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.

Corticotropin-releasing hormone inhibits in vitro oocyte maturation in mice.

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.

Update on the role of ovarian corticotropin-releasing hormone.

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.

Early hCG addition to rFSH for ovarian stimulation in IVF provides better results and the cDNA copies of the hCG receptor may be an indicator of successful stimulation.

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.

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.

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.

Nevirapine induces growth arrest and premature senescence in human cervical carcinoma cells.

OBJECTIVE: The aim of this study was to assess the impact of nevirapine on a cervix carcinoma cell line. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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.

Deleted in Azoospermia-Like (DAZL) gene-expressing cells in human amniotic fluid: a new source for germ cells research?

OBJECTIVE: To evaluate whether amniotic fluid cells contain a germ-like cell subpopulation. DESIGN: Experimental study. SETTING: University hospital. PATIENT(S): None. INTERVENTION(S): Cells from human amniotic fluid samples were analyzed for messenger RNA expression of Deleted in Azoospermia-Like gene (DAZL) and Oct-4 by reverse transcriptase polymerase chain reaction. DAZL and C-kit protein expression was assessed by flow cytometry. Immunocytochemistry also was performed to determine DAZL-, stage-specific embryonic antigen 4 (SSEA-4)-, and Oct-4-positive cells. MAIN OUTCOME MEASURE(S): DAZL gene expression in amniotic fluid cells. RESULT(S): Reverse transcriptase polymerase chain reaction, flow cytometric, and immunocytochemical analyses revealed that human amniotic fluid consists of a distinct cell population that expresses DAZL, C-kit, SSEA-4, and Oct-4. CONCLUSION(S): Our results suggest that human amniotic fluid represents a new source for the isolation of human DAZL-, C-kit-, SSEA-4-, and Oct-4-positive stem cells without raising the ethical issues associated with human embryonic research.

Luteal estrogen supplementation in stimulated cycles may improve the pregnancy rate in patients undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer.

OBJECTIVE: To evaluate the effect of estradiol addition to progesterone supplementation during the luteal phase on pregnancy and implantation rates in patients undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) cycles. METHODS: In this prospective, randomized study, carried out in an IVF unit of a university hospital, we studied patients who were undergoing IVF/ICSI with controlled ovarian hyperstimulation using a gonadotropin-releasing hormone agonist/human recombinant gonadotropin long protocol. The main outcome measures were the pregnancy and implantation rates measured in the two groups. RESULTS: Our results suggest higher pregnancy and implantation rates in IVF/ICSI-ET cycles that were supplemented with estradiol in the luteal phase. CONCLUSIONS: Estradiol supplementation during the luteal phase in women undergoing IVF/ICSI-ET has a beneficial effect on the outcome without (at least, as seems from this study) having any adverse effects.

Thyroid tuberculosis.

Although the association of thyroid disorders with tuberculosis has been known for a long time, the diagnosis of thyroid tuberculosis is rare. Differential diagnosis can be very difficult without fine needle aspiration. The clinical course of the disease may resemble toxic goiter or acute thyroiditis or may follow a subacute or chronic pattern without specific symptomatology. We describe a 49-year old male patient with thyroid tuberculosis presenting as thyroid enlargement, fever, increased erythrocyte sedimentation rate, normal thyroid function tests, and a chest x-ray indicating the presence of a lesion with distinct calcification in the lower lobe of the right lung. Tuberculosis of the thyroid gland, although very rare, should be considered as a possible diagnosis when localized swelling, cold abscess or thyroid nodule with or without a cystic component are present.

Estradiol and progesterone supplementation during luteal phase improved the receptivity of the endometrium in a patient with a history of diethylstilboestrol exposure in-utero.

BACKGROUND: Diethylstilboestrol (DES) exposure in-utero has been shown to have negative effects on pregnancy. DES-exposed women are at increased risk of early spontaneous pregnancy loss, ectopic gestation and infertility. DESIGN: A 34-year old woman with a 6-year history of primary infertility is presented. The patient underwent in vitro fertilization (IVF) treatment without success. To improve the quality of the endometrium following IVF treatment, E2 and progesterone supplementation was added to the usual therapeutic regimen. The pregnancy progressed uneventfully and a normal female was born. CONCLUSIONS: This case indicates that the administration of E2 and progesterone in DES-exposed women might improve endometrium receptivity and consequently pregnancy outcome.

Treatment options of polycystic ovary syndrome in adolescence.

The polycystic ovary syndrome (PCOS) is defined as a syndrome of hyperandrogenism and chronic anovulation in the absence of other underlying pituitary or adrenal disorders. PCOS often presents in adolescence and is the commonest cause of menstrual disorders and hirsutism. Premature adrenarche and hirsutism that occur before puberty have been associated with PCOS. The etiology of the syndrome is as yet uncertain. Theories have focused on intrinsic defects in the ovarian and adrenal steroidogenesis, and on the role of insulin resistance in modifying reproductive function. Insulin resistance associated with PCOS confers a markedly increased risk of impaired glucose tolerance and type 2 diabetes mellitus in adolescent girls, as in premenopausal women. Therapeutic considerations focus on the management of menstrual irregularities and hyperandrogenism, as well as on the prevention of the metabolic consequences of the syndrome.

Oocyte maturation in assisted reproductive techniques.

Human oocyte maturation is a long process during which nuclear maturation occurs resulting in germinal vesicle breakdown (transition from prophase I to metaphase II) and extrusion of the first polar body. During oocyte maturation, in parallel with nuclear maturation, a number of events take place in the oocyte cytoplasm that assist fertilization and early embryonic development. So far several attempts have been made to mature human oocytes in vitro. The main patient group to which in vitro maturation (IVM) has been applied is polycystic ovarian syndrome. In a concise review we present the techniques used for the IVM of oocytes and the role of hormones and growth factors in IVM and subsequent fertilization and early embryonic development.

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.

Oxytocin receptor is differentially expressed in mouse endometrium and embryo during blastocyst implantation.

The oxytocin (OT)-oxytocin receptor (OTR) system of the mammalian uterus has mainly been studied in relation to its involvement in the onset of labor. The aim of this study was to elucidate the in vivo expression and localization pattern of OTR in the mouse endometrium and embryo during implantation, as well as OTR mRNA expression in the in vitro developing mouse embryo. The expression of OTR or OT was detected immunohistochemically in uterine tissue sections of 5- to 8-week-old female mice between days 4 and 10 of an established pregnancy. In addition, the expression of OTR mRNA was detected by means of reverse transcription polymerase chain reaction (RT-PCR) in mouse oocytes and embryos up to the blastocyst stage. The mean ratios of normalized expression levels of OTR gene in all samples were also calculated. The recorded increase in OTR mRNA immediately after fertilization could mean a possible role of OT in this process, as OTR mRNA gradually decreased after the four-cell stage of pre-embryonic development. The differential expression of OTR during embryonic apposition and embryonic invasion/placentation in the mouse uterus suggests a potential role of OT in the implantation process of the mouse. It is possible that the interaction of OTR with the hormones included in the ovulation induction regiments utilized today in in vitro fertilization (IVF) could be affecting the receptivity/quality of the implanting endometrium.

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.

Effects of GH and IGF-I on the in vitro maturation of mouse oocytes.

OBJECTIVE: A number of hormones and growth factors have been reported to affect the in vitro maturation of oocytes. Their exact effects on follicular growth and oocyte maturation and the mechanisms involved are still unclear. In the present study, we have investigated the effects of Growth Hormone (GH) and Insulin-like Growth Factor 1 (IGF-1) on the in vitro maturation of mouse oocytes. DESIGN: Immature preovulatory oocytes without cumulus cells (denuded), were obtained from 4- to 8-week old female mice and were cultured in Ham's F-10 medium. GH and IGF-1 were added separately or in combination in gradually increasing concentrations in the culture media, while medium-only containing samples were employed as controls. Oocyte development was assessed daily for three days and maturation was considered to be completed when the first polar body appeared. RESULTS: In control samples, 44+/-3% (mean+/-SE) of denuded oocytes formed a polar body. The achieved maturation rate after the addition of either GH or IGF-1 or GH plus IGF-1 was significantly higher than in controls. The highest maturation rates were achieved after the addition of 0.2 microg/ml GH (76%+/-5%), 50 ng/ml IGF-1 (69%+/-5%) and a combination of 0.2 microg/ml GH plus 10 ng/ml IGF-1 (75%+/-5%). CONCLUSIONS: The data suggest that GH and IGF-1, alone or in combination, affect mouse oocyte maturation significantly. The lack of a synergistic effect on oocyte cultures when both hormones were added indicates that both hormones act through the same signaling pathway.