SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 246A

246A

Reproductive Sciences Vol. 25, Supplement 1, March 2018

Objective: To establish a quantitative method to measure the levels of
H3K9me3 relative to histone H3 in human sperm cells using Western
blot analysis and to assess if variations in these levels exist.
METHODS: After routine work-up using density centrifugation, surplus
sperm samples were collected from 15 patients with normospermia
undergoing IVF treatment. Purified sperm fractions were lysed at a
concentration of 200x106/ml in RIPA buffer with 5 mM DTT, followed
by sonication. Sperm protein extracts were separated on a 4-12% SDSPage gel and for 12 samples, both Histone H3 and H3K9me3 levels could
be detected by Western Blot analysis. Quantification was performed
using ImageJ and the H3K9me3/H3 ratio was calculated for these sperm
samples. For one patient the pregnancy outcome was missing.
RESULTS: The H3K9me3/H3 ratio in our study population varied
between 0,02-0,16. The range in sperm samples from treatments resulting
in pregnancy was more narrow (range 0,048-0,081; n=3), than in the nonpregnant group (range 0,022-0,162; n=8) (Figure 1).
*Figure(s) will be available online.
CONCLUSION: Using Western blot analysis, we were able to determine
H3K9me3 levels relative to histone H3 in human spermatozoa. We found
the H3K9me3/H3 ratio to vary as much as 8-fold between these IVF
patients. This variation may impact on cHC formation on the paternal
chromosomes, with potential consequences for genomic stability in the
embryo. More data are needed to confirm these findings.

F-197
TNFa and MFG-E8: Novel Biomarkers to Predict Implantation
Failure. Liang Yu,1 Tamar Alkon,2 Sergio Oehninger,1 Silvina Bocca*.1
1
Eastern Virginia Medical School, Norfolk, VA, United States; 2Institute
Mexicano de Alta Tecnologia Reprod, Mexico City, Mexico.
INTRODUCTION: Endometrial regulators have been recognized as
crucial for normal implantation. Among them, a novel gene, MFG-E8,
has been discovered by us with upregulation around the window
of implantation. We hypothesize that TNFα (released by invading
trophoblast to induce a physiological inflammatory response) and MFG-E8
cooperatively maintain the integrity of the normal endometrium, and that
in patients with implantation failure (IF) or with unexplained recurrent
pregnancy loss (RPL), excessive TNFα increases the maternal shedding
of MFG-E8, disrupting the normal protective effect of this protein.
METHODS: This is a prospective controlled pilot clinical study aiming
to quantify and correlate MFG-E8 and TNFα in serum and in endometrial
biopsies of women ages 21-35y in 3 groups: 10 fertile controls (C, egg
donors), 2 IF (unexplained IF following 2 or more IVF cycles) and 2
RPL (at least 2 unexplained first trimester miscarriages). IRB approval
was obtained and participants signed informed consent. Each participant
had 2 blood drawings at menstrual cycle days 2-3 (proliferative
phase) and around the window of implantation (urinary LH+7-8 days,
secretory phase). In both cases, serum MFG-E8, TNFα, estradiol (E) and
progesterone (P) were quantitated. An endometrial biopsy was obtained
on urinary LH+7-8 days and protein and mRNA expression of MFG-E8
and TNFα were assessed by Western and real-time PCR.
RESULTS: Serum E levels in the C group had a significant increase
in the secretory phase (p<0.05) that was not seen for the IF or the RPL
groups. Serum P levels were significantly higher in the secretory phase
in C and IF (both p<0.05), but not in RPL. C subjects had significantly
higher serum MFG-E8 levels than IF and RPL subjects (p<0.05). Mean
serum TNFα levels were significantly higher in the proliferative phase for
the IF and RPL groups compared to the C subjects (both p<0.05; Fig. 1).
The endometrial relative gene and protein expression of both MFG-E8
and TNFα did not differ amongst groups.
CONCLUSION: Our preliminary data shows that MFG-E8 and TNFα
serum levels may discriminate fertile from poor obstetrical outcome
patients which could provide basis for a simple and quick test to measure
serum markers in order to prospectively identify these groups of patients.
*Figure(s) will be available online.

Scientific Abstracts

F-198
Med12 is a Maternal Effect Gene That Doesn't Affect Ovarian
Folliculogenesis. Jyoti Goad†,2 Xiang Yang†,2 Priya Mittal,1 Carlos A
Castro,2 Gabriel Rajkovic,2 Aleksandar Rajkovic*.2 1St Jude Children's
Research Hospital, Memphis, TN, United States; 2University of Pittsburgh,
Pittsburgh, PA, United States.
INTRODUCTION: Mediator complex subunit 12 (MED12) is located
on the X chromosome and is involved in transcriptional regulation of the
RNA polymerase II complex. Recent studies from our lab and others have
shown that gain of function mutation in Med12 is associated with uterine
leiomyomas and that Med12 deficiency causes granulosa cell dysfunction.
However, the role of Med12 in the germline has not been explored to date.
Objective: To identify the physiological role of Med12 in mammalian
oocytes
METHODS: We utilized two well-established cre mouse models Gdf9Cre and Zp3-cre to generate oocyte-specific Med12 knockouts mice and
determine their effect on oogenesis and folliculogenesis.
RESULTS: We used oocyte-specific, Gdf9-Cre and Zp3-Cre transgenic
mice, to ablate Med12 from primordial and primary follicles, respectively.
Gdf9-Cre males crossed with Med12fl/fl females did not produce pups
that carried Gdf9-Cre. Leaky, paternal Gdf9-Cre expression is known
to occur, and the sperm carrying Gdf9-Cre transgene probably leads to
efficient recombination of Med12 floxed sites in the fertilized egg with
subsequent embryo death. The lack of Med12fl/+ Gdf9-Cre pups argues
for the importance of early embryonic bi-allelic Med12 expression for
successful embryogenesis to occur. We also utilized Zp3-Cre, which is
not expressed in the male germline. We successfully generated Med12fl/fl
Zp3-Cre female pups by mating Med12fl/y Zp3-cre males with Med12fl/
fl females. Med12fl/fl Zp3-Cre females were infertile when mated with
stud males. The ovarian histology of Med12fl/fl Zp3-Cre females showed
normal folliculogenesis and the presence of normal corpora lutea,
indicating successful ovulation. These results indicate that postnatal
Med12 expression in the oocyte is not important for oogenesis, but is
essential for early embryo development. Med12 is therefore a maternal
effect gene.
CONCLUSION: Collectively our data identifies Med12 as a maternal
effect gene. Med12 is one of the few maternal effect genes expressed
from the X chromosome.

F-199
Dynamic Changes in Gene Regulation in Human Endometrium.
Jenny N Fung,2 Sally Mortlock,2 Jane E Girling,1 Sarah J HoldsworthCarson,1 Wan Tinn Teh,1 Martin Healey,1 Peter AW Rogers,1 Grant W
Montgomery*.2 1The University of Melbourne, Melbourne, Australia;
2
The University of Queensland, Brisbane, Australia.
INTRODUCTION: Human endometrium is a dynamic tissue essential
for establishment and maintenance of pregnancy. Gene expression varies
markedly across the menstrual cycle and expression levels for many genes
are under genetic control. To better understand mechanisms underlying
gene regulation in the endometrium, we analysed gene expression and
mapped expression quantitative trait loci (eQTLs) in endometrial tissue
from 229 women with natural menstrual cycles.
METHODS: Whole-transcriptome profiles were characterized using
Illumina HT-12 v4.0 Expression Chips. DNA samples from blood from
the same individuals were genotyped on Illumina HumanCoreExome
chips. We performed eQTL mapping with ~5,000,000 genotyped and
imputed SNPs and the 15,262 probes. SNPs influencing gene expression
and located within a 250kb window of the associated probe were defined
as cis-acting eQTLs.
RESULTS: Dynamic changes in expression of individual genes across
the cycle include alterations in both mean expression and transcriptional
silencing. Significant effects of cycle stage on mean expression levels
were observed for 2,427 of the 15,262 probes with detectable expression
in at least 90% of samples. In addition, 9,626 probes were expressed
in some (range 1-90%), but not all samples. Novel analysis identified
that transcriptional silencing of many of these genes (2,697/9,626)
is significantly influenced by cycle stage. Pathway analysis supports
similar biological control of altered expression levels and transcriptional

Table of Contents for the Digital Edition of SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018