Influence of follicle size on gene expression in cumulus cells of women stimulated for IVF.

Introduction: It is known from studies in animals and human that follicle
size is related to oocyte developmental competence. Recently gene
expression analysis in cumulus complexes (CC) demonstrated a variance
between the different cumulus oocyte complexes (COC) from each patient
and these within patient expression variances were related to oocyte
competence.
The variability in CC gene expression within a patient might thus be dependent
on follicle size. In this study, follicle size, measured as the follicle
fluid volume aspirated at oocyte retrieval, was related to CC gene expression
of 8 oocyte quality genes. The aim of the study was to set up a multiple regression
model, allowing for correction of between-patient variances, considering
patient-dependent and stimulation-dependent variables.
Material and Methods: From 17 ICSI patients (7 GnRH agonist/HP-menotropin
and 10 GnRH antagonist/recombinant FSH) follicular fluid volume
was recorded and related to 85 cumulus oocyte complexes (COC) enclosing
MII oocytes. CC were collected from all COCs and were handled individually
from the moment of pick-up on. Two groups of variables were recorded.
A first group comprised the patient related factors: age, BMI, ovarian hyper
stimulation protocol, ovarian responsiveness (number of COCs at pick up/daily
gonadotrophin doses) and serum levels of LH, FSH, estradiol and progesterone
on day of hCG. A second group, oocyte-specific variables, contained the
embryology data on day3: number of cells, fragmentation rate and an overall
embryo quality score (1 to 4).
The cumulus cells of 40 oocytes (from 4 agonist/HP-hMG and 4
antagonist/
rFSH stimulated patients) were analysed by real-time PCR for activated
leukocyte
cell adhesion molecule (ALCAM), prostaglandin-endoperoxide
synthase 2 (PTGS2), gremlin 1 (GREM1), versican (VCAN), syndecan
4 (SDC4), transient receptor potential cation channel, subfamily M, member
7 (TRPM7), calmodulin 2 (CALM2) and inositol 1,4,5-trisphosphate 3-kinase
A (ITPKA).
Of 45 additional oocytes (3 agonist HP-hMG and 6 antagonist rFSH stimulated
patients) CC were analysed for SDC4, TRPM7, CALM2 and ITPKA. All
were normalized to UBC and B2M expression.
A stepwise regression model selection (with 3 forward and 1 backwards
step) was applied to identify the three main variables related to gene expression
levels using either oocyte related parameters and a general patient nominator or
oocyte and patient-specific variables.
Results: A first screening with 8 genes (40 COCs) showed that CALM2,
TRPM7, ITPKA (p <0.05) and SDC4 (p > 0.05) retained the variable follicle
fluid volume as determining factor of the gene expression level in a model
including oocyte related parameters and a general patient factor correcting for
between patient variation. Therefore the experiment was extended with extra
patients for those genes (total 85 COCs).
For the 85 COC dataset, specific patient characteristics were also allowed
into the models and for the four genes follicle volume was one of the three
main parameters determining the differences in gene expression. For TRPM7,
CALM2 and SDC4 the follicle volume was the only oocyte-specific variable
retained
next to two patient related variables, whereas ITPKA variance depended
only on oocyte-specific variables.
The correlation between follicle volume and gene expression in CC was
inverse and strongest for ITPKA (p <0.05, model p <0.01).
Conclusions: Follicle size could, at least partially, explain the within patient
variance in expression in the cumulus complexes for four genes, but the correlations
were not very strong. We therefore hypothesize that within patient
expression variation might be more dependent on oocyte than on follicle size
characteristics.

Jaar van publicatie:2011

Trefwoorden:follicle size, cumulus cells