O ρόλος του CRH στην in vitro ανάπτυξη αρχέγονων ωοθυλακίων και η δράση αυτού στην εμφύτευση

Doctoral Dissertation uoadl:1308552 169 Read counter

Unit:
Τομέας Υγείας - Μητέρας - Παιδιού
Library of the School of Health Sciences
Deposit date:
2015-05-25
Year:
2015
Author:
Ντινοπούλου Βασιλική
Dissertation committee:
Καθηγητής Δημήτριος Λουτράδης
Original Title:
O ρόλος του CRH στην in vitro ανάπτυξη αρχέγονων ωοθυλακίων και η δράση αυτού στην εμφύτευση
Languages:
Greek
Summary:
Introduction
The hormone CRH is a neuropeptide which is the main regulator of HPA
axis. It is detectable in most female reproductive tissues and interacts with
two receptors, the CRH-R1 and the CRH-R2. Τhe purpose of this study was to
investigate the effect of hormone CRH and the inhibitor, antalarmin, in the in
vitro maturation of preantral mouse follicles in early embryonic development
but also in steroidogenesis, in producing glycoprotein hormones and a peptide
hormone. We additionally performed Real-Time PCR to study the expression of
genes encoding the CRH-R1 and CRH-R2 in preantral mouse follicles and stage
mouse embryos 2-, 4- and 8-cells and mix of morulae-blastocysts. In this study
we also chose to evaluate the effect of CRH in the implantation, in association
with the expression of CRH, CRH-R1, CRH-R2 and integrins in women participating
in the IVF program, and had more than two attempts of implantation failure.

Materials and Methods
Female mice F1 12-14 days were sacrificed and their follicles were
mechanically isolated and cultured for 11 days with or without the addition of
CRH at a concentration of 10-7 mol/L and CRH 10-7 mol/L and antalarmin 10-6
mol/ L. Every 48 hours the culture medium was renewed and was kept in the
freezer for later measurement of estradiol, progesterone, testosterone, b-hCG,
FSH, LH and prolactin by electrochemiluminescence immunoassay. After 10 days of
culture we added hCG and EGF for ovulation. On day 11 we estimated the COCs
and transferred them after we sacrificed F1 male mice for in vitro
fertilization. Embryos were cultured for 5 days in Ham's F10/BSA at a ratio of
9/1 in order to assess early fetal development. Finally, total RNA was isolated
from the preantral follicles in culture days 3,5,7,9 and 11 as well as
embryonic stages 2, 4- and 8-cells and mix morulae-blastocysts in order to do
Real-Time RT-PCR to study the expression of the CRH-R1 and CRH-R2 receptors.
The clinical experimental protocol consisted of three stages to match
three menstrual cycles of seven women who participated in this clinical study.
The first menstrual cycle was natural cycle and was performed as the control
cycle for each woman and two biopsies were taken, five and eight days after
hCG. The second menstrual cycle was also natural but at this cycle we isolated
the PBMCs, we treated them with CRH and hCG, we infused them intrauterine and
finally endometrial biopsies were taken again. Then we isolated genetic
material and by Real Time PCR we studied the expression of CRH, CRH-R1, CRH-R2
and integrins αvβ3 and αvβ5 before and after injection with CRH. In the third
menstrual cycle the patients were taking the appropriate medications to
stimulate ovulation. Simultaneously we isolated the PBMCs, we treated them with
CRH and hCG, we infused them intrauterine with the difference that in this
cycle we finally did embryo transfer to evaluate pregnancy rates.

Results
We cultured 732 follicles in the control group, 1306 follicles in group
CRH 10-7 and 1202 follicles in group CRH 10-7/antalarmin 10-6. The maturation
rates for the control group were 33.6%, while for the group CRH 10-7 26.18%,
showing a statistically significant reduction (p<0.001). Also there was a
statistically significant difference between the control group and the group
CRH 10-7 at all stages of development of preimplantation embryos and between
the CRH 10-7 group and CRH 10-7/antalarmin 10-6 group. By hormonal assessment
it was found that CRH group had significantly lower levels of estradiol,
progesterone, testosterone and beta-hCG as days culture increased in comparison
with the other two groups which gave statistically significant differences. The
Real-Time RT-PCR showed that receptors CRH-R1,-R2 were detected at all stages
of preantral follicles while regarding the preimplantation embryos, only at the
mix morulae-blastocysts was detected the CRH-R1.
The clinical protocol was compared the expression of CRH, CRH-R1,
CRH-R2 and integrins αvβ3 and αvβ5 in the endometrium before and after infusion
with CRH, 5 days after hCG and 8 days after hCG. There was a tendency of
reduction of CRH expression which was not statistically significant, while the
reduction of the receptors CRH-R1, R2 was statistically significant. For
integrins there was a trend of increased expression without being statistically
significant. Also there was no statistical significance in women's pregnancy
rates (3/7 were pregnant, 42,9%, p> 0,05 MC Nemar test) when compared with the
non-existence of pregnancy in previous cycles before injection with CRH.

Conclusions
According to this study, CRH had a negative effect on follicular growth
and maturation, as also it was found a statistically significant difference in
the development of preimplantation embryos. The addition of specific CRH-R1
antagonist, antalarmin in in vitro cultures with CRH yielded higher survival
rates in all embryonic stages, suggesting that antalarmin reverses the
inhibitory effect of CRH. We have shown that the presence of CRH in a in vitro
culture of preantral mouse follicles suppressed stereoeidogenesi while the
CRH-R1 receptor was detected in all stages of culture of preantral follicles as
in embryonic stages of morulae and blastocysts. Our results demonstrated the
negative effect of CRH in the rates of early embryonic development in vitro
fertilized oocytes. Since CRH is the principal hormone "stress", we could use
this knowledge to optimize the quality of oocytes and early embryos during IVF
techniques, controlling the levels of stress of the patients.
Furthermore, according to this preliminary clinical pilot study there
was a trend of increase of the expression of integrins αvβ3 and αvβ5 and a
trend of decrease in CRH expression in the endometrium before and after
infusion with CRH. But there was a statistically significant decrease in
expression of the CRH-R1 receptors and CRH-R2. Further trials should be
performed in order to increase the number of women and prove the effectiveness
of treatment with CRH in women with repeated implantation failure.
Keywords:
CRH, Antalarmin, Preantal follicles, Ivg, Implantation
Index:
No
Number of index pages:
0
Contains images:
Yes
Number of references:
457
Number of pages:
285

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