacea purpurea intake during pregnancy on fetal growth and tissue angiogenic activity
Ewa Barcz, Ewa Sommer, Jadwiga Nartowska, Barbara Balan, Joanna Chorostowska-Wynimko, Ewa Skopinska-Rozewska
I Dept. of Obstetrics and Gynecology, Medical University of Warsaw, Poland
The process of angiogenesis and control of blood vessels sprouting are fundamental to human health, as they play key roles in many physiological and pathological conditions. Intake of different pharmaceuticals with antiangiogenic activity by pregnant women may lead to severe developmental disturbances as it was described in case of thalidomide. It may also cause immunomodulatory effects as it was shown for antibiotics, theobromine, caffeic acid or catechins on the pregnant mice model. At present, Echinacea purpurea-based phytoceuticals are among the most popular herbals in the marketplace. Many compounds of Echinacea extracts (polysaccharides, alkamides, polyphenols, glycoproteins) exert immunomodulatory, anti-oxidative and anti-inflammatory activity. Echinacea is one of the most powerful and effective remedies against many kinds of bacterial and viral infections. In previous studies we shown significant inhibitory effect of the Echinacea purpurea based remedy on tumour angiogenic activity using cutaneous angiogenesis test, and an inhibitory effect on L-1 sarcoma growth was observed .The aim of the present study was to establish whether pharmaceuticals containing alcoholic extracts of Echinacea purpurea given to pregnant mice influence angiogenic activity and tissue VEGF and bFGF production of their fetuses. We showed that angiogenic activity of tissue homogenates was increased in Esberitox group and diminished in case of Immunal forte as compared to standard diet group. In case of Echinapur group we did not find significant differences in angiogenic activity. VEGF and bFGF concentration were lower in all groups compared to the control. In the case of Echinapur and Esberitox number of fetuses in one litter were slightly lower as compared to control group, but the difference is on the border of statistical significance. In conclusion, there is some possibility that pharmaceuticals containing Echinacea purpurea might influence fetal development in human also, because they may interfere with embrional angiogenesis , and should not be recommended for pregnant women.
Histological and immunohistological aspects of the ovarian cycle of the algerian wild sand rat, Psammomys obesus Cretzschmar, 1828
Amina Boubekri, Therese Gernigon-Spychalowicz, Farida Khammar, Jean-Marie Exbrayat
Universite des Sciences et de la Technologie Houari Boumedicne, Faculte des Sciences Biologiques, Laboratoire de Recherche sur les Zones Arides, Alger, Algerie
The sand rat, Psammomys obesus, is largely used as a model for studying several metabolic disorders. In order to perform breeding laboratory conditions, the reproductive function of this species was investigated. Using histological and immunohistochemical techniques, several aspects of the ovaries were studied throughout the sexual cycle. During the ovarian cycle, the different stages of folliculogenesis, from primordial to Graafian follicle, have been shown; the differentiation of both granulosa and theca cells, the formation of the antrum, cumulus oophorus and corona radiate were described. Broken follicles and corpora lutea have been observed, confirming a spontaneous ovulation in isolated females. Steroid activities were analysed using immunohistochemical techniques. Estrogen, androgen and progesterone hormones were visualized in the different compartments of the ovary.
Comparative study of the effects of pre and post natal administration of a thyroid drug on testicular activity in adult rat
Z. Hamouli-Said, F. Tahari, F. Hamoudi, F. Hadj-Bekkouche
L.B.P.O/Section Endocrinology, Faculty of Biological Sciences, BP 32, U.S.T.H.B., 16123 Bab-Ezzouar, Algiers, Algeria
Thyroid hormone is known to play a critical role in growth and development of rat testes with a specific effect on the differentiation of Sertoli cells leading to a normal evolution of germ cells. In the present study, we aimed to compare the effect of induced hypothyroidism during fetal and post-natal life on the structure and function of the testis in adult. Pregnant or lactating mothers were treated with 6-propyl-2-thiouracil (PTU) during 21 days and weight gain of pups was steady until adult age. Plasma hormonal levels were determined by RIA and morphology of testis was studied on sections stained with Masson's trichrome. Pre and early post natal hypothyroidism resulted in an impairment of body development and a diminution of thyroid hormone levels of treated rats. No significant effect on testicular development has been observed when hypothyroidism is induced in fetal life while it was associated with reduction in testis weight, diameter of seminiferous tubules and hormonal levels and delay in maturation of germ cells, when induced during early post natal life.
During seminiferous tubule maturation testosterone and synergistic action of FSH with estradiol support germ cell survival while estradiol alone has pro-apoptotic effect
Renata Walczak-Jedrzejowska, Jolanta Slowikowska-Hilczer, Katarzyna Marchlewska, Elzbieta Oszukowska, Krzysztof Kula
Department of Andrology and Reproductive Endocrinology, Medical University of Lodz, Poland
During establishment of spermatogenesis at the prepubertal age, an early germ cells apoptotic wave occurs likely aimed to remove abnormal germ cells and to maintain a proper cell number ratio between maturating germ cells and Sertoli cells. Here we assessed Sertoli and germ cell apoptosis in relation to morphological parameters of Sertoli cell maturation in neonatal rats under the influence of testosterone, estradiol and FSH given alone or in combinations. From postnatal day (PND) 5th to 15th male rats were daily injected with: 1) 2.5 mg of testosterone propionate (TP), or 2) 12.5 &mgr;g of 17beta-estradiol benzoate (EB), or 3) TP+EB, or 4) 7.5 IU of human purified FSH (hFSH), or 5) hFSH+EB or solvents (control-C). Autopsy was performed on PND 16th. Sertoli cell nuclei area and incidence of seminiferous tubule lumen formation (LF) were taken as markers of Sertoli cell maturation. Sertoli and germ cell apoptosis was assessed using TUNEL method. In comparison with C, the area of Sertoli cell nuclei was significantly reduced after EB (25.7±2.0 vs. 30.9±1.6 &mgr;m2 for C, p<0.001) and increased after hFSH+EB (33.1±2.3 &mgr;m2, p<0.05). Incidence of LF was completely arrested by steroid hormone treatments given separately, significantly inhibited after TP+EB (median: 0.0%, vs. 2.0% for C p<0.05) and significantly enhanced after hFSH+EB (median: 51.0%, p<0.001). hFSH alone did not influence LF. Incidence of TUNEL positive Sertoli cells significantly increased after EB (median: 2.9% vs. 0.5% for C, p<0.05) or TP+EB (median: 2.2%, p<0.01) and was not affected by other treatments. Incidence of TUNEL positive germ cells increased significantly after EB alone (median: 4.4% vs. 2.5%, for C, p<0.01 ) and was significantly decreased by hFSH+EB (median: 0.5%, p<0.01). Conclusions: 1) Administration of testosterone or estradiol to immature rats inhibits Sertoli cell maturation. 2) Estradiol stimulates Sertoli and germ cell apoptosis while testosterone has no effect. 3) Testosterone eliminates estradiol - induced germ cell apoptosis when both hormones act in concert. 4) FSH in concert with estradiol, but neither one of the hormone alone, accelerate Sertoli cell differentiation and effectively inhibit germ cell apoptosis. 5) During seminiferous tubule maturation testosterone and the synergistic action of FSH with estradiol support germ cell survival while estradiol alone has an inhibitory, pro-apoptotic effect.
Biotechnological and clinical outcome of in vitro fertilization in non-obese patients with polycystic ovarian syndrome
Przemyslaw Ciepiela, Tomasz Baczkowski, Pawel Brelik, Anna Antonowicz, Krzysztof Safranow, Rafal Kurzawa
Department of Reproductive Medicine and Gynecology, Pomeranian Medical University, Szczecin-Police, Poland
Polycystic ovarian syndrome (PCOS) is a hormonal and metabolic disorder which poses problems with controlled ovarian stimulation (COH). It has been also postulated that PCOS patients have oocytes and embryos with poorer quality which affects IVF results. The of the study was to verify IVF outcome in non-obese patients with PCOS. IVF results of 71 non-obese PCOS patients with 243 non-obese non-POCS patients, regardless of stimulation protocol, from years 2004-2006 were compared. Results: Biotechnological results of PCOS patients in opposition to non-PCOS patients were respectively as follows: higher average number (10.19 vs. 7.61; p=0.001) and percentage (82.34% vs. 76.25%; p=0.025) of retrieved mature M2 oocytes; similar (77.01% vs. 76.75%; p=0.835) fertilization rate with higher average number of embryos (7.633 vs. 5.650 p=0.003); higher average number (4.830 vs. 3.304; p=0.001) and percentage (65.66% vs. 60.57%; p=0.006) of embryos with optimal Z1 and Z2 pronuclei pattern according to Scott; higher average number of class A embryos (3.57 vs. 2.34; p=0.001). Similar number of embryos were transferred in both groups (2.408 vs. 2.485, p=0.552). Clinical results in PCOS and non-PCOS patients were as follows: similar stimulation duration (10.53 days vs. 10.31 days; p=0.639) with significant less gonadotropin total usage (1866.54 IU vs. 2276.18 IU; p=0.001). Also clinical pregnancy per transfer (57.75% vs. 41.98%; p=0.021) and delivery per transfer (45.07% vs. 32.51%; p=0.066) were more often in PCOS patients with comparable miscarriages (12.68% vs. 6.58%; p=0.131) and ectopic pregnancy (0.00% vs. 2.06%; p=0.591) rates, respectively. Conclusion: PCOS in non-obese patients is linked with good biotechnological and clinical IVF outcome.
Immunophenotypic profiles of peripheral blood lymphocytes on the day of embryo transfer in women undergoing in vitro fertilization
Tomasz Baczkowski, Rafal Kurzawa
Department of Reproductive Medicine and Gynecology, Pomeranian Medical University, Szczecin-Police, Poland
Evaluation of different types of lymphocyte subpopulations in the peripheral blood has unknown and controversial significance in diagnosis of infertility. The aim of the study was to evaluate selected blood lymphocytes in patients treated with intracytoplasmic sperm injection (ICSI). Materials and methods: women were divided into three groups: (1) control fertile group (n=18), (2) infertile women that achieved (n=32), and (3) did not achieve a pregnancy after ICSI (n=26). The following types of leukocytes were analyzed by three-colour flow cytometry by detection of specific CD antigens: lymphocytes T (CD3+), B (CD19+ and CD5+CD19+), T and B (CD5+), NK cells (CD56+CD16-, CD56-CD16+, CD56+CD16+, CD56brightCD16-, CD56dimCD16+). Additionally, the antigen of early activation (CD69) was evaluated on T, B and NK cells. The results were presented as a percentage and total counts of all lymphocytes. Results: The percentage of total NK cells (CD56+CD16+, CD56+CD16- and CD56-CD16+) did not differ between pregnant and non pregnant women and was lower comparing to control group. Fractions of CD56-CD16+ cells were higher in pregnant vs. non-preg-nant women. The percentages of CD56brightCD16- NK cells were higher in control group comparing to both ICSI treated groups. Other fractions of lymphocyte subpopulations, including activated cells (with CD69 expression) did not differ between the analyzed groups. Total counts of CD56-CD16+ cells were higher in pregnant vs. non-pregnant group, and the CD56brightCD16- cells was more abundant in control group vs. women with unsuccessful ICSI. Conclusions: Testing of peripheral blood NK cells and the others lymphocytes has limited value as a prognostic factor in ICSI treated patients. The antigen of early lymphocytic activation (CD69) has not any predictive value in prognosis of ICSI outcome.
Pronuclear scoring as a predictor of embryo quality in in vitro fertilization program
Magdalena Depa-Martynow, Piotr Jedrzejczak, Leszek Pawelczyk
Division of Infertility and Reproductive Endocrinology, Department of Gynecology and Obstetrics, Karol Marcinkowski University of Medical Sciences in Poznan, Poland
Many strategies have been proposed for the selection of viable embryos for transfer in human assisted reproduction. These have included morphological scoring criteria for 20, 28, 44 and 68 h after insemination. The embryo selection is based on morphology, degree of fragmentation and development to the 8-cell. All have shown some correlation with implantation. However, the overall success of these methods is still limited, with over 50% of all transferred embryos failing to implant. Pronuclear zygote morphology has gained much attention recently due to its positive value in predicting implantation and pregnancy. This prospective study involved 178 conventional IVF patients only. The key aspects of pronuclear scoring and namely the presence of a cytoplasmic halo were related to day 3 of development and morphology in a retrospective study. The Z-score and the presence/absence of a halo had significant effect on the rate of development on day 3 embryo. Low Z-score result in slow development and poor morphology. The absence of a halo also resulted in slow and poor development, low morphology, increased fragmentation.
Quality of life and marital sexual satisfaction in women with polycystic ovary syndrome
Agnieszka Drosdzol, Violetta Skrzypulec, Barbara Mazur, Romana Pawlinska-Chmara
Woman's Health Chair, the Medical University of Silesia, Katowice, Poland
Polycystic ovary syndrome affects 5-10% of women in the developed world, making it the most common endocrine disorder among women of reproductive age. The symptoms typically associated with polycystic ovary syndrome: amenorrhea, oligomenorrhea, hirsutism, obesity, subfertility, anovulation and acne can lead to a significant reduction in female life quality.The aim of the study was to evaluate the effect of polycystic ovary syndrome on quality of life and marital sexual satisfaction. Fifty women with polycystic ovary syndrome were qualified to the study as the research group. The control group consisted of fourty healthy women. A specific questionnaire was used as a research tool in this study. It included the socio-demographic part, polycystic ovary syndrome's symptomatology and validated scales: Polish version of Short Form-36 Health Survey (SF-36) and Index of Sexual Satisfaction (ISS). The mean age of researched women was 28.9±5.6 years, and in the control group - 30.5±5.3 years (p>0.05). Quality of life parameters for women with polycystic ovary syndrome were lower than for the controls in the aspect of: general health (p<0.01), limitations due to physical health (p<0.05), limitations due to emotional problems (p<0.001), social functioning (p<0.01), energy/fatigue (p<0.001) and emotional wellbeing (p<0.01). Studied women showed worse marital sexual functioning (p<0.05). Marital sexual dysfunctions were diagnosed in 28.6% of women with polycystic ovary syndrome and in 10.5% of healthy women (p<0.05). Polycystic ovary syndrome decreases quality of life and marital sexual functioning among women. A negative effect of hirsutism severity on general well-being and marital sexual life is also observed.
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Diagnosing and management of iatrogenic moderate and severe ovarian hyperstymulation syndrome (OHSS) in clinical material S88 S91
Artur Jakimiuk, Anna Fritz, Wojciech Grzybowski, Irena Walecka, Piotr Lewandowski
Department of Obstetrics and Gynecology in Central Clinical Hospital of Ministry of Interior Affairs, Warsaw, Poland
Severe ovarian hyperstymulation syndrome is a rare but potentially life-threatening complication in patients undergoing assisted reproductive techniques (ART). The pathogenesis of this condition is likely to be multifactorial. The aim of the retrospective study was to present management in moderate and severe iatrogenic ovarian hyperstymulation syndrome (OHSS) in clinical material. The study group was 19 women, admitted to the Department of Obstetrics and Gynecology in Central Clinical Hospital of Ministry of Interior and Administration in Warsaw from large outpatient infertility center "Novum" in Warsaw with moderate and severe OHSS between 14.07.2004 and 8.11.2005. Laboratory tests and ultrasound examination of the ovarian size and ascites were performed, abdominal circumference was measured. Patients were treated with rehydration with intravenous crystalloids and colloids, diuretics, antibiotics, anticoagulants and ultrasound-guided paracentesis if symptoms of ascites become severe (ascites causes pain and compromised pulmonary function). Oral intake of water was restricted, monitoring of fluid intake and output, and daily monitoring of body weight was performed. During treatment controlled laboratory tests were done. In one patient occurred intra-abdominal hemorrhage from ovarian rupture and laparotomy with oophorectomy was performed. The ovarian hyperstimulation syndrome is still a difficult diagnostic and therapeutic problem and more studies are required to elucidate pathophysiology of OHSS. Because of still unknown etiology treatment is empirical and in most of cases bases on experience of medical team. Thus, the management in individual patients varies according to the severity of ovarian hyperstymulation syndrome and its complications.tymulation syndrome and its complications.
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Evaluation of protamines 1 and 2 transcript contents in spermatozoa from asthenozoospermic men
Bartosz Kempisty, Magdalena Depa-Martynow, Margarita Lianeri
During mammalian spermatogenesis, the chromatin structure undergoes substantial condensation. The key role in this process is played by protamines 1 and 2 (PRM1, PRM2). We attempted to compare the levels of PRM1 and PRM2 transcripts in mature spermatozoa of normospermic and asthenozoospermic men. Human ejaculates from normozoospermic (n=70) and asthenozoospermic (n=100) donors were purified by centrifugation through discontinuous Percoll density gradient. RNA was isolated from spermatozoa according to the Chomczyński and Sacchi method, treated with DNase I, and reverse-transcribed into cDNA. Using reverse transcription and real-time quantitative polymerase chain reaction analysis, we found a reduction in the levels of PRM1 and PRM2 transcripts in spermatozoa from asthenozoospermic men, as compared to controls (P<0.001). Our findings indicate that a reduction in contents of PRM1 and PRM2 transcripts in spermatozoa may be linked with asthenozoospermia.
Clomiphene citrate challenge test and serum anti-Mullerian hormone levels in women with menstrual irregularities and/or infertility
Tomasz Milewicz, Jozef Krzysiek, Barbara Blakala, Magdalena Krzyczkowska-Sendrakowska, Stanislaw Radowicki, Olga Kacalska, Marta Zabinska
Departament of Gynecological Endocrinology, Collegium Medicum, Jagiellonian University Cracow, Poland
Aim: Evaluation of CCCT results and anti-Mullerian hormone (AMH) plasma levels in women with menstrual cycle irregularities and/or infertility. Patients and methods: 70 patients with menstrual cycle disturbances were recruited to the study. Clomiphene citrate challenge test (CCCT) was carried out in each patient enrolled. At day 3 of the cycle plasma basic levels of FSH, estradiol, progesterone, IGFBP-1, TSH, prolactin, DHEAS and anti-Mullerian hormone were measured. At day 10 of the cycle plasma levels of FSH and estradiol were evaluated. Plasma levels of hormones were measured by commercially available ELISA kits. Results: 50 women with normal result of CCCT (group A) had higher mean plasma level of AMH compared to 20 women with abnormal result of CCCT (group B). Mean plasma FSH level at day 3 of the cycle was lower in group A compared to group B. There were no other statistically significant differences in mean values of assessed parameters between groups A and B. Taking into account all patients enrolled to the study AMH correlated significantly with patients' age and plasma levels of FSH at day 3 and day 10 of the cycle). Basic AMH plasma levels in group A correlated negatively with plasma levels of FSH at day 3 and day 10. In group B plasma levels of FSH at day 10 of the cycle also correlated with basic AMH plasma levels. Plasma levels of estradiol at day 10 of the cycle were related inversely with basic AMH plasma levels in group A, but directly in group B. Conclusion: It should be recommended to perform the CCCT before infertility treatment. Evaluation of the anti-Mullerian hormone plasma level reflects the results of the CCCT.
Evaluation of superoxide dismutase activity and its impact on semen quality parameters of infertile men
Marek Murawski, Jolanta Saczko, Anna Marcinkowska, Agnieszka Chwilkowska, Marian Gryboś, Teresa Banaś
1st Department of Gynecology and Obstetrics, Medical University of Wroclaw, Poland
The evaluation of superoxide dismutase (SOD) activity, as one of the most important antioxidative defence enzymes, in seminal plasma of patients consulting for male infertility was presented in the article. The study included also the determination of its influence on selected human semen quality parameters. The material represents semen samples obtained from 15 men, which were divided into two groups: Group I (n=10) including patients consulting for infertility and Group II (n=5) containing healthy sperm donors as a control. All of the semen samples were cryopreserved and stored in liquid nitrogen. The frozen samples were thawed at the same time and then SOD activity was determined spectrophotometrically. The analysis of the investigations results indicates a significantly lower semen SOD activity detected in oligoasthenozoospermic patients, comparing to the activity found in normospermic men. The study showed a positive correlation between SOD activity in seminal plasma and semen quality parameters - sperm concentration and overall motility, which are regarded as the most important for normal fertilizing ability of the spermatozoa. Significantly lower SOD activity in seminal plasma of infertile patients, comparing to healthy sperm donors, as well as positive correlation and beneficial impact of SOD activity on human semen quality parameters seem to confirm the observations, that decreased seminal plasma scavenger antioxidant capacity, particularly in form of low SOD activity, can be responsible for male infertility. This trial shows that SOD activity survey in seminal plasma could be a useful tool for determining sperm fertilization potential and could improve the diagnosis of male infertility.
Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia
M. Piasecka, D. Gaczarzewicz, M. Laszczynska, A. Starczewski, A. Brodowska
Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p< 0.01) as compared to men with normal sperm motility (n=54). Sperm DNA fragmentation negatively correlated (n=94) with sperm motility, concentration, and integrity of the sperm plasma membrane (HOS-test). Two categories of patients were distinguished: (1) patients (23 out of 94 subjects) with 4% of TUNEL-positive cells and (2) patients (71 subjects) with >4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation as well as by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.
Antigens HLA-G, sHLA- G and sHLA- class I in reproductive failure
Olimpia Sipak-Szmigiel, Elzbieta Ronin-Walknowska, Cezary Cybulski, Tomasz Plonka, Jan Lubinski
Chair for Obstetrics and Gynecology, Department for Feto-Maternal Medicine, Pomeranian Medical University in Szczecin, Szczecin, Poland
It can be supposed that relation between HLA-G polymorphism and sHLA-G protein expression are associated with successful embryo implantation and pregnancy maintenance. The aim of the study was the estimation specific differences in expression of sHLA-G and sHLA- class I antigens in women with reproductive failure in comparison with fertile women. The study sample enrolled 80 women, divided into 2 groups. The study group (B) enrolled 60 women with reproductive failure including 20 women with 3 recurrent spontaneous abortions in the first trimester of pregnancy (RSA), 20 women with empty sac (ES) and 20 women with 3 consecutive in-vitro fertilization failures (IVFf). The control group (C) enrolled 20 fertile women with at least 2 children. Soluble HLA- class I antigens (sHLA-I) and soluble HLA-G (sHLA-G) were determined using ELISA test kits from IBio Vendor Labolatory Medicine, Inc. HLA-G allele found in individuals in our study were identified by comparing the obtained bp sequences of exon 2., 3. and 4. with bp sequences of HLA-G antigen published at the Nolan Research Institute website. The highest concentration of sHLA-I is noted among women with HLA-G 10401 allele which differed significantly for the mean sHLA-I concentration calculated for all the remaining alleles (p<0.0001). The most prevalent alleles were: HLA-G 10101, 10102 and 10108 with sHLA-I concentrations among women bearing those alleles significantly lower in comparison to the HLA-G 10401 carriers (p<0.001). Allele 10101 and 10102 was related to the lower significantly plasma sHLA-I concentrations than 10108 allele (p<0.02). Lowest mean sHLA-G values were observed in the IVFf group with significant difference from the remaining groups (p<0.05). To conclude, sHLA-G molecules is associated to certain HLA-G alleles and imply that sHLA-G levels are under genetic control. Low concentration sHLA-G seems to be prognostically important in IVF failure.
TGF superfamily and MMP2, MMP9, TIMP1 genes expression in the endometrium of women with impaired reproduction
Jana Skrzypczak, Przemysław Wirstlein, Mateusz Mikolajczyk, Grzegorz Ludwikowski, Tomasz Zak
Department of Gynecology and Obstetrics, Division of Reproduction, University of Medical Sciences in Poznan, Poland
During the putative "implantation window", a period of maximal endometrial receptivity that spans 7-9 days after ovulation, a series of changes on the structural and molecular level occur that render the endometrium susceptible to implantation for the human embryo. Many members of the TGF&bgr;s are expressed by human endometrium at different stages of menstrual cycle. Also studies regarding the MMP2 gene expression and activity of MMP2 in the implantation window have shown a higher expression and activity of MMP2 in women with impaired fertility. We have examined by RT-PCR the expression of TGF&bgr;2 and MMP2, MMP9 and TIMP1 in 28 patients with idiopathic infertility, 16 patients with unexplained recurrent miscarriage and 16 control women were enrolled in this study. Seven to nine days after ovulation endometrial biopsy by Pipelle or hysteroscopy was performed to assess the expression of TGF&bgr;2 , MMP2, MMP9 and TIMP1.We found that in endometria from women with idiopathic infertility TGF&bgr;2 expression was 2.8 fold higher than in endometria from control group and 2.1 fold higher in endometrial samples from women with unexplained recurrent miscarriage compared to the control group. The MMP2, MMP9 and TIMP1 expression in endometrial samples revealed no significant differences between the study groups and control group. There was a statistically significant negative correlation between TGF&bgr;2 and MMP9 expression in endometria from women in control group. The present investigations suggest that dysregulated TGF&bgr;2, MMP2, MMP9 and TIMP1 expression are associated with infertility and early pregnancy loss. However the exact mechanism of how overexpression of endometrial TGF&bgr;and MMPs interferes with implantation may be more complex.
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Perinatal outcome analysis of twin pregnancies at the Department of Obstetrics and Gynecology Central Clinical Hospital of Ministry of Interior and Administration in Warsaw in the years 2005-2006
Tatiana Szymborska, Krzysztof Kaminski, Krzysztof Niemiec, Artur Jakimiuk
Department of Obstetrics and Gynecology in Central Clinical Hospital of Ministry of Interior Affairs, Warsaw, Poland
A group of 59 twin pregnant women who gave birth at the Department of Obstetrics and Gynecology Central Clinical Hospital of Ministry of Interior and Administration in Warsaw in the years 2005-2006. The patients have been divided into four groups: spontaneous twin pregnant women (n=16), twin pregnant women after in- vitro fertilization (IVF) (n=11), twin pregnant women after in-vitro fertilization with intracytoplasmic sperm injection (ICSI) (n=29) and twin pregnant women after ICSI and transfer of frozen embryos (n=3). In one case intrauterine death of one of twins in the 34th week of gestation has been noticed. The cause of the death was umbilical cord wrapped around his neck. The gestation was ended with cesarean section and Apgar score of the second twin was 8 in the fifth minute. In one case there was an urgent indication for a cesarean delivery of children with a very low birth weight (because of intrauterine infection, preterm labor in progress) and in three cases at least one of twins with a low birth weight. Among the group 19 women (32%) have given birth prematurely. The Apgar score in the first, third and fifth minute has been statistically significant and inversely proportional dependent only on the gestational age. There were no differences in birth weight among study groups regardless the way of conception. Only two spontaneous twin pregnant patients have had a vaginal labor. By the remaining 57 patients there has been an elective cesarean section in thirty five cases and there has been an urgent indication for cesarean section in twenty two cases.
Circulating monocyte chemoattractant protein-1 in women with gestational diabetes
Beata Telejko, Mariusz Kuzmicki, Anna Zonenberg, Karolina Niedziolko-Bagniuk, Agnieszka Nikolajuk, Jacek Szamatowicz, Maria Gorska
Department Endocrinology, Diabetology and Internal Medicine, Medical University of Bialystok, Poland
Monocyte chemoattractant protein 1 (MCP-1) has been implicated as a key factor in the recruitment and activation of peripheral blood leukocytes in atherosclerotic lesions and adipose tissue. Elevated levels of circulating MCP-1 have been found in patients with type 1 and type 2 diabetes, as well as with coronary artery disease. In this study we compared serum MCP-1 concentrations between pregnant women with normal glucose tolerance (NGT), gestational diabetes mellitus (GDM) and non-pregnant healthy women. The group studied consisted of 62 patients with GDM (mean age 30.1 ± 5.0 years) at 29.0 ± 3.5 week of gestation, 64 pregnant women with NGT (mean age 30.0 ± 4.7 years) at 29.2 ± 2.9 week of gestation and 34 non-pregnant healthy women (mean age 29.8 ± 4.7 years). Serum MCP-1 concentration was measured using an enzyme - linked immunosorbent assay. Median MCP-1 concentrations did not differ significantly between women with GDM (median 342.3 [interquartile range 267.9-424.4] pg/ml) and NGT (338.0 [274.7-408.2] pg/ml), but were markedly lower than those found in non-pregnant women (485.2 [409.6-642.4] pg/ml, p<0.0001). After adjusting for glucose, the difference between pregnant and non-pregnant women remained highly significant (p<0.0001). In GDM patients MCP-1 levels correlated significantly with fasting glucose (r=0.2665, p=0.0363), insulin (r=0.4330, p=0.0004), HOMA-IR (r=0.4402, p=0.0003), ISQUICKI (r=-0.4402, p=0.0003), HbA1c (r=0.2724, p=0.0322), as well as with prepregnancy and current BMI (r=0.3501, p=0.0057 and r=0.3250, p=0.0106, respectively). Multiple regression analysis revealed that MCP1 concentrations were significantly predicted only by plasma glucose ( beta=0.3489, p=0.00004). Our results suggest that MCP1 levels are decreased in pregnant women, irrespective of their glucose tolerance status.
The differences in RCAS1 and DFF45 endometrial expression between late proliferative, early secretory, and mid-secretory cycle phases
Tadeusz Popiela, Lukasz Wicherek, Michal Radwan, Jerzy Sikora, Tomasz Banas, Pawel Basta, Magdalena Kulczycka, Marek Grabiec, Bogdan Obrzut, Jaroslaw Kalinka
Department of Gynecology, Obstetrics and Oncology of the Jagiellonian University, Krakow, Poland
RCAS1 expression is related to the regulation of activated immune cells and to connective tissue remodeling within the endometrium. DFF45 seems to play an important role in the apoptotic process, most likely by acting through the regulation of DNA fragmentation. Its expression changes within the endometrium seem to be related to the resistance of endometrial cells to apoptosis. The aim of the present study was to evaluate RCAS1 and DFF45 endometrial expressions during ovulation and the implantation period. RCAS1 and DFF45 expression was assessed by the Western-blot method in endometrial tissue samples obtained from 20 patients. The tissue samples were classified according to the menstrual cycle phases in which they were collected, with a division into three phases: late proliferative, early secretory, and mid-secretory. The lowest level of RCAS1 and the highest level of DFF45 endometrial expression was found during the early secretory cycle phase. Statistically significantly higher RCAS1 and statistically significantly lower DFF45 endometrial expression was identified in the endometrium during the late proliferative as compared to the early secretory cycle phase. Moreover, statistically significantly higher RCAS1 and statistically significantly lower DFF45 expression was found in the endometrium during the mid-secretory as compared to the early secretory cycle phase. The preparation for implantation process in the endometrium is preceded by dynamic changes in endometrial ECM and results from the proper interaction between endometrial and immune cells. The course of this process is conditioned by the immunomodulating activity of endometrial cells and their resistance to immune-mediated apoptosis. These dynamic changes are closely related to RCAS1 and DFF45 expression alterations.
Features of impaired seminiferous tubule differentiation are associated with germ cell neoplasia in adult men surgically treated in childhood because of cryptorchidism
Anna Guminska, Jolanta Slowikowska-Hilczer, Wojciech Kuzanski, Marek Sosnowski, Elzbieta Oszukowska, Katarzyna Marchlewska, Renata Walczak-Jedrzejowska, Jerzy Niedzielski, Krzysztof Kula
Department of Andrology and Reproductive Endocrinology, Medical University of Lodz, Poland
Seminiferous tubule differentiation was related to the occurrence of germ cell neoplasia in 38 men, aged 17-47, treated surgically in childhood for cryptorchidism. Tissues from 46 testes obtained from biopsies taken as a neoplastic preventive procedure or whole testes removed because of GCT were evaluated quantitatively. Paraffin sections were treated with antibodies against placental like alkaline phosphatase (PLAP), a marker of germ cell neoplasia, and cytokeratin 18 (CK-18), a marker of immature Sertoli cells. Quality of spermatogenesis and number Leydig cells were assessed with a score count. Seminiferous tubules diameter, thickness of basal membrane and size of intertubular spaces were measured with image analysis software. In 17.4% of testes spermatogenesis was normal (9.9 points) (N) and neoplasia was not found there. In the other 38 specimens (83%) spermatogenesis was abnormal (A). When spermatogenesis was arrested or when germ cells were absent (3.7±1.8 points), neoplastic lesions were found in 13.1% of the specimens. In A group 5.1±7.1% of tubules contained immature Sertoli cells, while in N they were not found. Tubular diameter was significantly lower in A (161.5±31.8 &mgr;m) than in N (184.6±24.3 &mgr;m) and the percentage of seminiferous tubules with the thickening of tubular basal membrane was also greater in A. Intertubular spaces were significantly larger in A (49.9±18.6%) in comparison to N group (32.6±12.5%). Mean number of Leydig cells was similar in both groups. To conclude, in most of the formerly cryptorchid testes, despite surgical treatment, impaired seminiferous tubules differentiation is predominant. Germ cell neoplasia is present in testes with retarded seminiferous tubules differentiation. Retardation of seminiferous tubule differentiation consists of inhibited spermatogenesis, presence of tubules with immature Sertoli cells, decreased tubular diameter, increased thickness of basal membrane and enlarged intertubular spaces. Examination of testicular biopsy with respect to the state of seminiferous tubule differentiation may be helpful to predict the appearance of germ cell neoplasia in adult men with cryptorchidism in anamnesis. Orchiopexy of cryptorchid testes may not prevent the occurrence of features of testicular dysgenesis and the associated germ cell neoplasia.
Can vitamin A modify the activity of docetaxel in MCF-7 breast cancer cells?
Ewa Czeczuga-Semeniuk, Dorota Lemancewicz, Slawomir Wolczynski
Department of Reproduction and Gynecological Endocrinology, Medical University of Białystok, Białystok, Poland
Docetaxel is one of the most effective chemotherapeutic agents in the treatment of breast cancer. On the other hand, the vitamin A family compounds play the essential roles in many biological processes in mammary gland. The aim of our study was to investigate the effect of all-trans retinol, carotenoids (&bgr;-carotene, lycopene) and retinoids (9-cis, 13-cis and all-trans retinoic acid) on the activity of docetaxel and to compare these effects with the estradiol and tamoxifen actions on human ER(+) MCF-7 breast cancer cell line. The evaluation was based on [3H] thymidine incorporation and the proliferative activity of PCNA and Ki 67 positive cells. In our study, the incorporation of [3H]thymidine into cancer cells was inhibited to 50% by 0.2, 0.5 and 1 &mgr;M of docetaxel in the 24-hour culture and addition of estradiol (0.001 μM) didn't influence the results. However, addition of tamoxifen caused a statistically significant decrease of the percentage of the proliferating cells in the culture medium with 0.2 and 0.5 &mgr;M of docetaxel (38.99 ± 2.84%, p<0.01 and 40.67 ± 5.62%, p<0.01) in comparison to the docetaxel only group. The above-mentioned observations were also confirmed with the use of the immunohistochemical investigations. Among the examined vitamin A family compounds, the simultaneous application of &bgr;-carotene
Increased expression of gap junction protein - connexin 32 in lymph node metastases of human ductal breast cancer
Luiza Kanczuga-Koda, Mariola Sulkowska, Mariusz Koda, Ryszard Rutkowski, Stanislaw Sulkowski
Departments of Medical and General Pathomorphology, Medical University of Bialystok, Poland
Gap junctions are specialized cell membrane channels composed of connexins (Cxs), which mediate the direct passage of small molecules between adjacent cells. They are involved in the regulation of cell cycle, cell signaling and differentiation as well as probably invasion and metastasis. Up to now, Cx32 status in human breast cancer has not been studied. Consequently, the aim of the present study was the evaluation of the expression of connexin 32 (Cx32) in primary breast tumors (PTs) and matched-paired metastases to lymph nodes (MLNs) in correlation with selected clinicopathological features. Tissue samples from 79 women were examined by immunohistochemistry, using the streptavidin-biotin-peroxidase complex technique for Cx32. Cytoplasmic expression of Cx32 was detected in 31 of 79 breast cancers (39.2%). Both epithelial and myoepithelial cells of normal ducts adjacent to the tumor did not express Cx32. Increased expression of studied Cx was observed in metastases to lymph nodes relative to primary tumors. Additionally, Cx32-negative primary tumors developed Cx32-positive metastases. Statistical comparisons of Cx32 expression in the matched pairs indicate that this protein significantly increased in lymph node metastases compared to primary tumors (p<0.001). The expression of Cx32 in primary breast cancer was not statistically associated with age of patients, tumor size, lymph node status, but we observed a tendency toward association between Cx32 expression and histological differentiation. In conclusion, transformed cells may have an ability to produce Cxs also atypical for normal cells. Increased expression of Cx32 in metastases to the lymph nodes might reflect alteration in connexin gene transcription during breast carcinogenesis and finally, it may be a sign of more malignant phenotype of cancerous cells.
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Mechanism of collagen biosynthesis up-regulation in cultured leiomyoma cells
Monika Zbucka, Wojciech Miltyk, Tomasz Bielawski, Arkadiusz Surazynski, Jerzy Palka, Slawomir Wolczynski
Department of Reproduction and Gynecological Endocrinology, Medical University in Bialystok, Poland
Uterine leiomyoma is the most common tumour in women with a reported incidence of 25-30%. The tumors are benign, composed of smooth muscle cells with variable amount of collagen - rich fibrous tissue. It is well established that accumulation of extracellular matrix in leiomyoma is key feature of tissue fibrosis. However, the pathogenesis of leiomyoma is still unclear. The aim of this study was to evaluate the metabolism of collagen in cultured leiomyoma cells and in control myometrium cells. The effect of estradiol, selective modulators of estrogen receptors (raloxifene, tamoxifen) and estrogen receptor down regulator (ICI 182.780) on collagen biosynthesis (measured by 5-[3H]-proline incorporation assay and measurement of prolidase activity) and collagen degradation (measured by metalloproteinase activity assay) was studied. It was found that collagen biosynthesis is strongly stimulated by low doses of estradiol (5 nM) in leiomyoma cells while it is not changed in control myometrium cells. An increase in estradiol concentration to 10 nM results in drastic decrease of this process both in leiomyoma as well as control cells. Although raloxifene and tamoxifen only slightly affected collagen biosynthesis in control myometrium cells, they significantly inhibited the process in leiomyoma cells. There was no coordinate correlation between collagen biosynthesis and prolidase activity suggesting that regulation of this process may take place at transcriptional level. Both estrogen and SERMs were found to inhibit MMP-2 in leiomyoma as well as in control myometrium cells. The data suggest that stimulatory action of estrogen on collagen biosynthesis and inhibitory effect on MMP-2 activity in uterine leiomyoma may contribute to accumulation of this protein in ECM of this tissue.
Expression of leptin and its receptor in female breast cancer in relation with selected apoptotic markers
Mariusz Koda, Mariola Sulkowska, Luiza Kanczuga-Koda, Katarzyna Jarzabek, Stanislaw Sulkowski
Department of General Pathomorphology, Medical University of Bialystok, Poland
Leptin and its receptor may be engaged in pathogenesis of breast cancer among various human tumors. In vitro investigations showed leptin-mediated escalation of estrogen synthesis and boosted activity of estrogen receptor ERα. Furthermore, leptin induced growth of malignant cells, counteracted apoptosis and stimulated cell migration as well as overexpression of angiogenic factors and degrading enzymes that split network of intercellular matrix. On the other side, leptin has been reported to favor apoptosis, lately. Proapoptotic effect of leptin action was revealed in interstitial cells of bone marrow and adipocytes. Our past reports provide evidences for overexpression of leptin and its receptor in breast cancer in comparison with benign mammary lesions. In current study we aimed at assessment of eventual relationships between leptin, leptin receptor and selected protein regulators of apoptosis in breast cancer. We applied immunohistochemistry for leptin, leptin receptor, anti-apoptotic Bcl-2 and Bcl-xL as well as pro-apoptotic Bak and Bax expression assessment in 106 cases of human breast cancers. The immunoreaction was graded and statistically evaluated. Expression of leptin was positively correlated with Bcl-xL, Bak and Bax (p<0.001, r=0.614; p<0.001, r=0.518; p<0.001, r=0.511, respectively). Statistical significances were noted between expression of leptin receptor and Bcl-xL or Bax (p=0.011, r=0.210; p<0.001, r=0.313, respectively). No correlation was encountered between leptin and Bcl-2, either leptin receptor and Bcl-2 or leptin receptor and Bak. On the basis of obtained results, leptin system could interfere in balance among expressions of pro- and anti-apoptot-ic proteins and regulate cell turnover and - by means of it - facilitate breast cancer progression.