Tom Adriaenssens

Human Oocytes Reversibly Arrested in Prophase I by Phosphodiesterase Type 3 Inhibitor In Vitro

Abstract This study addresses the role of cAMP hydrolytic isoenzyme phosphodiesterase type 3 (PDE 3) modulation on human oocyte maturation in vitro. Presence of phosphodiesterase type 3 A (PDE 3A) mRNA was confirmed in human germinal vesicle-stage (GV) oocytes. Making use of a selective PDE 3 inhibitor, Org 9935 (10 μM), oocytes retrieved from immature follicles were arrested in prophase I with a high efficiency for up to 72 h. Cumulus oocyte complexes (COCs) were retrieved in the follicular phase of the cycle before or after exposure to endogenous LH or hCG administration in vivo and randomly distributed into maturation medium with or without the PDE 3 inhibitor. Previous exposure of small follicles to LH activity in vivo had no influence on the arresting capacity of the PDE 3 inhibitor. Reversal from pharmacological arrest leads to a progression through meiosis in a normal time frame with formation of a well-aligned metaphase plate. Ultrastructure analysis of COC derived from follicles between 8 and 12 mm showed that the induced extension of prophase I arrest in vitro resulted in cytoplasm changes but not in apparent nuclear changes during culture.

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.

A Reproducible Two-Step Culture System for Isolated Primary Mouse Ovarian Follicles as Single Functional Units

Abstract A reproducible two-step culture system for isolated mouse ovarian follicles smaller than 100 μm (type 3a follicles) was designed. First, isolated follicles were grown in single droplets of α-minimal essential medium (MEM) without (deoxy)ribonucleosides at a lower concentration of fetal bovine serum (FBS; 1%) for 6 days with mechanical prohibition of thecal cell attachment. Growing follicles reaching at least 100 μm were transferred to α-MEM medium enriched with a higher concentration (5%) of FBS to allow attachment and were cultured subsequently for an additional 12 days. Overall, more than 85% of the follicles survived the first culture step, and oocyte growth and granulosa cell proliferation had increased by 25% (P < 0.05). Follicle survival at Day 18 was related to initial follicle diameters at isolation. Average meiotic maturation rates and estrogen secretion were lower compared to those of cultures starting with early preantral follicles of 100–130 μm. Although reverse transcription-polymerase chain reaction analysis revealed the presence of LH-receptor mRNA in thecal cells, an exogenous androstenedione replacement resulted in an increase of estrogen production, suggesting substrate insufficiency. The time needed to grow from early preantral stages to in vitro ovulation is strongly dependent on the initial follicle diameter at isolation. Morphological characteristics of cultured follicles were suggestive for combined transforming growth factor β deficiencies during in vitro culture.

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.

Unaltered imprinting establishment of key imprinted genes in mouse oocytes after in vitro follicle culture under variable follicle-stimulating hormone exposure

Imprinted genes are differentially methylated during gametogenesis to allow parental-specific monoallelic expression of genes. During mouse oogenesis, DNA methylation at imprinted genes is established during the transition from primordial to antral follicle stages. Studies in human and mouse suggest aberrant imprinting in oocytes following in vitro maturation and after superovulation with high doses of gonadotrophines. The exact mechanisms leading to aberrant imprinting are unknown. We examined the methylation status of differentially methylated regions of key imprinted genes (by bisulphite sequencing) in mouse metaphase II oocytes, grown in a long-term pre-antral follicle culture system and matured in vitro, in the presence of a physiological (10 IU/L) and a high (100 IU/L) recombinant FSH dose. Our results showed a normal DNA methylation at the studied regulatory sequences of Snrpn, Igf2r and H19, demonstrating that 1) prolonged culture and in vitro maturation do not per se modify the establishment of imprinting in oocytes and 2) supraphysiological FSH doses do not induce aberrant DNA methylation at the studied regulatory sequences in this system.

Effects of Low Methyl Donor Levels in Culture Medium During Mouse Follicle Culture on Oocyte Imprinting Establishment

Imprinted genes are differentially methylated during gametogenesis to allow parent-of-origin-specific monoallelic expression. We previously demonstrated establishment of normal imprinting at four key imprinted genes in mouse metaphase II oocytes after in vitro follicle culture. Commercially available culture media feature a wide range of methyl donor levels. The aim of the present study was to examine the effect of low methyl donor (methionine, vitamin B12, folic acid, choline, and vitamin B6) levels during follicle culture on acquisition of DNA methylation at imprinted genes in mouse oocytes. Follicle culture performed under low methyl donor levels led to decreased antral follicle development (mean [SD] antral follicle rate, 87.5% [12.6%] vs. 97.7% [4.3%] in control conditions; P < 0.05) and to a dramatic decrease in polar body (PB) oocyte rate (mean [SD] PB oocyte rate, 38.7% [25.5%] vs. 96.1% [7.1%]; P < 0.001). The methylation status of differentially methylated regions (DMRs) of four key imprinted genes was studied (by bisulphite sequencing) in normal-looking PB and germinal vesicle breakdown-arrested oocytes obtained from follicle culture under low methyl donor levels. DMRs of Snrpn, Igf2r, and H19 showed no alteration in DNA methylation, but at Mest DMR in PB oocytes, we found a significant reduction in DNA methylation compared to that in control follicle culture (DNA methylation, 89.9% and 98.2%, respectively; P = 0.0014). In conclusion, restriction of methyl donors during follicle culture led to a dramatic decrease in PB oocyte rate but induced no or only minor DNA methylation alterations at the studied regulatory sequences of key imprinted genes in oocytes.

Quantification of oocyte-specific transcripts in follicle-enclosed oocytes during antral development and maturation in vitro

Oocyte cytoplasmic maturation is influenced by the quantity of synthesized RNA and proteins accumulated and stored during growth. Transcriptional repression and degradation of transcripts occur during oocyte nuclear maturation, and prolonged transcriptional arrest might compromise RNA stores for early development. RNA quantification of key genes in oocytes might be valuable when setting up in vitro cultures that lack the normal hormonal interplay found in vivo. This study quantifies gene expression levels in relation to follicle culture time and time of oocyte maturation in a mouse model. RNA levels of Gdf-9, Bmp-15, Mater, Zar-1, Npm-2 and Fgf-8 were measured in germinal vesicle oocytes along fixed times during in vitro follicle development. For all genes, the highest mRNA levels were detected in oocytes in the pre-antral follicle stage. Antrum formation was associated with a progressive shutdown in transcription leading to mRNA values lower than those in vivo preovulatory oocytes by extending period of in vitro culture. In contrast to in vitro-matured oocytes, the in vivo oocytes from 22- and 29-day-old prepubertal animals obtained after pregnant mares serum gonadotrophin and human chorionic gonadotrophin priming did not down-regulate transcripts upon maturation stimulus except for Mater. These findings show that oocyte gene expression patterns under in vitro conditions can, at certain times, mimic what is reported to occur under in vivo conditions. Moreover, they also show that meiotically competent oocytes kept in a prolonged transcriptionally inactive stage express altered levels of key transcripts compared with in vivo in both immature and mature oocytes.

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.

Different Follicle-Stimulating Hormone Exposure Regimens During Antral Follicle Growth Alter Gene Expression in the Cumulus-Oocyte Complex in Mice

Follicle-stimulating hormone (FSH) and oocyte-secreted factors influence granulosa cell differentiation and follicle development. Whereas FSH stimulates the expression of mural cell transcripts, oocyte-secreted factors regulate specific cumulus cell genes and suppress the appearance of mural cell transcripts. This study addresses the extent to which clinically relevant changes in FSH doses applied during antral follicle development in vitro could alter the expression of oocyte and cumulus cell transcripts. A 12-day culture system in which mouse ovarian preantral follicles can grow to preovulatory follicles was used. The following three FSH regimens were considered: 1) continuous exposure to an FSH level of 10 mIU/ml (control), 2) decreasing concentrations of FSH (low FSH), and 3) an FSH level of 25 mIU/ml (high FSH) as soon as the antrum is formed. Transcripts in oocytes (Gdf9, Bmp15, and Fgf8) and in cumulus cells (Amh, Lhcgr, Ar, and Pfkp) were quantified by real-time PCR. Under high FSH, the three oocyte transcripts were upregulated, while in cumulus cells a shutdown of the Amh signal and substantial increases in Lhcgr and Ar expression were measured. In contrast, low FSH tended to reduce Lhcgr to levels comparable to those in vivo. Levels of Pfkp were not affected by FSH doses. These results demonstrate that a 2.5-fold increase in FSH changes both oocyte and cumulus cell transcript levels. Conversely, a decrease in FSH does not affect transcript levels but seems to limit inappropriate Lhcgr expression. Modulating FSH within physiological ranges during the antral phase of culture alters cumulus cell differentiation.

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.