Name: methods for studying uterine contributions to pregnancy establishment in an ovariectomized mouse model
Uploaded: 2023-04-07T12:50:00
Description: Watch this Scientific Journal Video about Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model at JoVE.com

This work was made possible through the Victorian State Government Operational Infrastructure Support and Australian Government National Health and Medical Research Council (NHMRC) IRIISS. This work was supported by the Monash University Faculty of Medicine, Nursing and Health Science Platform Access Grant to A.L.W. (Winship-PAG18-0343) to access the Monash Reproductive Services Platform. A.L.W. is supported by DECRA funding DE21010037 from the Australian Research Council (ARC). J.N.H. and L.R.A. are supported by an Australian Government Research Training Program Scholarship. L.R.A. is supported by a Monash Graduate Excellence Scholarship. K.J.H. is supported by an ARC Future Fellowship FT190100265.

All animals were housed in temperature-controlled, high-barrier facilities (Monash University Animal Research Laboratory) with free food and water access and a 12 h light-dark cycle. All the procedures were performed in accordance with approval from the Monash Animal Research Platform Ethics committee (#21908, 17971) and performed in accordance with the National Health and Medical Research Council Code of Practice for the care and use of animals.
1. Surgical preparation
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This article provides step-by-step instructions on how to perform OVX and provide exogenous hormones for studies focused on understanding the uterine contributions to pregnancy and fertility. Two detailed protocols are provided on two experimental applications of these methods, including performing embryo transfer and inducing decidualization artificially.
Whilst performing OVX can be challenging initially - especially for researchers new to rodent models - it is a relatively simple procedure ...

With the rising use of assisted reproductive technologies in recent decades, many barriers to conception have been overcome, allowing many couples to start families despite fertility problems1. Oocyte or sperm deficits can often be bypassed using in vitro fertilization or intracytoplasmic sperm injection; however, issues related to the uterus and endometrial receptivity remain an elusive &#34;black box&#34; of reproductive potential2.
Pregnancy is established when a high-quality embryo successfully interacts with a receptive endometrium (uterine lining). The chances of successful pregnancy in any given menstrual cycle are low, at around 30%3,4. Of those that are successful, only 50%-60% advance past 20 weeks of gestation, with implantation failure being responsible for 75% of pregnancies that do not reach 20 weeks3. Despite these figures dating back to the late 1990s, the field is yet to overcome the limitations caused by an inadequately receptive endometrium. This has resulted in stagnating - and sometimes declining - IVF success rates in recent years5,6.
Women with unexplained infertility often have a displaced window of receptivity or are unable to achieve receptivity for unknown reasons. Recently, the endometrial receptivity array was developed, which assesses the expression of hundreds of genes with the purpose of tailoring the timing of embryo transfer to an individual&#39;s window of receptivity7,8,9. However, the field still lacks an understanding of the pathogenesis of pregnancy complications that manifest after the implantation process is complete.
The female reproductive system is highly dynamic and under tight hormonal control. The hypothalamic-pituitary-gonadal (HPG) axis controls the release of luteinizing hormone and follicle-stimulating hormone, which regulate aspects of the ovarian cycle, including follicle maturation and estrogen and progesterone activity. In turn, the uterine menstrual cycle is regulated by estrogens and progesterone10,11. Thus, studying uterine biological mechanisms is complicated by ovarian influence. For example, when studying how cancer therapies may impact the uterus, it can be difficult to distinguish if any uterine phenotype observed (such as pregnancy loss or menstrual acyclicity) is the result of a direct insult to the uterus or a consequential effect from damage to the ovaries.
To comprehensively understand fertility, the uterine contributions to pregnancy must be characterized. Importantly, this understanding must extend beyond uterine function under ovarian control. This cannot be studied in humans; therefore, animal models are often employed. As such, ovariectomy (OVX) is commonly used to enable researchers to regulate rodent estrous cycles (analogous to the menstrual cycle) by supplying hormones exogenously. Additionally, OVX allows uterine responses to be studied independently of ovarian influence12. However, if hormones are not immediately supplied post-OVX, a menopause phenotype will eventuate, which needs to be carefully considered by the researchers.
OVX is frequently utilized in rodent models13,14,15,16,17 and is relatively easy to perform after adequate training. Methods vary depending on whether the ovary alone or the ovary and oviduct are removed, as well as depending on the age of the animal (adult, cycling animals have larger ovaries with a visible corpus luteum on their surface, meaning their ovaries are easier to visualize). Similarly, many methods of hormone supplementation exist, including subcutaneous injections14, slow-release pellets15, osmotic mini pumps18, and ovarian grafting.
In this article, detailed instructions are provided on how to perform ovariectomy and prepare three types of hormone supplementation, including subcutaneous injections, slow-release pellets, and osmotic mini pumps. Two detailed protocols are provided for experimental endpoints that benefit from OVX followed by exogenous hormone supplementation (embryo transfer and artificial decidualization). This article discusses the strengths and weaknesses of each approach with the goal of guiding researchers regarding how to perform studies to isolate the impacts on the uterus, specifically in the pregnancy and fertility fields of research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ALZET 1002 mini osmotic pumps</td>
			<td>BioScientifica</td>
			<td>1002</td>
			<td>Delivers 0.25 &#181;L/h for 14 days. Use for section 7 (Experimental procedure - Embryo transfer).</td>
		</tr>
		<tr>
			<td>ALZET 1003D mini osmotic pumps</td>
			<td>BioScientifica</td>
			<td>1003D</td>
			<td>Delivers 1 &#181;L/h for 14 days. Use for section 8 (Experimental procedure - Artificial decidualization).</td>
		</tr>
		<tr>
			<td>ALZET Reflex 7 mm clips</td>
			<td>BioScientifica</td>
			<td>0009971</td>
			<td>Either Reflex clips or Michel clips can be used for wound closure, depending on preference</td>
		</tr>
		<tr>
			<td>ALZET Reflex clip applicator</td>
			<td>BioScientifica</td>
			<td>0009974</td>
			<td>Either Reflex clips or Michel clips can be used for wound closure, depending on preference</td>
		</tr>
		<tr>
			<td>ALZET Reflex clip remover</td>
			<td>BioScientifica</td>
			<td>0009976</td>
			<td>Either Reflex clips or Michel clips can be used for wound closure, depending on preference</td>
		</tr>
		<tr>
			<td>Bupivicaine injection</td>
			<td>Pfizer</td>
			<td>NA</td>
			<td>Stock 0.5%. Use at 0.05% in saline</td>
		</tr>
		<tr>
			<td>Estradiol</td>
			<td>Sigma</td>
			<td>E8875</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Ilium</td>
			<td>NA</td>
			<td>Active constituent 0.5 mg/mL. Use 3.5 mL per 200 mL cage bottle, or as your institutions vet prescribes.</td>
		</tr>
		<tr>
			<td>Michel clips</td>
			<td>Daniels</td>
			<td>NS-000242</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi purpose sealant</td>
			<td>Dow Corning</td>
			<td>732</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-surgical embryo transfer (NSET) device</td>
			<td>ParaTechs</td>
			<td>60010</td>
			<td>Contains 6 mm speculum. Single use only.</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P0130</td>
			<td>Soluble in ethanol. Use for&#160; section 3 (Hormone preparation - subcutaneous injection) and&#160; section 4 (Hormone preparation - slow-release pellets)</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P7556</td>
			<td>Soluble in water. Use for section 5 (Hormone preparation - osmotic mini pumps)</td>
		</tr>
		<tr>
			<td>Refresh eye ointment</td>
			<td>Allergan</td>
			<td>NA</td>
			<td>42.5% w/v liquid paraffin, 57.3% w/v soft white paraffin</td>
		</tr>
		<tr>
			<td>Rimadyl Carprofen</td>
			<td>Zoetis</td>
			<td>NA</td>
			<td>Stock 50 mg/mL. Use at 5 mg/kg</td>
		</tr>
		<tr>
			<td>Rubber tubing</td>
			<td>Dow Corning</td>
			<td>508-008</td>
			<td>Washed in 100% ethanol and cut into 1 cm pieces. Inside diameter 1.57 mm &#177;&#160; 0.23 mm; outside diamater 3.18 mm &#177; 0.23 mm; wall 0.81 mm.</td>
		</tr>
		<tr>
			<td>Sesame oil</td>
			<td>Sigma</td>
			<td>S3547</td>
			<td></td>
		</tr>
		<tr>
			<td>Sofsilk Silk sutures size 3-0</td>
			<td>Covidien</td>
			<td>GS-832</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for studying uterine contributions to pregnancy establishment in an ovariectomized mouse model

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation and placenta formation and enable ongoing pregnancy. The limitations to pregnancy success caused by embryonic defects are well known and have been largely overcome in recent decades with the rise of in vitro fertilization (IVF) and assisted reproductive technologies. As yet, however, the field has not overcome the limitations caused by an inadequately receptive endometrium, thus resulting in stagnating IVF success rates. Ovarian and endometrial functions are closely intertwined, as hormones produced by the ovary are responsible for the endometrium's menstrual cyclicity. As such, when using rodent models of pregnancy, it can be difficult to ascertain whether an observed result is due to an ovarian or uterine deficit. To overcome this, an ovariectomized mouse model was developed with embryo transfer or artificial decidualization to allow the study of uterine-specific contributions to pregnancy. This article will provide instructions on how to perform ovariectomy and offer insights into various techniques for supplying exogenous hormones to support successful artificial decidualization or pregnancy following embryo transfer from healthy donors. These techniques include subcutaneous injection, slow-release pellets, and osmotic mini pumps. The key advantages and disadvantages of each method will be discussed, enabling researchers to choose the best study design for their specific research question.

A well-characterized model of artificial decidualization is described in this protocol paper (Figure 1A). Here, young adult female mice (8 weeks old) underwent surgical ovariectomy as described in section 1 and section 2. The mice were then rested for 2 weeks to ensure that the endogenous ovarian hormones dissipated before being supported with exogenous hormones as described in sections 3-7 and section 9. Artificial decidualization was induced by an intravaginal injecti...

Watch this Scientific Journal Video about Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model at JoVE.com

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

Pregnancy establishment is a dynamic process involving complex embryo and uterine crosstalk. The precise contributions of the maternal uterine environment to these processes remain an active area of investigation. Here, detailed protocols are provided to aid in designing in vivo animal models to address these research questions.

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation ...

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