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ABSTRACT: PURPOSE: To investigate two of the most studied estrogen receptor alpha polymorphisms (PvuII and XbaI) in combination, in order to evaluate their impact on an ART program outcome. METHODS: 203 normally ovulating women who underwent IVF or ICSI treatment were genotyped for PvuII and XbaI polymorphisms in ESR1 intron 1 using Real-Time PCR. The relationship between the presence of polymorphic alleles and the ovulation induction parameters and outcome was examined. RESULTS: Women were grouped according to the number of polymorphic alleles they carried in two groups (0-2 versus 3-4 polymorphic alleles). The presence of 3 or more polymorphic alleles was associated with significantly lower E2 levels on the day of hCG administration and a significantly lower rate of good quality embryos. CONCLUSION: There is an association between ESR1 polymorphisms and some ART parameters such as the level of E2 on the day of hCG administration and the quality of the embryos. These results underline the importance of ESR1 as a candidate gene for the prediction of ovarian response to IVF/ICSI protocols. Future research work concerning several more genes is necessary for a better evaluation of patients before entering an IVF/ICSI program.
Journal of Assisted Reproduction and Genetics 03/2013; · 1.84 Impact Factor
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ABSTRACT: BACKGROUND: RUNX2 is a transcription factor, whose expression has been recently identified in the mouse ovary. Regulation of RUNX2 expression and its function in the human ovary have not been determined yet. The aim of the present study is the investigation of the possible correlation between RUNX2 gene expression in cumulus cells and controlled ovarian stimulation and pregnancy outcomes after ART treatment. METHODS: A total of 41 patients undergoing ICSI treatment for male factor infertility were enrolled into a specific ART program, during which cumulus cells were collected. The expression of RUNX2 gene in cumulus cells was examined by real-time PCR. RESULTS: Concerning RUNX2 gene expression, 12 out of 41 women were detected with RUNX2 expression, with ratios ranging from 0.84 to 1.00, while 28 out of 41 women had no expression (ratio = 0). Only 1 woman presented a weak RUNX2 gene expression (ratio = 0.52). From 8 women that proceeded to pregnancy, 7 of them did not express RUNX2 gene in cumulus cells, while one was the woman with weak gene expression that also achieved pregnancy. The group of women without RUNX2 expression presented higher number of follicles (p = 0.013), higher number of retrieved oocytes (p = 0.016), higher basal LH serum levels (p = 0.016) and higher peak estradiol levels (p = 0.013), while the number of fertilized oocytes differed marginally between the two groups (p = 0.089). Moreover, RUNX2 expression was negatively associated with LH levels (OR = 0.22, p = 0.021) and E2 levels (OR = 0.25, p = 0.026). CONCLUSIONS: Consequently, based on the preliminary findings of the present pilot study a potential inhibitory mechanism of RUNX2 gene is observed in the ovary when high mRNA levels are detected, suggesting that RUNX2 could possibly be used as a candidate genetic marker in the monitoring of the outcome of an ART treatment.
Reproductive Biology and Endocrinology 11/2012; 10(1):99. · 2.05 Impact Factor
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ABSTRACT: Luteinizing hormone (LH) exerts its actions through its receptor (LHR), which is mainly expressed in theca cells and to a lesser extent in oocytes, granulosa and cumulus cells. The aim of the present study was the investigation of a possible correlation between LHR gene and LHR splice variants expression in cumulus cells and ovarian response as well as ART outcome.
Forty patients undergoing ICSI treatment for male factor infertility underwent a long luteal GnRH-agonist downregulation protocol with a fixed 5-day rLH pre-treatment prior to rFSH stimulation and samples of cumulus cells were collected on the day of egg collection. RNA extraction and cDNA preparation was followed by LHR gene expression investigation through real-time PCR. Furthermore, cumulus cells were investigated for the detection of LHR splice variants using reverse transcription PCR.
Concerning LHR expression in cumulus cells, a statistically significant negative association was observed with the duration of ovarian stimulation (odds ratio = 0.23, p = 0.012). Interestingly, 6 over 7 women who fell pregnant expressed at least two specific types of LHR splice variants (735 bp, 621 bp), while only 1 out of 19 women that did not express any splice variant achieved a pregnancy.
Consequently, the present study provide a step towards a new role of LHR gene expression profiling as a biomarker in the prediction of ovarian response at least in terms of duration of stimulation and also a tentative role of LHR splice variants expression in the prediction of pregnancy success.
Journal of Assisted Reproduction and Genetics 03/2012; 29(5):409-16. · 1.84 Impact Factor
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ABSTRACT: The expression of corticotropin-releasing hormone (CRH) receptor 1 messenger RNA in stages of follicle growth was examined by reverse transcriptase-polymerase chain reaction in long-term cultures of early preantral mouse follicles with and without CRH addition. Corticotropin-releasing hormone receptor 1 is present in stages of mouse follicle growth, whereas 10(-9), 10(-7), and 10(-6) mol/L CRH inhibits oocyte maturation in vitro, an effect reversed by antalarmin addition.
Fertility and sterility 01/2011; 95(4):1497-9.e1. · 3.97 Impact Factor
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ABSTRACT: Corticotropin-releasing hormone (CRH) is a 41-amino acid peptide synthesized by neurons of the parvocellular and paraventricular hypothalamic nuclei. Central CRH is the principal regulator of the stress system influencing several systems in the brain and influenced by them. It activates the secretion of glucocorticoids and indirectly regulates the immune system and the immune response. Peripheral CRH is secreted by postganglionic sympathetic and unmyelinated sensory afferent neurons and has been identified in several peripheral tissues and organs, including those of the reproductive system (ovary, endometrium, placenta, and testis). In the human ovary, receptors are detected in thecal and stromal cells and in follicular fluid. Ovarian CRH regulates ovarian steroidogenesis and is involved in follicular maturation, ovulation, and luteolysis. In this concise review we briefly discuss the role of ovarian CRH in reproduction, emphasizing its role in oocyte maturation.
Annals of the New York Academy of Sciences 09/2010; 1205:225-9. · 3.15 Impact Factor
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ABSTRACT: To understand cell cycle controls in the 8-Cell human blastomere.
Data from whole human genome (43,377 elements) microarray analyses of RNAs from normal 8-Cell human embryos were compiled with published microarrays of RNAs from human fibroblasts, before and after induced pluripotency, and embryonic stem cells. A sub database of 3,803 genes identified by high throughput RNA knock-down studies, plus genes that oscillate in human cells, was analyzed.
Thirty-five genes over-detected at least 7-fold specifically on the 8-Cell arrays were enriched for cell cycle drivers and for proteins that stabilize chromosome cohesion and spindle attachment and limit DNA and centrosome replication to once per cycle.
These results indicate that 8-cell human blastomere cleavage is guided by cyclic over-expression of key proteins, rather than canonical checkpoints, leading to rapidly increasing gene copy number and a susceptibility to chromosome and cytokinesis mishaps, well-noted characteristics of preimplantation human embryos.
Journal of Assisted Reproduction and Genetics 04/2010; 27(6):265-76. · 1.84 Impact Factor
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Peter Drakakis, Dimitris Loutradis,
Apostolos Beloukas,
Vana Sypsa,
Vasiliki Anastasiadou,
George Kalofolias,
Helen Arabatzi,
Erasmia Kiapekou,
Konstantinos Stefanidis,
Dimitris Paraskevis,
Antonis Makrigiannakis,
Angelos Hatzakis,
Aris Antsaklis
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ABSTRACT: A simple, safe and cost-effective treatment protocol in ovarian stimulation is of great importance in IVF practice, especially in the case of previous unsuccessful attempts. hCG has been used as a substitute of LH because of the degree of homology between the two hormones. The main aim of this prospective randomized study was to determine, for the first time, whether low dose hCG added to rFSH for ovarian stimulation could produce better results compared to the addition of rLH in women entering IVF-ET, especially in those women that had previous IVF failures. An additional aim was to find an indicator that would allow us to follow-up ovarian stimulation and, possibly, modify it in order to achieve a better IVF outcome; and that indicator may be the cDNA copies of the LH/hCG receptor. Group A patients (n = 58) were administered hCG and Group B rLH (n = 56) in addition to rFSH in the first days of ovarian stimulation. The number of follicles and oocytes and, most importantly, implantation and pregnancy rates were shown to be statistically significantly higher in the hCG group. This study has also determined, for the first time to our best knowledge, m-RNA for LH/hCG receptors in the lymphocytes of peripheral blood 40 h before ovum pick-up. cDNA levels of the hCG receptor after ovarian stimulation were significantly higher among women receiving hCG compared to those receiving LH. In addition, higher levels were encountered among women with pregnancy compared to those without, although this was not statistically significant due to the small number of pregnancies. It seems that hCG permits a highly effective and more stable occupancy of rLH/hCG receptors and gives more follicles and more oocytes. The determination of cDNA copies could be, in the future, a marker during ovulation induction protocols and of course a predictor for the outcome of ART in the special subgroup of patients with previous failures.
Reproductive Biology and Endocrinology 10/2009; 7:110. · 2.05 Impact Factor
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Konstantinos Stefanidis, Dimitris Loutradis,
Vasiliki Anastasiadou,
Panagiotis Beretsos,
Ritsa Bletsa,
Vasiliki Dinopoulou,
Kaliopi Lekka,
Evangelia Elenis,
Erasmia Kiapekou,
Stauros Koussoulakos,
Anastasios Fotinos,
Aris Antsaklis
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ABSTRACT: Oxytocin is a nine amino acid peptide involved in a wide spectrum of physiological functions; predominantly those concerning reproduction and differentiation are of interest. Oxytocin receptors are expressed at early developmental stages of mammals, suggesting that oxytocin might be involved in the determination of the germ stem cell line, at the very early stages of mammalian development. In this respect, the proximate aim of the present study was to confirm and further analyze the existence of oxytocin receptors at a very early level of cell commitment, that is, the determination of germ cells derived from embryoid bodies. To achieve our purpose we have cultured mouse embryonic stem cells under conditions inducing formation of embryoid bodies. In this work, ES cells were allowed to aggregate in a novel medium, "Stefanidis medium" from day 0 to day 14 until formed EBs. RNA was isolated from EBs and using RT-PCR we showed that EBs expressed Oct-4, OTR, OT, and DAZL. To demonstrate simultaneous expression immunocytochemistry was preformed, in which EBs showed strong immunoreactivity for both, OTR and DAZL molecular markers. We found that 35% of the cells displayed OTR, using flow cytometry. In addition, this novel medium showed to increase OTR mRNA. We propose, that at least in murine induced embryoid bodies there is simultaneous expression of oxytocin receptors and germ cell markers (DAZL) in many cells (expressing Oct-4). We thus conclude that, the oxytocin might indeed be a molecule playing a leading role in germ cell determination.
Bio Systems 09/2009; 98(2):122-6. · 1.27 Impact Factor
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ABSTRACT: Recently, it has been demonstrated that, in patients down-regulated by GnRH analogues (GnRHa), a short-term pre-treatment with recombinant LH (rLH), prior to recombinant FSH (rFSH) administration, increases the number of small antral follicle prior to FSH stimulation and the yield of normally fertilized embryos. However, no data exist in the literature regarding the potential beneficial effect of "hCG priming" in controlled ovarian hyperstimulation (COH) through a long GnRH-a protocol, which binds the same receptor (LH/hCGR), though it is a much more potent compared to LH. The primary aims of this study were to assess the effect of short-term pre-rFSH administration of hCG in women entering an ICSI treatment cycle on follicular development, quality of oocytes and early embryo development. The secondary endpoints were to record the effects on endometrial quality and pregnancy rate.
Patients with a history of at least one previous unsuccessful ICSI cycle were randomly assigned into two groups to receive treatment with either a long protocol with rFSH (control group) or a long protocol with rFSH and pre-treatment with hCG (hCG group). In particular, in the latter group, a fixed 7 days course of 200 IU/day hCG was administered as soon as pituitary desensitization was confirmed.
The mean number of oocytes retrieved was not significantly different between the two treatment groups, although the percentage of mature oocytes tended to be higher but not significantly different in hCG-treated patients. The percentage of patients with more than one grade 3 embryos was higher in the pre-treatment group, which also showed a higher pregnancy rate.
All the above clinical observations, in conjunction with previous data, suggest a point towards a beneficial "hCG priming" effect in controlled ovarian hyperstimulation through a long GnRH-a down-regulation protocol, particularly in patients with previous ART failures.
Reproductive Biology and Endocrinology 09/2009; 7:91. · 2.05 Impact Factor
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ABSTRACT: Prolactin (PRL), along with other hormones, plays a role in oocyte maturation, fertilization, and early embryonic development in mammals. In order to investigate the role of PRL on in vitro oocyte maturation from early follicular growth stages, as well as on fertilization and early embryonic development, we cultured preantral mouse follicles with and without PRL, followed by fertilization of the in vitro matured oocytes. Prolactin significantly improved the rate of oocyte maturation, fertilization, and early embryo development. Four isoforms of PRL-Receptor (R) have been found in whole ovaries of mice: one long (PRL-RL) and three short (-RS(1), -RS(2), and -RS(3)). We examined expression of the four PRL-R isoforms in preantral follicles, in cumulus-oocyte complexes (COCs) and in germinal vesicle GV stage oocytes by RT-PCR. Prolactin-RL, -RS(2) and -RS(3) mRNA, but not -RS(1), were expressed in preantral follicles, COCs, and GV stage oocytes. Our results indicate the prolactin pathway is functional in early preantral follicles, in COCs and in GV stage oocytes, and promotes oocyte maturation, meiosis, fertilization, and early embryonic development.
Cloning and Stem Cells 07/2009; 11(2):293-300. · 2.66 Impact Factor
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ABSTRACT: Corifollitropin alfa is being developed by Schering-Plough Corp as an injectable, long-acting follicle-stimulating hormone (FSH) agonist for the treatment of infertility. A single dose of corifollitropin alfa could initiate and sustain multifollicular growth in patients undergoing controlled ovarian stimulation, such as during in vitro fertilization or intracytoplasmic sperm injection. The agent comprises an alpha-subunit, which is identical to that of FSH, and a beta-subunit, which is produced by the fusion of the C-terminal peptide from the beta-subunit of chorionic gonadotropin to the beta-subunit of FSH. Corifollitropin alfa has a longer half-life compared with FSH and thus requires less frequent dosing. The drug was well tolerated and does not appear to be associated with any serious adverse events or the formation of antibodies. The initial results from a large, phase III, double-blind clinical trial indicated that the ongoing pregnancy rate achieved with corifollitropin alfa treatment was high and similar to the rate established with daily treatment of recombinant FSH. The number of oocytes retrieved following the administration of corifollitropin alfa was slightly higher compared with the number observed with daily recombinant FSH treatment. Thus, corifollitropin alfa has the potential to serve as a viable fertility agent and to gain a place in the infertility market.
Current opinion in investigational drugs (London, England: 2000) 05/2009; 10(4):372-80. · 3.31 Impact Factor
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ABSTRACT: There is considerable controversy concerning the correct management of gravid patients with cardiac valve prostheses.
We describe the uneventful course of pregnancy and delivery in a pregnant patient with a mechanical heart valve receiving anticoagulation treatment. A 35-year-old primigravida received anticoagulation throughout pregnancy because of mitral valve replacement at the age of 16 due to rheumatic disease. The antithrombotic regimen used was acenocoumarol (coumarin derivative) 2 mg/day throughout the 2nd and 3rd trimesters of pregnancy with replacement of acenocoumarol by enoxaparin sodium 60 mg twice daily (low-molecular-weight heparin - LMWH) in the 1st trimester during weeks 7-12. International normalization ratio (INR) values were kept above 2.5. At 36 weeks of gestation, acenocoumarol was substituted with unfractionated heparin and 2 days later the patient entered spontaneous premature labor and was emergently delivered via cesarean section (INR = 1.4). A healthy male infant was born 2,560 g, Apgar scores 8 and 10 at 1 and 5 min. No coumarin embryopathy was revealed and the postoperative course of the mother was uneventful.
Even though the use of acenocoumarol throughout pregnancy replaced by LMWH in the 1st trimester was proven safe and efficacious in this case report, large randomized controlled studies are still needed to make definitive recommendations about optimal antithrombotic therapy in pregnant patients with mechanical heart valves.
Fetal Diagnosis and Therapy 04/2009; 25(1):115-8. · 1.05 Impact Factor
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ABSTRACT: To understand the molecular pathways that control early human embryo development.
Improved methods of linear amplification of mRNAs and whole human genome microarray analyses were utilized to characterize gene expression in normal appearing 8-Cell human embryos, in comparison with published microarrays of human fibroblasts and pluripotent stem cells.
Many genes involved in circadian rhythm and cell division were over-expressed in the 8-Cells. The cell cycle checkpoints, RB and WEE1, were silent on the 8-Cell arrays, whereas the recently described tumor suppressor, UHRF2, was up-regulated >10-fold, and the proto-oncogene, MYC, and the core element of circadian rhythm, CLOCK, were elevated up to >50-fold on the 8-Cell arrays.
The canonical G1 and G2 cell cycle checkpoints are not active in totipotent human blastomeres, perhaps replaced by UHRF2, MYC, and intracellular circadian pathways, which may play important roles in early human development.
Journal of Assisted Reproduction and Genetics 04/2009; 26(4):187-95. · 1.84 Impact Factor
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ABSTRACT: To evaluate whether amniotic fluid cells contain cardiomyocyte- and neuron-like cells.
In this experimental study, cells from human amniotic fluid samples were analyzed for mRNA expression of microtubule-associated protein 2 (MAP-2), vimentin and oxytocin (OT) receptor via RT-PCR. Immunocytochemistry was also performed with MAP-2, OT receptor, vimentin and troponin I antibodies.
In all samples, MAP-2, vimentin and mRNA expression were detectable. OT receptor was also detectable. The cells showed strong immunoreactivity for molecular markers of neurogenic cells including MAP-2 and vimentin. The cells also showed strong immunoreactivity for molecular markers of cardiac muscle such as troponin I and for OT receptors. This report also shows that atosiban (an OT antagonist) added to culture medium of amniotic fluid cells did not induce neurogenic and cardiomyogenic differentiation.
The observed concurrent development of cardiomyocyte- and neuron-like cells suggests that amniotic fluid contains progenitor cells and there is bidirectional communication between both cell types.
Fetal Diagnosis and Therapy 03/2009; 25(1):62-6. · 1.05 Impact Factor
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ABSTRACT: Implantation of mammalian conceptus in uterine cavity is the result of evolutionary adaptation, through high level of physiological procedures to ensure its success. However the majority of pregnancy losses occur before or during implantation. It is expected that exploring and defining the molecular and physiological road map during the crucial time of implantation will enable us to decode and effectively treat fertility defects. Immunological, hormonal and molecular factors participate in the feto-maternal cross talk during implantation and designate the effectiveness of the process. The atypical expression of major histocompatibility complex and other protein-antigens, such as Fas/FasL and petformin in human trophoblast, the modified function of cellular constituents of the feto-maternal interface, as well as the specific role of some hormones and cytokines, represent substantive parameters of feto-maternal immunotolerance during implantation.
American Journal Of Reproductive Immunology 01/2009; 60(6):482-96. · 2.17 Impact Factor
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ABSTRACT: Implantation of mammalian conceptus in uterine cavity is the result of evolutionary adaptation, through high level of physiological procedures to ensure its success. However the majority of pregnancy losses occur before or during implantation. It is expected that exploring and defining the molecular and physiological road map during the crucial time of implantation will enable us to decode and effectively treat fertility defects. Immunological, hormonal and molecular factors participate in the feto-maternal cross talk during implantation and designate the effectiveness of the process. The atypical expression of major histocompatibility complex and other protein-antigens, such as Fas/FasL and petformin in human trophoblast, the modified function of cellular constituents of the feto-maternal interface, as well as the specific role of some hormones and cytokines, represent substantive parameters of feto-maternal immunotolerance during implantation.
American Journal Of Reproductive Immunology 11/2008; 60(6):482 - 496. · 2.17 Impact Factor
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ABSTRACT: Steroid hormones hold a major role in female fertility and their proper utilisation and monitoring in modern assisted reproduction protocols is important. Oocyte maturation and endometrial receptivity are the two major factors that appear to be related to a successful outcome in Assisted Reproductive Technology (ART). Many reports suggest that oocyte immaturity accounts for a considerable loss of efficiency in ART, mainly due to the poor quality of the obtained embryos and their inability to develop normally. Oestrogen appears to exert its effects on the cytoplasmic maturation of the oocyte, while progesterone has been shown to accelerate meiotic resumption. Moreover, ovarian stimulation appears to affect the normal luteal function and shifts in the window of implantation as a response to hormonal supplementation have also been observed. The ethical limitations in conducting in vivo studies of human implantation, have led to an indirect hormonal- and morphologic-oriented assessment of endometrial receptivity. The two main protocols of luteal support involve either progesterone supplementation or hCG administration, whereas the combined supplementation with oestradiol remains controversial. This brief review aims to summarize the current knowledge on steroidal actions during the above processes and to address their potential use in the improvement of current ART protocols.
The Journal of Steroid Biochemistry and Molecular Biology 10/2008; 112(1-3):1-4. · 3.05 Impact Factor
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ABSTRACT: The aim of this study was to assess the impact of nevirapine on a cervix carcinoma cell line.
HeLa cells were cultured in Dulbecco's modified Eagle medium supplemented with 20% fetal bovine serum at 37 degrees C and humidified 10% CO(2) in air. Nevirapine was purified from commercially available Viramune (Boehringer-Ingelheim), diluted in dimethyl sulfoxide (DMSO, Sigma-Aldrich) in 350 microMu final concentration and added to cell cultures 5 h after seeding. The same DMSO volume (0.2% final concentration) was added to controls.
We found that nevirapine treatment induces reversible growth arrest and produces morphological changes in treated cells. In contrast with previous reports the observed effects of nevirapine did not correlate with promotion of differentiation, but with induction of premature senescence. Premature senescence as a response to anti-tumour treatment is a common effect of the most anti-cancer chemotherapeutics. Nevirapine treated cells strongly accumulated SA-b-Gal activity and also expressed increased levels of p53 and p21 when analyzed via RT-PCR. In order to further explore the potent mechanisms of premature senescence induction we performed pChk2-Thr68 immunofluorescence analysis and found that nevirapine treated cells exhibited increased number of nuclear foci, indicating activated DNA damage response.
We propose that at least in HeLa cell line nevirapine treatment exerts an effect far from the differentiation process, by introducing the cells into premature senescence. Based on these data, the effects of RT inhibitors should be further investigated since they may represent an additional agent against human cancer.
Gynecologic Oncology 10/2008; 111(2):344-9. · 3.89 Impact Factor
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ABSTRACT: To present the options and the results in the management of poor responders in in-vitro fertilization.
There is no controlled ovarian hyperstimulation protocol which is best suited for all poor responders. Low dose gonadotropin-releasing hormone agonist regimes appear to be most advantageous. Prediction of compromised response prior to cycle initiation by a thorough assessment of ovarian reserve as well as a careful review of past responses could allow for a more appropriate selection of a controlled ovarian hyperstimulation protocol for each individual patient. Optimistic data have been presented by the use of high doses of gonadotropins, flare up gonadotropin-releasing hormone agonist protocols (standard or microdose), stop protocols, luteal onset of gonadotropin-releasing hormone agonist, and short protocols. Natural cycle also seems to be an appropriate strategy to be considered.
There is no universal definition for the 'poor responder'. Numerous strategies have been proposed to improve ovarian stimulation in poor responders, but none of them is the ideal for all such patients. More data from good quality controlled trials are needed.
Current Opinion in Obstetrics and Gynecology 08/2008; 20(4):374-8. · 2.38 Impact Factor
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ABSTRACT: The present clinical and molecular study aimed at investigating the presence of the genes encoding oxytocin receptor (OT-R) and Oct-4 in human amniotic fluid cells.
Amniotic fluid samples were obtained from amniocentesis. Cells from human amniotic fluid samples were analyzed for mRNA expression of OT-R and Oct-4 via reverse transcription-polymerase chain reaction (RT-PCR). Immunocytochemistry was also performed with OT-R and Oct-4 antibodies.
RT-PCR from 10 independent amniocentesis samples demonstrated the expression of OT-R and Oct-4 mRNA. The cells also showed strong immunoreactivity for molecular markers of OT-R and Oct-4.
OT-R and Oct-4 are expressed in human amniotic fluid cells. The role of oxytocin in the physiology and pathophysiology of amniotic fluid cells remains to be settled.
Gynecological Endocrinology 06/2008; 24(5):280-4. · 1.58 Impact Factor
