Ingrid Segers

Cumulus cell gene expression is associated with oocyte developmental quality and influenced by patient and treatment characteristics

BACKGROUND Gene expression of cumulus cells (CC) could predict oocyte developmental quality. Knowledge of the genes involved in determining oocyte quality is scanty. The aim was to correlate clinical and biological characteristics during ovarian stimulation with the expression of 10 selected genes in CC. METHODS Sixty-three ICSI patients were stimulated with GnRH-agonist plus highly purified hMG (n = 35) or recombinant FSH (n = 28). Thirteen variables were analyzed: Age, BMI, duration of stimulation, serum concentrations of progesterone, 17beta-estradiol, FSH and LH on day of hCG, Ovarian Response, Oocyte Maturity, 2 pronuclei and three embryo morphology related variables: > or =7 cells, Low Fragmentation, Good Quality Embryos score. Expression of HAS2, VCAN, SDC4, ALCAM, GREM1, PTGS1, PTGS2, DUSP16, SPROUTY4 and RPS6KA2 was analyzed in pooled CC using quantitative PCR, and the relationship to the 13 variables was evaluated by multivariable analysis. RESULTS All 10 genes are expressed at oocyte retrieval, with PTGS1, SPROUTY4, DUSP16 and RPS6KA2 described in human ovary for the first time. The three variables that correlated most often with differential expression were Age, BMI and serum FSH level. Significant correlation was found with Oocyte Maturity (VCAN, P < 0.005), Low Fragmentation (RPS6KA2, P < 0.05), Embryos with > or =7 cells (ALCAM and GREM1, P < 0.05). The expression of the other genes was also correlated to oocyte developmental quality but to a less extent. SDC4, VCAN, GREM1, SPROUTY4 and RPS6KA2 showed gonadotrophin preparation-dependent expression and/or interactions (all P < 0.05). CONCLUSION The expression of ovulation related genes in CC is associated with patient and treatment characteristics, oocyte developmental potential and differs with the type of gonadotrophin used.

Cumulus cell gene expression predicts better cleavage-stage embryo or blastocyst development and pregnancy for ICSI patients

BACKGROUND Cumulus cell (CC) gene expression is suggested as a non-invasive analysis method to predict oocyte competence. There are, however, important between-patient differences in CC gene expression. These can be compensated when expression results are combined with patient and cycle characteristics using a multiple regression analysis model. METHODS From ICSI patients stimulated with GnRH antagonist and recombinant FSH (n= 25) or GnRH agonist and highly purified menotrophin (n= 20), CC were collected and oocytes were individually fertilized and cultured. CC were analyzed for the expression of Syndecan 4 (SDC4), Prostaglandin-endoperoxide synthase 2 (PTGS2), Versican (VCAN), Activated leukocyte cell adhesion molecule, Gremlin 1, transient receptor potential cation channel, subfamily M, member 7 (TRPM7), Calmodulin 2 and Inositol 1,4,5-trisphosphate 3-kinase A (ITPKA) using quantitative PCR. Results were analyzed in relation to the stimulation protocol. Within-patient variation in gene expression was related to oocyte maturity and developmental potential. Models predictive for normal embryo or blastocyst development and pregnancy in single embryo transfer cycles were developed. RESULTS Mature oocytes have higher PTGS2 and lower VCAN expression in their cumulus. All genes except VCAN had a positive correlation with good embryo or blastocyst morphology and were used to develop predictive models for embryo or blastocyst development (P< 0.01). Specific models were obtained for the two stimulation protocols. In both groups, better cleavage-stage embryo prediction relied on TRPM7 and ITPKA expression and pregnancy prediction relied on SDC4 and VCAN expression. In the current data set, the use of CC expression for pregnancy prediction resulted in a sensitivity of >70% and a specificity of >90%. CONCLUSIONS Multivariable models based on CC gene expression can be used to predict embryo development and pregnancy.

New candidate genes to predict pregnancy outcome in single embryo transfer cycles when using cumulus cell gene expression

OBJECTIVE To relate the gene expression in cumulus cells surrounding an oocyte to the potential of the oocyte, as evaluated by the embryo morphology (days 3 and 5) and pregnancy obtained in single-embryo transfer cycles. DESIGN Retrospective analysis of individual human cumulus complexes using quantitative real-time polymerase chain reaction for 11 genes. SETTING University hospital IVF center. PATIENT(S) Thirty-three intracytoplasmic sperm injection patients, of which 16 were pregnant (4 biochemical and 12 live birth). INTERVENTION(S) Gene expression analysis in human cumulus complexes collected individually at pickup, allowing a correlation with the outcome of the corresponding oocyte. Multiparametric models were built for embryo morphology parameters and pregnancy prediction to find the most predictive genes. MAIN OUTCOME MEASURE(S) Gene expression profile of 99 cumulus complexes for 11 genes. RESULT(S) For embryo morphology prediction, TRPM7, ITPKA, STC2, CYP11A1, and HSD3B1 were often retained as informative. Models for pregnancy-biochemical or live birth-complemented or not with patient and cycle characteristics, always retained EFNB2 and CAMK1D together with STC1 or STC2. Positive and negative predictive values of the live birth models were >85%. CONCLUSION(S) EFNB2 and CAMK1D are promising genes that could help to choose the embryo to transfer with the highest chance of a pregnancy.

Timing of Nuclear Maturation and Postovulatory Aging in Oocytes of In Vitro-Grown Mouse Follicles with or Without Oil Overlay

Meiotic maturation of the oocyte is a timed sequence of events induced by the ovulatory LH surge. In vitro maturation of oocytes is known to alter the meiotic time course. This study documented the timing of meiosis in oocytes grown in vitro for 12 days, from the preantral follicle stage onward, and the influence of an oil overlay. In the oil-free culture, the stability of the metaphase II spindle was further explored to determine the postovulatory aging events. After the maturation stimulus, in vitro-grown oocytes were collected at 2-h intervals spanning the period of meiosis (0-18 h) and at 3-h intervals during early postovulatory aging (18-27 h). Stage of maturation was assessed both morphologically and by detailed spindle analysis and chromosome alignment. Results revealed that oil overlay did not impair the competence of cultured oocytes to proceed to meiosis II, but delayed meiosis I progression. Oil overlay during culture causes a different hormonal exposure of the follicles by a differential segregation into the oil overlay. The use of a progesterone receptor antagonist, however, did not induce a delay in meiotic progression. Aging effects in oil-free cultured follicles were detected 5 h after the establishment of the metaphase II spindle, comparable to their in vivo grown counterparts. The predominant effect of aging was an interphase-like appearance of the cytoskeleton. So an optimal time window for fertilization after in vitro follicular growth was determined to be 16-21 h after maturation induction.

Acquisition and loss of oocyte meiotic and developmental competence during in vitro antral follicle growth in mouse

OBJECTIVE To analyze the effect of various culture periods and FSH on oocyte competence in cultured mouse follicles. DESIGN Controlled in vitro laboratory experiment. SETTING Academic research environment. INTERVENTION(S) DNA staining, maturation induction, parthenogenesis, and IVF were performed after various periods of oocyte culture. Additionally, FSH modulation during preantral follicle growth was studied. MAIN OUTCOME MEASURE(S) Nucleolar conformation, meiotic resumption, activation potential, and preimplantation development were recorded. RESULT(S) Surrounded nucleolus (SN) conformation was found in oocytes cultured for 8 days and correlated by time with meiotic competence. Culture for 12 days increased oocyte diameter (day 8 69.8 mum vs. day 12 72.4 mum) and meiotic competence (day 8 63% vs. day 12 81% polar body oocytes), but decreased fertilization rate (day 8 75% vs. day 12 53% 2-cell embryos). Equal activation potential (49%) on both days indicated that impaired sperm entry could not account for lower fertilization rate after 12 days. Depriving early follicular growth of FSH delayed antrum formation but not SN conformation. CONCLUSION(S) Fertilization of cultured oocytes is most efficient close to the completion of SN. Omitting FSH from early follicle growth could not postpone the early onset of SN, which seems to be imposed by the time spent in vitro.

Expression Patterns of Poliovirus Receptor, Erythrocyte Protein Band 4.1-Like 3, Regulator of G-Protein Signaling 11 and Oxytocin Receptor in Mouse Ovarian Cells During Follicle Growth and Early Luteinization In Vitro and In Vivo

ABSTRACT Poliovirus receptor (Pvr), erythrocyte protein band 4.1-like 3 (Epb4.1l3), regulator of G-protein signaling 11 (Rgs11), and oxytocin receptor (Oxtr) expression were quantified in in vitro- and in vivo-grown mouse follicles. The expression of all genes was increased during antral growth in in vitro-grown cumulus cells, whereas only Rgs11 and Oxtr were increased and Pvr and Epb4.1l3 were decreased in in vivo grown cumulus cells. In vivo mural granulosa cells showed the highest expression of Pvr, Rgs11, and Oxtr. The in vitro granulosa + theca compartment responded to human chorionic gonadotropinduring early luteinization by either an upregulation (Pvr, Oxtr) or downregulation (Epb41l3, Rgs11). Oocytes expressed Epb4.1l3, not Rgs11, and Pvr only in in vitro-grown oocytes. Translation into protein was confirmed for Epb4.1l3 in in vitro-grown follicles and in vivo-grown cumulus-oocyte complexes. Protein 4.1B was present during antral growth in cumulus, granulosa cells, and oocytes. Hypothetical functions of Epb4.1l3 and Pvr involve cell adhesion regulation and Rgs11 could be involved in cAMP production in the follicle. Oxtr is known to be important during and after the ovulatory stimulus, but, as in bovine, was also regulated during folliculogenesis. High expression of Pvr and Epb4.1l3 with culture duration in cumulus cells might mark inappropriate differentiation into a mural granulosa-like cell type and function as negative follicle development marker. Rgs11 and Oxtr are both in vivo and in vitro upregulated in cumulus cells during antral follicle growth and might be considered positive markers for follicle development.

Differences in Collagen Expression in Cumulus Cells after Exposure to Highly Purified Menotropin or Recombinant Follicle-Stimulating Hormone in a Mouse Follicle Culture Model

Abstract Extracellular matrix (ECM) formation by cumulus cells is an important process that determines fertilization and embryo quality. Several collagen types are present in the ovarian follicular ECM and are related to proliferation, steroidogenesis, and luteinization. In vitro mouse follicles can optimally grow and provide developmentally competent oocytes with 10 IU/L recombinant follicle-stimulating hormone (rFSH). As a model for superovulation, experiments with 100 IU/L rFSH or 100 IU/L highly purified menotropin (HP-hMG) exposure during antral growth were undertaken. Col4a1, Col4a2, and Col6a2 expression levels were analyzed at three time points during antral growth and at a 4-h interval up to 16 h after ovulation induction using quantitative PCR. The presence and induction of the collagen mRNA and protein were confirmed in cumulus from in vivo- and in vitro-grown follicles, and TGFBs 1 and 2 were assayed as potential regulators. The study revealed that exposure to 100 IU/L FSH, as in both superovulation conditions, significantly influenced the follicle morphology and slowed down nuclear maturation and mucification (P < 0.05). This coincided with an increased expression of the three collagens in the cumulus-oocyte complex at the end of antral growth and in the first hours following the ovulatory dose of human chorionic gonadotropin (P < 0.05). The increased expression might reflect a differentiation but is most likely due to a precocious luteinization of the cumulus. Growth in HP-hMG resulted in higher Tgfb1 mRNA and protein levels, fewer COCs with an increased collagen expression and with a more synchronous nuclear maturation. This suggests that the presence of luteinizing hormone activity tempered the effect of the elevated FSH dose.

Pregnancy prediction in single embryo transfer cycles after ICSI using QPCR: validation in oocytes from the same cohort.

Cumulus cell (CC) gene expression is being explored as an additional method to morphological scoring to choose the embryo with the highest chance to pregnancy. In 47 ICSI patients with single embryo transfer (SET), from which individual CC samples had been stored, 12 genes using QPCR were retrospectively analyzed. The CC samples were at the same occasion also used to validate a previously obtained pregnancy prediction model comprising three genes (ephrin-B2 (EFNB2), calcium/calmodulin-dependent protein kinase ID, stanniocalcin 1). Latter validation yielded a correct pregnant/non-pregnant classification in 72% of the samples. Subsequently, 9 new genes were analyzed on the same samples and new prediction models were built. Out of the 12 genes analyzed a combination of the best predictive genes was obtained by stepwise multiple regression. One model retained EFNB2 in combination with glutathione S-transferase alpha 3 and 4, progesterone receptor and glutathione peroxidase 3, resulting in 93% correct predictions when 3 patient and treatment cycle characteristics were included into the model. This large patient group allowed to do an intra-patient analysis for 7 patients, an analysis mimicking the methodology that would ultimately be used in clinical routine. CC related to a SET that did not give pregnancy and CC related to their subsequent frozen/thawed embryos which ended in pregnancy were analyzed. The models obtained in the between-patient analysis were used to rank the oocytes within-patients for their chance to pregnancy and resulted in 86% of correct predictions. In conclusion, prediction models built on selected quantified transcripts in CC might help in the decision making process which is currently only based on subjective embryo morphology scoring. The validity of our current models for routine application still need prospective assessment in a larger and more diverse patient population allowing intra-patient analysis.

A differential cytokine expression profile is induced by highly purified human menopausal gonadotropin and recombinant follicle-stimulating hormone in a pre- and postovulatory mouse follicle culture model

OBJECTIVE To compare the differential effects of highly purified (HP) hMG or recombinant FSH (rFSH) on cytokine expression before and after ovulation in an in vitro mouse ovarian follicle model. DESIGN A prospective laboratory in vitro study. SETTING A university-based reproductive biology laboratory. MATERIAL(S): Mechanically isolated mouse preantral follicles from 14-day-old prepubertal mouse ovaries (F1 hybrids: C57BL/6JxCBA/ca). INTERVENTION(S) Randomly distributed mouse early preantral follicles were exposed to two hyperstimulation conditions with either HP-hMG or rFSH. An ovulatory stimulus was given using hCG/epidermal growth factor. Conditioned media from the two culture conditions were collected on the days before and after in vitro ovulation. Conditioned media were compared for their relative cytokine profile content as measured by a cytokine antibody array analysis. MAIN OUTCOME MEASURE(S) Relative concentrations of 62 cytokines in conditioned media before and after ovulation. RESULT(S) Statistically significant increase in the production of a number of cytokines was found after HP-hMG stimulation compared with rFSH: 14 and 24 pre- and post-rhCG, respectively. Cytokines with the largest significant difference (more than 5 times) before and after ovulation included thymus-expressed cytokine (TECK), sTNFRI, and SDF-1alpha. The cytokines that are most strongly related to oocyte and embryo quality and implantation and that have been related to oocyte yield and maturation were significantly higher with HP-hMG. CONCLUSION(S) The significant differences in follicular cytokine production induced by HP-HMG and rFSH before and after in vitro ovulation might explain the difference in treatment outcome.

Gene expression differences induced by equimolar low doses of LH or hCG in combination with FSH in cultured mouse antral follicles

In a natural cycle, follicle growth is coordinated by FSH and LH. Follicle growth stimulation in Assisted Reproductive Technologies (ART) requires antral follicles to be exposed to both FSH and LH bioactivity, especially after GNRH analog pretreatment. The main aim was to detect possible differences in gene expression in granulosa cells after exposing the follicle during antral growth to LH or hCG, as LH and hCG are different molecules acting on the same receptor. Effects of five gonadotropin treatments were investigated for 16 genes using a mouse follicle culture model. Early (day 6) antral follicles were exposed to high recombinant FSH combined or not with equimolar concentrations of recombinant LH (rLH) or recombinant hCG (rhCG) and to highly purified human menopausal gonadotropin (HP-hMG) for 6 h, 12 h, or 3 days. Expression differences were tested for genes involved in steroidogenesis: Mvk, Lss, Cyp11a1, Hsd3b1, Cyp19a1, Nr4a1, and Timp1; final granulosa differentiation: Lhcgr, Oxtr, Pgr, Egfr, Hif1a, and Vegfa; and cytokines: Cxcl12, Cxcr4, and Sdc4. Lhcgr was present and upregulated by gonadotropins. Nr4a1, Cxcl12, and Cxcr4 showed a different expression pattern if LH bioactivity was added to high FSH in the first hours after exposure. However, no signs of premature luteinization were present even after a 3-day treatment as shown by Cyp19a1, Oxtr, Pgr, and Egfr and by estrogen and progesterone measurements. The downstream signaling by rhCG or rLH through the LHCGR was not different for this gene selection. Granulosa cells from follicles exposed to HP-hMG showed an enhanced expression level for several genes compared with recombinant gonadotropin exposure, possibly pointing to enhanced cellular activity.