Peter Drakakis

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

OBJECTIVEnTo study the expression of the FSH and LH receptors in human oocytes and preimplantation embryos and their potential roles in early human development.nnnDESIGNnClinical and molecular studies.nnnSETTINGnUniversity hospital IVF center.nnnPATIENT(S)nFemale volunteers undergoing intracytoplasmic sperm injection at the IVF unit of the Athens University Hospital. All patients gave written informed consent.nnnINTERVENTION(S)nOvarian stimulation was performed with exogenous gonadotropin administration. Intracytoplasmic sperm injection was performed on mature oocytes.nnnMAIN OUTCOME MEASURE(S)nUnfertilized 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.nnnRESULT(S)nTranscripts 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.nnnCONCLUSION(S)nMessenger 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.

Poor responder protocols for in-vitro fertilization : options and results

Purpose of review To present the options and the results in the management of poor responders in in-vitro fertilization. Recent findings 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. Summary 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.

Pharmacogenetics in Ovarian Stimulation—Current Concepts

Ovarian response to follicle‐stimulating hormone (FSH) action differs considerably among women; this has prompted researchers to determine which factors modify this response. The challenge is to identify the genes that affect the response to FSH stimulation by the application of pharmacogenetics to assisted reproduction techniques (ARTs). Recently, new insights have been gained in the investigation of the variability in the gene that encodes the FSH receptor (FSHR) gene or genes of the estrogen pathway. Several polymorphisms of the FSHR gene have been discovered, but Ser680Asn and Thr307Ala are the two most studied. The Ser680Asn polymorphism of the FSHR gene has been found to influence the ovarian response to FSH stimulation in women undergoing in vitro fertilization (IVF), and in women with the genotype Ser/Ser, the FSHR appears to be more resistant to FSH action. The clinical implications of this finding are highly important; the ultimate goal is to apply genetic markers as routine diagnostic tests before ovarian stimulation to predict ovarian response, determine the required FSH dose, and avoid the possible complications related to FSH stimulation.

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.

hCG priming effect in controlled ovarian stimulation through a long protocol.

BackgroundRecently, 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.MethodsPatients 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.ResultsThe 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.ConclusionAll 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.

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.

The effect of CRH and its inhibitor, antalarmin, on in vitro growth of preantral mouse follicles, early embryo development, and steroidogenesis.

In vitro growth systems of preantral follicles allow studying the effect of various endocrine, paracrine, and autocrine factors on follicular growth and oocyte maturation. CRH is a 41-amino-acid neuropeptide responsible for endocrine, autonomic, immunological, and behavioral responses of mammals to stress and has two receptors, CRH receptor type 1 (CRH-R1) and CRH-R2. Antalarmin, a CRH-R1 antagonist, has been used to elucidate the role of CRH in stress, inflammation, and reproduction. The present study describes in vitro growth of mouse preantral follicles, early embryo development, and steroidogenesis in the presence of CRH and its antagonist antalarmin. We cultured 732 follicles in control media, 1306 in CRH 10(-7) mol/liter, and 1202 in CRH 10(-7) plus antalarmin 10(-6) mol/liter. The culture medium was assayed on alternate days for 17β-estradiol, progesterone, and β-human chorionic gonadotropin. Total RNA was extracted from preantral follicles as well as early preimplantation embryos and was assessed by real-time RT-PCR for the expression of CRH-R1 and CRH-R2 mRNAs. Hormone analysis showed that the CRH group had lower levels of 17β-estradiol, progesterone, and β-human chorionic gonadotropin as the culture progressed, in comparison with the other two groups. RT-PCR demonstrated the presence of CRH-R1 and CRH-R2 in all stages of preantral follicle culture. Morula/blastocyst-stage embryos expressed only CRH-R1. In conclusion, CRH has an inhibitory effect on in vitro fertilized oocytes, resulting from cultured preantral follicles at all stages of preimplantation embryo development. Furthermore, the presence of CRH in the culture medium inhibits steroidogenesis by preantral mouse follicles cultured in vitro.

Cervical Cancer Cell Line Secretome Highlights the Roles of Transforming Growth Factor-Beta-Induced Protein ig-h3, Peroxiredoxin-2, and NRF2 on Cervical Carcinogenesis

Cancer cells acquire unique secretome compositions that contribute to tumor development and metastasis. The aim of our study was to elucidate the biological processes involved in cervical cancer, by performing a proteomic analysis of the secretome from the following informative cervical cell lines: SiHa (HPV16+), HeLa (HPV18+), C33A (HPV−), and HCK1T (normal). Proteins were analyzed by 2D gel electrophoresis coupled to MALDI-TOF-MS. Enrichment of secreted proteins with characteristic profiles for each cell line was followed by the identification of differentially expressed proteins. Particularly, transforming growth factor-beta-induced protein ig-h3 (Beta ig-h3) and peroxiredoxin-2 (PRDX2) overexpression in the secretome of cancer cell lines was detected and confirmed by Western blot. Bioinformatics analysis identified the transcription factor NRF2 as a regulator of differentially expressed proteins in the cervical cancer secretome. NRF2 levels were measured by both Western blot and Multiple Reaction Monitoring (MRM) in the total cell extract of the four cell lines. NRF2 was upregulated in SiHa and C33A compared to HCK1T. In conclusion, the secreted proteins identified in cervical cancer cell lines indicate that aberrant NRF2-mediated oxidative stress response (OSR) is a prominent feature of cervical carcinogenesis.

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.

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.