الفهرس 
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Abstract
A crucial microenvironmental component for embryonic development is hypoxia. A key transcription factor called hypoxia inducible factor (HIF) reacts to low oxygen tension by causing the production of hypoxia-related genes like VEGF and erythropoietin. HIF regulates a variety of physiological processes, including metabolism, cell death, and angiogenesis, through the activation of these genes.
The Hypothesis of this study is to make sure that although that the hypoxia has a great role for successful implantation during pregnancy but when it will be excessive induced it has a negative impact on the receptivity and implantation causing implantation failure for the pregnant female and it causes many defects in the structure of the uterus for non-pregnant female.
Experimental design
Eight to fourteen-week-old C57Bl/6N mice were kept in a controlled environment at 22 ± 2°C with a 12-hour light/dark cycle for a week to acclimatize them to the environment. The mice were given access to food and water. Then the experiment is divided to two parts:
Part 1. Non-pregnant females were weighed; animals were divided into two groups (n=6 per group); the control group received normoxic mice with atmospheric oxygen levels at 21%; the hypoxic group received 10.5% hypoxia for 72 hours; and the mice were sacrificed after being exposed to the hypoxic environment for 72 hours.
Part 2. Overnight, males (aged 3 months) were paired singly with females. Weight the animals, and then divide them into the two groups (n=6 per group) as follows: control (one at atmospheric oxygen 21%) and hypoxic group (10.5% hypoxia for 72h), on the morning of the presence of a vaginal plug (ULVAC DA-60S, Japan). The implantation process was then examined after the sacrifice of pregnant mice at E3.5 and E4.5.
This research was carried out between October 2021 and August 2022 at Nagoya City University’s Graduate School of Medical Sciences’ Comparative and Experimental Medicine (DCEM) department in Japan.
The present study aimed to:
Investigate the effects of external hypoxia on female mice that were both pregnant and not pregnant, with a focus on how it impacts the overexpression of the HIF gene and how it related to the following factors:
1. In non-pregnant female mice, the various endometrial structures (epi, stroma, and myometrium).
2. The pregnant female’s implantation window and the early stages of blastocyst development.
3. Some receptivity and implantation genes are expressed (Ihh, Wn4, Spink3, LIF, Hand2, Hoxa10, and Hoxa11).
4. Progesterone levels in the first trimester of pregnancy.
The methods we employed to accomplish these goals is listed below:
Blood samples is collected from Normoxic and Hypoxic animals for estimation of progesterone level using ELISA.
Animals are sacrificed at E3.5 and E4.5 firstly flushing uterus for collection of blastocysts then collect one ovary and uterine horn RNA extraction, and others are fixed in 4% PFA for histological and immunohistochemical analysis. Then the following steps were done:
1. Histological analysis (H&E). The tissues are cutted by microtome about 0.5μm then deparaffinization using different xylol and ethanol then stained with Hematoxyline and Eosin, the images were taken using Nanozoomer.
2. Immunohistochemistry (IHC). By using different antibodies as Ki67 for uterine epithelial cell, Caspase3 for apoptosis in ovarian tissues
3. Molecular analysis. RNA isolation from uterine and ovarian tissues, Total RNA was reversely transcribed into first-strand cDNA for use in real-time PCR analysis and Estimate expression of genes of interest using quantitative PCR as.
A. Expression of HIF2 and Vegfa.
B. Expression of some receptivity and implantation genes (Ihh, Wn4, Spink3, LIF, Hand2, Hoxa10, and Hoxa11).
C. Expression of ovarian gene responsible for progesterone production (HSD-3β).