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.

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worldwide+decline+of+IVF+birth+rates+and+its+probable+causes.">Worldwide decline of IVF birth rates and its probable causes.</a> Human Reproduction Open. 2019 (3), (2019).</li><li>Diaz-Gimeno, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomic+diagnostic+tool+for+human+endometrial+receptivity+based+on+the+transcriptomic+signature.">A genomic diagnostic tool for human endometrial receptivity based on the transcriptomic signature.</a> Fertility &amp; Sterility. 95 (1), 50-60 (2011).</li><li>Amin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalized+embryo+transfer+outcomes+in+recurrent+implantation+failure+patients+following+endometrial+receptivity+array+with+pre-implantation+genetic+testing.">Personalized embryo transfer outcomes in recurrent implantation failure patients following endometrial receptivity array with pre-implantation genetic testing.</a> Cureus. 14 (6), e26248 (2022).</li><li>Patel, J. A., Patel, A. J., Banker, J. M., Shah, S. I., Banker, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalized+embryo+transfer+helps+in+improving+in+vitro+fertilization/ICSI+outcomes+in+patients+with+recurrent+implantation+failure.">Personalized embryo transfer helps in improving in vitro fertilization/ICSI outcomes in patients with recurrent implantation failure.</a> Journal of Human Reproductive Sciences. 12 (1), 59-66 (2019).</li><li>Khan, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+differences+between+functionalis+and+basalis+endometria+in+women+with+and+without+adenomyosis.">Biological differences between functionalis and basalis endometria in women with and without adenomyosis.</a> European Journal of Obstetrics, Gynecology, and Reproductive Biology. 203, 49-55 (2016).</li><li>Richards, J. S., Ren, Y. A., Candelaria, N., Adams, J. E., Rajkovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+follicular+theca+cell+recruitment,+differentiation,+and+impact+on+fertility:+2017+update.">Ovarian follicular theca cell recruitment, differentiation, and impact on fertility: 2017 update.</a> Endocrine Reviews. 39 (1), 1-20 (2018).</li><li>Corciulo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+administration+for+physiological+estrogen+replacement+in+mice.">Pulsed administration for physiological estrogen replacement in mice.</a> F1000Research. 10, 809 (2021).</li><li>Greaves, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mouse+model+of+endometriosis+mimics+human+phenotype+and+reveals+insights+into+the+inflammatory+contribution+of+shed+endometrium.">A novel mouse model of endometriosis mimics human phenotype and reveals insights into the inflammatory contribution of shed endometrium.</a> The American Journal of Pathology. 184 (7), 1930-1939 (2014).</li><li>Griffiths, M. J., Alesi, L. R., Winship, A. L., Hutt, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+embryo+transfer+model+to+study+uterine+contributions+to+pregnancy+in+vivo+in+mice.">Development of an embryo transfer model to study uterine contributions to pregnancy in vivo in mice.</a> Reproduction &amp; Fertility. 3 (1), 10-18 (2022).</li><li>Cousins, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+from+a+mouse+model+that+epithelial+cell+migration+and+mesenchymal-epithelial+transition+contribute+to+rapid+restoration+of+uterine+tissue+integrity+during+menstruation.">Evidence from a mouse model that epithelial cell migration and mesenchymal-epithelial transition contribute to rapid restoration of uterine tissue integrity during menstruation.</a> PLoS One. 9 (1), e86378 (2014).</li><li>Cousins, F. 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R., Reyes, E., Chukwuocha, R., Tokuda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticosteroid+and+immune+responses+of+mice+following+mini-osmotic+pump+implantation.">Corticosteroid and immune responses of mice following mini-osmotic pump implantation.</a> Immunopharmacology. 20 (3), 187-190 (1990).</li><li>Barton, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+steroid+hormones+in+oviductal+function.">Roles of steroid hormones in oviductal function.</a> Reproduction. 159 (3), R125-R137 (2020).</li><li>Lee, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+regulates+embryonic+survival+during+delayed+implantation.">Autophagy regulates embryonic survival during delayed implantation.</a> Endocrinology. 152 (5), 2067-2075 (2011).</li><li>Hamatani, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+analysis+identifies+molecular+pathways+distinguishing+blastocyst+dormancy+and+activation.">Global gene expression analysis identifies molecular pathways distinguishing blastocyst dormancy and activation.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (28), 10326-10331 (2004).</li><li>Cui, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcervical+embryo+transfer+in+mice.">Transcervical embryo transfer in mice.</a> Journal of the American Association for Laboratory Animal Science. 53 (3), 228-231 (2014).</li></ol>

The authors declare no competing financial or other interests.

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 once appropriately trained and practiced. The key steps in the procedures include closely monitoring the animals while they are under anesthesia and ensuring there is no ovarian tissue left behind. In some models, the oviduct may be left intact. However, it should be noted that the oviduct is a hormone-responsive tissue with abundant estrogen and progesterone receptors19. The surgical protocol for removing the ovary and oviduct is much simpler compared to removing just the ovary, as the former can be completed with the naked eye. To remove just the ovary and leave the oviduct in place in the latter case, a dissecting microscope is required, as this is a much more intricate procedure. Consequently, the operating time may be extended, as the animal needs to be moved between the dissecting microscope stage and the operating field for different parts of the procedure, such as suturing the internal body wall.
The analgesic protocols detailed here are standard and approved by the Monash University Animal Ethics committee, so they may vary depending on the individual institution&#39;s ethics committee requirements or preferences. It should be noted that no analgesia was provided for the artificial decidualization procedure, as typical non-steroidal anti-inflammatories interfere with the decidualization process. If researchers wish to provide analgesics at the time of artificial decidualization, this should be taken into consideration.
This work presents three methods of hormone delivery to supplement ovarian hormones following OVX, and each method has its own strengths and weaknesses (Table 1). Subcutaneous injections of hormones in oil are common in the literature14,16,17. This technique has many strengths, including the fact that no surgical procedure is required, and, thus, no formal training in rodent surgery or gas anesthesia is required. This makes subcutaneous injection an accessible option for almost all research groups. Injections are also affordable and easy to carry out. Practically, however, they have some limitations, particularly in models of pregnancy. To maintain pregnancy in an OVX animal, hormone supplementation with progesterone must be given daily to support the pregnancy. It may be possible to stop the daily injections once the placenta is sufficiently developed to take over as the main source of progesterone, though this has not been trialed in the protocols presented here. Anecdotally, it is possible for hormones in oil to leak from the injection site following subcutaneous injection. In part, this may be due to the needle size required (26 G) to easily dispense something as viscous as sesame oil. Therefore, this leakage needs to be monitored and recorded when performing injections in oil in order to correlate this with the experimental outcomes.
Slow-release pellets are preferable to subcutaneous injections, as they are cost-effective and simple to make in-house. However, they require multiple overnight steps, which should be considered when planning experimental timelines. These pellets secrete approximately 500 &#181;g daily (as assessed during a time course incubation in cell culture medium and subsequent progesterone ELISA). It should be noted this is a lower concentration compared to the daily subcutaneous injections described above, and this is due to the consistency in the delivery of progesterone from the pellet. As aforementioned, oil injections can leak out of the injection site, thus reducing the overall concentration delivered. In previous studies, these pellets have only been active in vivo for up to 10 days after they are surgically inserted. Therefore, in pregnancy studies, it remains unclear if it may be necessary to insert a second pellet at mid-gestation or whether the placenta could sufficiently provide endocrine support for the pregnancy by that stage. These pellets are, therefore, optimal for short-term models of pregnancy, including the artificial decidualization protocol presented here, as well as pregnancy studies up to 10 days post-embryo transfer. While slow-release pellets negate the need for daily animal handling and injections, some low-dose estrogen injections are still required to balance the progesterone receptor feedback loop. This strategy has been used previously20,21.
Lastly, osmotic mini pumps are the most accurate hormone delivery method and are commercially available, but they are the most expensive option. Osmotic mini pumps can deliver a set concentration of hormone daily for up to 28 days, depending on the model selected. Similar to the slow-release pellets, while osmotic mini pumps avoid the need for daily animal handling, some low-dose estrogen injections are still required.
The artificial decidualization protocol described here allows the study of an early pregnancy milestone independent of ovarian and embryonic influence. While humans undergo decidualization with every menstrual cycle, rodents only decidualize during pregnancy establishment. Thus, this model has immense value for studying human-like pregnancy milestones in a manipulable rodent model. The procedure detailed herein is relatively non-invasive, as it uses a non-surgical embryo transfer device (NSET) to deliver sesame oil directly to the uterine horn via the vagina and cervix. Though this procedure is less invasive than other methodologies, it can become quite expensive when using commercial NSETs. In comparison, other published models of artificial decidualization require a surgical procedure to perform intrauterine injections of oil17. This requires a surgical setup similar to that described in section 1 and steps 2.1-2.11. However, in animals that have been ovariectomized previously, it can be more challenging to identify and expose the uterine horn. There can also be adhesions formed from the previous surgical procedure for ovariectomy. Thus, while it may be more cost-efficient to perform surgical intrauterine injections to induce decidualization, the surgical and anesthesia time is substantially longer than the alternative of using NSETs. There are established protocols for in-house fabricated alternatives22 to commercially available NSETs, which are much more cost-effective.
While the embryo transfer procedure is described here, we have previously published this model and its success rates across different strains of mice14. Moreover, while the embryo transfer method described here uses a surgical approach, NSETs could also be integrated into this procedure.
Future directions in this area should include studies focused on the specific uterine contributions to the establishment and maintenance of pregnancy. This knowledge is critical for furthering our understanding of idiopathic infertility, implantation failure, and pregnancy complications. Expanding our knowledge in these areas is also fundamental for improving clinical outcomes for IVF/ICSI patients, as well as our comprehension of pregnancy as a biological process.
In conclusion, OVX is a simple procedure that can be integrated into animal models to study the uterine contributions to pregnancy and fertility. Models in the future will benefit from integrating OVX and exogenous hormone delivery so that comparisons can be made between ovarian-specific and uterine-specific contributions to fertility and pregnancy.

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><tbody><tr><td>ALZET 1002 mini osmotic pumps</td><td>BioScientific</td><td>1002</td><td>Delivers 0.25 &amp;micro;L/h for 14 days. Use for section 7 (Experimental procedure - Embryo transfer).</td></tr><tr><td>ALZET 1003D mini osmotic pumps</td><td>BioScientific</td><td>1003D</td><td>Delivers 1 &amp;micro;L/h for 14 days. Use for section 8 (Experimental procedure - Artificial decidualization).</td></tr><tr><td>ALZET Reflex 7 mm clips</td><td>BioScientific</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>BioScientific</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>BioScientific</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 400 mL cage water bottle, or as your institution&#039;s 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&amp;nbsp; section 3 (Hormone preparation - subcutaneous injection) and&amp;nbsp; 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 1 mg/ml (for 5 mg/kg dose)</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 &amp;plusmn;&amp;nbsp; 0.23 mm; outside diamater 3.18 mm &amp;plusmn; 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 injection of sesame oil, and then the animals were rested until tissue collection, as described in section 9. In this study, artificial decidualization was performed in C57BL6/J mice, a commonly used mouse strain. At the time of tissue collection, the body weight was recorded, and the uterus was dissected and well-trimmed before being weighed (Figure 1B). The extent of the decidual response was recorded by expressing the uterine weight as a ratio of the body weight. In this study, 80% of the C57BL6/J mice decidualized (0.01012 &#177; 0.001515, n = 15), while 20% of the animal uteri did not decidualize (0.002108 &#177; 0.0001764, n = 3) (Figure 1C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64763/64763fig01v2.jpg" /> 
Figure 1: Schematic and representative results. (A)&#160;Schematic timeline for experimentally inducing artificial decidualization in a mouse model. Abbreviations: OVX = ovariectomy; E2 = estradiol (100 ng days 1-3, 5 ng days 7-9); P4 = progesterone. Note: A P4 pellet was used to generate the results presented here. Alternative methods for progesterone delivery include daily subcutaneous injections and mini-osmotic pumps. (B)&#160;Representative images of non-decidualised (ND) and decidualised (D) uteri from young adult C57BL6/J mice. Scale bars = 5 mm.(C)&#160;Comparison of the uterine weight to body weight (UW:BW) ratio in non-decidualized and decidualized animals. Data are mean &#177; SEM; Mann-Whitney test, **p = 0.003; ND: n = 3, D: n = 15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64763/64763fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hormone delivery method</td>
			<td>Strengths</td>
			<td>Weaknesses</td>
		</tr>
		<tr>
			<td rowspan="2">Subcutaneous injections</td>
			<td>No surgical intervention required</td>
			<td>Repeated daily handling</td>
		</tr>
		<tr>
			<td>Accessible technique that does not require surgical training (as compared to pellet implantation)</td>
			<td>Hormones in oil can leak out of the injection site, therefore amount absorbed by each animal can vary</td>
		</tr>
		<tr>
			<td rowspan="4">Slow release pellets</td>
			<td>No need for daily handling</td>
			<td>Surgical procedure required</td>
		</tr>
		<tr>
			<td>Can be made in house</td>
			<td>Not commercially available</td>
		</tr>
		<tr>
			<td>Affordable alternative to osmotic mini pumps</td>
			<td></td>
		</tr>
		<tr>
			<td>Small and very well tolerated by animals</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">Osmotic mini pumps</td>
			<td>No need for daily handling</td>
			<td>Surgical procedure required</td>
		</tr>
		<tr>
			<td>Commercially available</td>
			<td>Expensive</td>
		</tr>
		<tr>
			<td>Most accurate delivery method</td>
			<td>Much larger than slow release pellets</td>
		</tr>
	</tbody>
</table>
Table 1: Strengths and weaknesses of the hormone delivery methods.

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 ...

methods-for-studying-uterine-contributions-to-pregnancy-establishment

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

1.6K Views.  Monash University. Het is dus moeilijk om de rol van baarmoeder- en ovariële hormonale invloeden op de zwangerschap te scheiden, omdat beide nauw met elkaar verbonden zijn. Dit protocol stelt ons dus in staat om de baarmoederbijdragen direct te onderzoeken. Hoewel sommige van deze technieken zoals ovariëctomie vrij invasief zijn, zijn ze relatief eenvoudig uit te voeren na oefening en training, waardoor ze geschikt zijn voor onderzoekers die nieuw zijn in muismodellen.Deze methode kan dus worden gebrui...

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

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States1. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available2. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat3 and later transferred to the mouse4. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease5,6. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis2,7. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system8, hormones9,10 and environmental factors11,12 affect endometriosis as well as the effects of endometriosis on fertility13 and pain14.

A description of the surgical induction of endometriosis in mice and rats by auto-transplantation of uterine tissue to the arterial cascade of the intestinal mesentery.

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of ...

Medicine

Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of decidualization leads to various endometrial disorders, such as infertility, recurrent miscarriage, and preterm birth. The development and use of the endometrial decidualization model in reproductive studies have been a highlight for reproductive researchers for a long time. The mouse has been extensively used in studying reproduction and decidualization. There are three well-established mouse models regarding decidualization, namely natural pregnancy decidualization (NPD), artificial decidualization (AD), and in vitro decidualization (IVD). Among them, AD is considered a reliable model for mouse decidualization, which is easy to implement and close to NPD. This paper focuses on a modified method of the generation and application process of the mouse artificial decidualization model with ovariectomy to avoid ovarian effects, which can obtain highly reproducible results with small within group variances. This method provides a good and reliable animal model for the study of endometrial decidualization.

Here, we describe the method of generating an artificial decidualization model using the ovariectomized mouse, a classic endometrial decidualization experiment in the research field of endometrial decidualization.

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of ...

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.

Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

Biology

A Syngeneic Murine Model of Endometriosis using Naturally Cycling Mice

Endometriosis is a leading cause of pelvic pain and infertility. It is defined by the presence of endometrial tissue in extrauterine locations. The development of novel therapies and diagnostic tools for endometriosis has been limited due in part to challenges in studying the disease. Outside of primates, few mammals menstruate, and none develop spontaneous endometriosis. Rodent models are popular but require artificial induction of endometriosis, with many utilizing either immunocompromised mice or surgically induced disease. Recently, more attention has been given to models involving intraperitoneal injection. We present a murine model of endometriosis that integrates several features of existing endometriosis models into a novel, simplified system that relies on microscopic quantification in lieu of subjective grading. In this model, we perform hormonal stimulation of donor mice, intraperitoneal injection, systematic abdominal survey and tissue harvest, and histologic quantification that can be performed and verified at any time after necropsy. This model requires minimal resources and training; does not require expertise by lab technicians in murine survival surgery or in the identification of gross endometriotic lesions; can be used in immunocompromised, immunocompetent, and/or mutant mice; and reliably creates endometriotic lesions that are histologically consistent with human endometriotic disease.

Many rodent models of endometriosis are limited by technical complexity, reproducibility, and/or need for immunocompromised animals or special reporter mice. We present a simplified system of lesion induction using any experimental mouse with an independently verifiable, objective scoring system and with no requirement for ovariectomy or survival surgery.

Endometriosis is a leading cause of pelvic pain and infertility. It is defined by the presence of endometrial tissue in extrauterine locations. The ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. Here, we present an easy and inexpensive method that would be instrumental for testing new drugs decreasing uterine contractility. This method utilizes the unique ability of the entire mouse reproductive tract to exhibit spontaneous motility when maintained ex vivo in a Petri dish containing oxygenated Krebs buffer. This spontaneous motility resembles the wave-like myometrial activity of the human uterus, referred to as endometrial waves. To demonstrate the effectiveness of the method, we employed a well-known uterine relaxant drug, epinephrine. We demonstrate that the spontaneous motility of the entire mouse reproductive tract can be quickly and reversibly inhibited by 1 &#181;M epinephrine in this Petri dish model. Documenting the changes of uterine motility can be easily done using an ordinary smart phone or a sophisticated digital camera. We developed a MATLAB-based algorithm allowing motion tracking to quantify spontaneous uterine motility changes by measuring the rate of uterine horn movements. A major advantage of this ex vivo approach is that the reproductive tract remains intact throughout the entire experiment, preserving all intrinsic intrauterine cellular interactions. The major limitation of this approach is that up to 10-20% of uteri may exhibit no spontaneous motility. Thus far, this is the first quantitative ex vivo method for assessing spontaneous uterine motility in a Petri dish model.

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...

Development of a Novel Endometrioma-Simulating Mouse Model

Establishment of an Experimental Mouse Model of Endometrioma to Study its Related Infertility

Endometrioma (OMA), a subtype of endometriosis characterized by the formation of endometriotic cysts in the ovaries, affects 17-44% of individuals diagnosed with endometriosis. Women with OMA often experience compromised fertility, yet the exact mechanisms underlying OMA-associated infertility remain unclear. Notably, existing animal models simulate superficial peritoneal endometriosis (SUP) and deep infiltrating endometriosis (DIE), leaving a notable gap in research focused on OMA. In response to the gap of knowledge, this paper introduces a pioneering OMA-simulating mouse model and provides a comprehensive description of the techniques and procedures employed in the model. With a high success rate of 83% and ovarian lesion specificity, this model holds significant promise for advancing our understanding of OMA, particularly in the context of infertility. It offers a valuable platform for conducting targeted research into OMA-associated fertility challenges, potentially paving the way for improved diagnostic and therapeutic strategies in the field of reproductive medicine.

This study introduces a novel mouse model specifically designed to simulate endometrioma, a clinically significant subtype of endometriosis. By utilizing C57BL/6J mice, the model aims to elucidate the pathophysiological mechanisms underlying endometrioma-related infertility, offering a refined tool to bridge the current knowledge gap in reproductive medicine.

Endometrioma (OMA), a subtype of endometriosis characterized by the formation of endometriotic cysts in the ovaries, affects 17-44% of individuals ...

Cervical Manipulation Induced Pseudopregnancy in Mice for Assisted Reproduction

Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination

For successfully maintaining pregnancy with embryo transfer or artificial insemination,&#160;female recipient mice must be induced into a pseudopregnant state. Female mice are traditionally paired overnight with vasectomized males, and the following morning, the presence of a copulation plug is assessed. To increase the efficiency of producing pseudopregnant females, a cervical manipulation technique has been standardized to be used in combination with non-surgical embryo transfer or artificial insemination techniques in mice. The blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated for 30 s by contact with a trimmer. The procedure is quick and does not require anesthesia or analgesia. This technique increases the reliability and predictability of producing pseudopregnant females and entirely eliminates the requirement for vasectomized males. For CD1 mice, the efficiency of pseudopregnancy induction using cervical manipulation was 83% for females in estrus (N = 76) but only 38% of females in estrus were plugged by vasectomized males (N = 24). Artificial insemination in CD1 mice was performed by estrus synchronization with hormones, cervical manipulation, and the uterine transfer of sperm. Artificial insemination recipients receiving cervical manipulation (N = 76) had a pregnancy rate of 72% and an average litter size of 8.3 pups. This method can also be used to produce pseudopregnant females for non-surgical embryo transfer. Therefore, inducing pseudopregnancy by cervical manipulation is a convenient and efficient alternative to mating with a vasectomized male when performing non-surgical assisted reproduction techniques. Using cervical manipulation provides 3Rs (replacement, reduction, and refinement) benefits for assisted reproduction techniques by reducing the number of animals required and eliminating the necessity for surgically altered males.

Author Spotlight: Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination  

The cervical manipulation method presented can induce pseudopregnancy in mice without the need for breeding females with vasectomized males. The induction of pseudopregnancy is required for the success of non-surgical embryo transfer and non-surgical artificial insemination, both of which are also presented.

For successfully maintaining pregnancy with embryo transfer or artificial insemination,&#160;female recipient mice must be induced into a pseudopregnant ...

Developing an Ovariectomized Mouse Model with Elevated FSH Level

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes, including decreased bone mass, increased blood lipids, and increased visceral adiposity. Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition. Many studies have shown that FSH in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. In this study, C57BL/6 female mice were ovariectomized and supplemented with estradiol valerate (OVX + E2) to eliminate the effect of the hypothalamic-pituitary-gonadal axis. The OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels. Thus, we successfully generated an experimental mouse model to mimic the early stage of menopause transition, characterized by elevated serum FSH levels. The OVF model has the advantages of being stable, low cost, and easy to operate, which is suitable for studies to explore the extragonadal actions of FSH. Here, we describe detailed protocols for the mouse OVF model.

Author Spotlight: Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model 

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological ...

Optimized Thin Endometrium Mouse Model with Low Mortality and Minimal Adhesions

Establishing a Mouse Model of Thin Endometrium

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory treatment options are still urgently needed. Several animal models of TE have been developed, but the mouse model involving abdominal surgery and injection of 95% ethanol presents a formidable challenge due to the high mortality rate and risk of intrauterine adhesions if not performed correctly. Here, we describe a detailed protocol that produces reliable and reproducible TE with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus with varying infusion times. The results showed that all of the mice successfully developed TE with infusion times ranging from 1-3 min, characterized by a typical reduction in endometrial thickness and the number of glands, as well as excessive endometrial fibrosis. These findings suggest that this mouse model is suitable for studying thin endometrium and can serve as a platform for developing future TE treatments.

This article describes a detailed protocol for producing a reliable and reproducible thin endometrium with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus within 1-3 min.

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory ...

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

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