Detection of RUNX2 gene expression in cumulus cells in women undergoing controlled ovarian stimulation.

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.

Three Dimensional Power Doppler evaluation of human endometrium after administration of oxytocine receptor antagonist (OTRa) in an IVF program.

PURPOSE: To compare endometrial and subendometrial morphological changes and vascularity as measured by 3D Power Doppler sonography, based on a specific scoring system between women subjected or not to oxytocine receptor antagonist (OTRa) during IVF cycles. METHODS: Twenty-six women were divided into groups according to OTRa (Atosiban tractocide) administration. The first group (control n = 13 women) was examined with 3D Power Doppler 3 days after embryo transfer. The second group (n = 13 women) was administered 7.5 mg intravenous tractocide 2 days after embryo transfer and a 3D Power Doppler was performed after a day. RESULTS: The control group presented the following ultrasonographic characteristics: (a) echogenic endometrium in all cases, (b) endometrial thickness >7 mm in all cases (84.6%), (c) endometrial volume >2.31 cm(3) in 5 cases (38.5%), (d) abnormal sub-endometrial halo in 3 cases (23.1%), (e) endometrial blood flow in 6 cases (46.2%) and (f) complex vessel's architecture in 2 cases (15.4%). In women who underwent OTRa administration were observed: (a) echogenic endometrium in 1 case (7.7%), triple line endometrium in 12 cases (92.3%), (b) endometrial thickness >7 mm in all cases, (c) endometrial volume >2.31 cm(3) in 11 cases (84.6%), (d) abnormal sub-endometrial halo in 3 cases (23.1%), (e) endometrial blood flow in 11 cases (84.6%) and (f) complex vessel's architecture in 6 cases (46.2%). CONCLUSIONS: Women who have taken OTRa presented an endometrium with characteristics more predictive of implantation.

Comparison of the Fetal Fraction of Cell-Free DNA in In-Vitro Fertilization (IVF) Versus Natural Conception Evaluation of the Fetal Fraction With IVF Parameters.

Background As the offspring of assisted reproduction techniques (ARTs) have become a substantial proportion of the population, increased attention has been placed on the safety of ART. Investigators have focused on identifying a tool that combines molecular or biological tests that can predict the outcomes of in-vitro fertilization (IVF) or intracytoplasmic sperm injection and the resulting pregnancy after ART-mediated embryo implantation. This study aimed to answer the following questions: is there a difference between natural conception and IVF pregnancies regarding fetal fraction (FF) of cell-free DNA (cfDNA) in maternal age, birth weight, gender, and gestational age? Is there a difference between FF concentration regarding the parameters of IVF as possible predictive factors affecting the outcomes of IVF? Methodology This study included 31 women with singleton pregnancies conceived via IVF who underwent cell-free fetal DNA (cffDNA) screening for trisomy 13, 18, and 21; sex determination; and FF. The control group included 55 women who experienced natural conception. For all women, anthropometric characteristics such as age, weight, height, and body mass index (BMI) were recorded. For the IVF group, early follicular phase values of follicle-stimulating hormone, luteinizing hormone, prolactin, anti-müllerian hormone, thyroid-stimulating hormone, and estradiol were recorded. Results The natural conception and IVF groups were similar regarding maternal age, BMI of the mother, gender, birth weight, and gestational age. FF was not significantly different between the natural conception and IVF groups (10 (3.8) vs. 9 (2.6); p = 0.144). The results were similar after adjusting for maternal age via regression analysis. cfDNA was not associated with maternal age, birth weight, gender, or gestational age in the entire study sample or separately for the natural conception and IVF groups. No significant correlation was found between cfDNA and IVF parameters. Conclusions The FF is an important factor for non-invasive prenatal testing (NIPT) accuracy. Several studies have found a reduction in FF in pregnancies following ART compared with natural conception, while other studies have presented no differences in the FF. All researchers agree on the importance of NIPT; however, knowledge on how the FF is affected in ART pregnancies compared with naturally conceived pregnancies is very limited. In this study, no difference in FF for the IVF group compared with natural conception women was observed. The cffDNA concentrations in maternal serum do not appear to be affected in IVF conception. We suggest that FF is an independent factor compared with IVF parameters.

Genome-wide microarray evidence that 8-cell human blastomeres over-express cell cycle drivers and under-express checkpoints.

PURPOSE: To understand cell cycle controls in the 8-Cell human blastomere. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Evidence that human blastomere cleavage is under unique cell cycle control.

PURPOSE: To understand the molecular pathways that control early human embryo development. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Oxytocin receptor- and Oct-4-expressing cells in human amniotic fluid.

BACKGROUND/AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Effect of prolactin in the absence of hCG on maturation, fertilization, and embryonic development of in vitro matured mouse oocytes.

Oocyte maturation is a complex process involving both the progression of meiotic cycle and the reprogramming of cytoplasmic events. The aim of this study was to investigate the effects of prolactin (PRL) in the in vitro maturation (IVM) of preantral mouse oocytes, in the absence of human chorionic gonadotrophin (hCG). Mouse preantral follicles were collected from female mice without prior hormonal ovarian stimulation and were cultured in the presence of varying concentrations of PRL (20, 100, 200, and 300 ng/mL) for 12 days. A group of in vitro matured oocytes were assessed for polar body (PB) formation, while the rest were fertilized and embryonic development was recorded. The maturation of preantral mouse follicles, as well as their fertilization and cleavage rates, observed when the culture medium was supplemented with middle- and high-range doses of PRL was beneficially affected. This effect was considerably high, although the culture media lacked hCG, a hormone extensively used in modern ovulation induction regiments, as well as in IVM media.

Effect of recombinant-LH and hCG in the absence of FSH on in vitro maturation (IVM) fertilization and early embryonic development of mouse germinal vesicle (GV)-stage oocytes.

During in vitro maturation (IVM), intrinsic and extrinsic factors must co-operate properly in order to ensure cytoplasmic and nuclear maturation. We examined the possible effect of LH/hCG in the process of oocyte maturation in mice with the addition of recombinant LH (r-LH) and hCG in our IVM cultures of mouse germinal vesicle (GV)-stage oocytes. Moreover, the effects of these hormones on fertilization, early embryonic development and the expression of LH/hCG receptor were examined. Nuclear maturation of GV-stage oocytes was evaluated after culture in the presence of r-LH or hCG. Fertilization rates and embryonic development were assessed after 24h. Total RNA was isolated from oocytes of different stages of maturation and from zygotes and embryos of different stages of development in order to examine the expression of LH/hCG receptor, using RT-PCR. The in vitro nuclear maturation rate of GV-stage oocytes that received hCG was significantly higher compared to the control group. Early embryonic development was increased in the hCG and LH cultures of GV oocytes when LH was further added. The LH/hCG receptor was expressed in all stages of in vitro matured mouse oocytes and in every stage of early embryonic development. Addition of hCG in IVM cultures of mouse GV oocytes increased maturation rates significantly. LH, however, was more beneficial to early embryonic development than hCG. This suggests a promising new technique in basic science research or in clinical reproductive medicine.

Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells.

Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that it is an independent circadian rhythm-competent equivalence group poised to signal its environment, defend against maternal immune rejection, and begin the rapid commitment events of early embryogenesis.

Messenger RNA expression for the follicle-stimulating hormone receptor and luteinizing hormone receptor in human oocytes and preimplantation-stage embryos.

OBJECTIVE: To study the expression of the FSH and LH receptors in human oocytes and preimplantation embryos and their potential roles in early human development. DESIGN: Clinical and molecular studies. SETTING: University hospital IVF center. PATIENT(S): Female volunteers undergoing intracytoplasmic sperm injection at the IVF unit of the Athens University Hospital. All patients gave written informed consent. INTERVENTION(S): Ovarian stimulation was performed with exogenous gonadotropin administration. Intracytoplasmic sperm injection was performed on mature oocytes. MAIN OUTCOME MEASURE(S): Unfertilized oocytes and zygotes and embryos at the 2-cell, 4-cell, morula, and blastocyst stage were selected for study. A polymerase chain reaction methodology was used to analyze human oocytes and embryos. Messenger RNA was reverse transcribed and amplified with FSH and LH receptor specific primers. RESULT(S): Transcripts for the FSH receptor were detected in oocytes and zygotes and embryos at the 2-cell, morula, and blastocyst stage, while no message was detected in embryos at the 4-cell stage. Transcripts for the LH receptor were observed in oocytes and zygotes and morula- and blastocyst-stage embryos, whereas no message was detected in embryos at the 2-cell and 4-cell stage. CONCLUSION(S): Messenger RNA for the FSH and LH receptors was observed in oocytes and preimplantation embryos at different stages, indicating a physiological role in the oocyte maturation process and early embryonic development in the human.

Effect of PRL on in vitro follicle growth, in vitro oocyte maturation, fertilization and early embryonic development in mice.

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.

Embryoid bodies from mouse stem cells express oxytocin receptor, Oct-4 and DAZL.

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.

Follicle-stimulating hormone receptor gene mutations are not evident in Greek women with premature ovarian failure and poor responders.

BACKGROUND/AIMS: This clinical and molecular study aimed to investigate the presence of follicle-stimulating hormone (FSH) receptor gene mutations in women with premature ovarian failure (POF) and poor responders to in vitro fertilization treatment. METHODS: DNA was extracted from blood samples for subsequent polymerase chain reaction (PCR). PCR was followed by restriction fragment length polymorphism and direct sequencing. RESULTS: No inactivating mutations reported so far were identified in exons 6, 7, and 10 in women with POF and poor responders. CONCLUSION: FSH receptor gene mutations are not frequent in Greek patients with POF as is the case in the rest of the world except for cases with ovarian dysgenesis in Finland.

Prolactin receptor mRNA expression in oocytes and preimplantation mouse embryos.

Prolactin was first identified as an anterior pituitary lobe hormone, responsible for the regulation of mammary gland growth and development. Prolactin receptors have been localized in a number of peripheral tissues, including tissues involved in reproduction. Studies with knockout animals have shown that prolactin receptor deficient mice present reproductive defects, whereas prolactin promotes the developmental potential of preimplantation mouse and rat embryos in vitro. To better understand the role of prolactin in the process of reproduction and early embryo development in mice, the expression of the four transcript variants of prolactin receptor was examined in the first stages of mouse embryo development. Prolactin long receptor mRNA was expressed in all stages examined, that is in cumulus cells, oocytes, zygotes, 2-cell embryos, 4-cell embryos, morulae and blastocysts. Prolactin receptor type S1 mRNA was observed only in cumulus cells, while S2 mRNA was present in cumulus cells, oocytes, zygotes and 2-cell embryos. S3 mRNA was expressed only in cumulus cells and oocytes. These results indicate that different isoforms of prolactin receptors may be present in the various stages of mouse preimplantation embryo and may play an important role in the control of its growth and development.

Effect of human hydrosalpinx fluid on the development of mouse embryos and role of the concentration of growth factors in culture medium with and without hydrosalpinx fluid.

The aim of this study was to investigate the effect of human hydrosalpinx fluid (HF) on the development and blastulation of mouse embryos and the role of the concentration of growth factors in culture medium with and without HF. In total, 2100 mouse embryos were cultured. Female mice were induced to superovulate and then mated with males. Two-cell-stage embryos were recovered from the oviduct and cultured in Ham's F-10 medium with bovine serum albumin and HF. Epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) were analyzed by quantitative enzyme immunoassay. Mean blastulation index of 1.11, 0.97 and 0.98 was found at HF concentration of 5%, 20% and 30%, respectively (p = 0.8). The mean value of EGF in the control culture medium without HF was 11.2 pg/ml, which was statistically significantly different from that in culture medium containing HF (p < 0.001). The mean value of IGF-I in the control group without HF was 1.30 pg/ml and was not statistically significantly different from that in culture medium containing HF. Development of the two-cell-stage embryos was not affected at low (< 30%) HF concentrations. In conclusion, the present study demonstrates that even apparently normal blastulation is affected by any concentration of HF because of low embryonic EGF.