This study is funded by the Theme-based Research Scheme granted by the Research Grants Council of the Hong Kong Government of Special Administrative Region (T13-602/21-N).

All experimental procedures conducted in this study were approved and regulated under protocol number 21-203-MIS_[C] by the Ethical Committee of the Chinese University of Hong Kong.
1. Animal acclimatization and selection
<ol>
	<li>Mice preparation
	<ol>
		<li>Obtain 18 C57BL/6J mice (primiparous females, 8-9 weeks old: 6 donors, 12 recipients) from a reputable supplier. Verify the health certificates.</li>
		<li>Maintain all mice in a controlled housing environment: 22-24 &...

The prevalence of OMA in the global female population underscores a critical health concern38. Beyond the general symptoms of endometriosis, OMA brings additional challenges for fertility, including severe pelvic pain, potential for ovarian torsion, and other notable implications39,40. While the understanding of OMA pathogenesis and progression is largely shrouded in mystery, the knowledge vacuum not only impedes the formulation of targete...

Endometrioma (OMA) is the most predominant subtype of endometriosis, observed in approximately 17-44% of individuals diagnosed with endometriosis1,2. It is characterized by the formation of endometriotic cysts within the ovaries. These cysts, colloquially termed &#34;chocolate cysts&#34;, derive their name from their distinctive brown, tar-like consistency3. In addition to OMA, endometriosis can also present as superficial peritoneal endometriosis (SUP) and deep infiltrating endometriosis (DIE). SUP refers to the lesions on the peritoneal lining, whereas DIE refers to lesions penetrating more than 5 mm underneath the peritoneal surface4. The heterogeneity in lesion location, appearance, and depth among these endometriosis subtypes results in diverse clinical symptoms and variable disease severity5,6. OMA is particularly associated with more severe endometriosis stages and has been implicated in infertility, pelvic adhesions, and an increased ovarian cancer risk1,7,8,9.
The association of endometriosis, especially OMA, with infertility is a clinical issue of grave concern. Endometriosis is present in 25-50% of infertile women, with 30-50% of women diagnosed with endometriosis experiencing infertility10,11,12,13,14. While the exact OMA-associated infertility mechanisms remain elusive, some hypotheses have been raised. One suggests that endometriosis leads to a chronic inflammatory state, which can disrupt normal ovarian function and impair oocyte quality15,16. Another proposes abnormal iron overload in the ovarian follicular fluid, which is believed to be associated with oocyte maturation disorder14. Other studies address the disruptions in hormonal regulation17, oocyte quality18, and embryo development19.
Historically, rodent models have played a crucial role in deepening our understanding of endometriosis, particularly in studying its pathology, impact on fertility, and mechanisms of pain20,21,22,23. Rodents, particularly rats and mice, have been extensively utilized as animal models in exploring the cause-and-effect relationships, underlying mechanisms, potential diagnostic techniques, and therapeutic interventions for this disease22,24,25,26. It is important to recognize that all animal models have their specific uses and limitations. The choice of a particular model depends on the specific outcome or aspect being measured or studied27. For instance, a rat model demonstrated that stress could exacerbate endometriosis manifestations and influence inflammatory parameters, providing insights into the disease&#39;s potential triggers28.
In the context of endometriosis research, there are various rodent models employed. One commonly used approach is the homologous model, which involves the transplantation of rodent uterine tissue into the same species&#39; peritoneal cavity or mesenteric vessels29. This model offers advantages in terms of immunocompetence and suitability for long-term studies30. However, limitations arise due to the partial disparity between the implanted ectopic mouse uterine tissue and the characteristics of human endometriotic lesions31. Another approach is the heterologous model, where a human endometrial biopsy is implanted into an immunosuppressed mouse32. This model allows the use of human ectopic endometrium as a donor tissue for lesion development, providing constructive validity32. However, it relies on immunosuppressed mice as recipients, which hinders a comprehensive assessment of the immune responses involved in the etiology of the disorder33. Nevertheless, it is important to acknowledge that the existing models primarily focus on mirroring the generalized condition SUP, inadequately capturing the unique characteristics of OMA and DIE34. Currently, there is a paucity of specific models available for studying DIE, with only a few models existing, including a recent rodent model developed by Yan et al.35. This scarcity underscores the need for further research and the development of models that accurately capture the specific characteristics of DIE. Additionally, our understanding of OMA, especially its nuanced association with fertility, also remains limited26,36.
Addressing the existing challenges, this paper presents a novel experimental homologous mouse model designed to specifically simulate OMA. It is aimed at providing unique insights into the pathological mechanisms underlying OMA-related infertility, thereby bridging the knowledge gap in this crucial area of reproductive medicine. In brief, C57BL/6J mice were used for their human-like reproductive traits. Following estrus cycle synchronization, uterine tissues from donor mice were minced and transplanted into the recipient mice&#39;s ovarian bursa at a 1:2 ratio. After a 4-week interval, both morphological and histological examinations of the ovarian lesions were conducted to validate OMA presence and evaluate any associated follicular atresia. With an 83% successful rate, this model offers researchers a reliable platform for OMA studies, emphasizing lesion development specifically in the ovaries for focused investigations.

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	<tbody>
		<tr>
			<td>1 cc syringe with 25 G needle</td>
			<td>BD Biosciences</td>
			<td>301320</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mm Tissue culture dish</td>
			<td>FALCON</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Buffered formalin</td>
			<td>Fisher Scientific</td>
			<td>SF100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>10x phosphate buffered saline (PBS) buffer</td>
			<td>Fisher Scientific</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G needle</td>
			<td>BD Biosciences</td>
			<td>305107</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 black braided silk non-absorbable suture</td>
			<td>Ethicon Inc.</td>
			<td>W500H</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive microscope slides</td>
			<td>Marienfeld</td>
			<td>810401</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine Injection&#160;</td>
			<td>Med-Vet International</td>
			<td>RXBUPRENOR5</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-headed cotton swab</td>
			<td>SANYO Co., LTD.</td>
			<td>HUBY-340</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Alfasan International b.v</td>
			<td>2203095-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtubes</td>
			<td>Corning&#160;</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Excelta Corporation</td>
			<td>2827-NH-35-SE-ND</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Major Pharmaceuticals</td>
			<td>10033691</td>
			<td></td>
		</tr>
		<tr>
			<td>Periodic Acid Schiff (PAS) Stain Kit&#160;</td>
			<td>Abcam</td>
			<td>Ab150680</td>
			<td></td>
		</tr>
		<tr>
			<td>Powder free sterile gloves</td>
			<td>Fisher Scientific</td>
			<td>19020558</td>
			<td></td>
		</tr>
		<tr>
			<td>Processing/embedding cassettes</td>
			<td>Fisher Scientific</td>
			<td>15-197-700A</td>
			<td></td>
		</tr>
		<tr>
			<td>Size 3 scalpel</td>
			<td>Fisher Scientific</td>
			<td>22-079-657</td>
			<td></td>
		</tr>
		<tr>
			<td>Small serrated semi-curved forceps</td>
			<td>Roboz Surgical Instruments Co.</td>
			<td>RS-5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Small surgical scissors</td>
			<td>Roboz Surgical Instruments Co.</td>
			<td>RS-5850</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard light microscope</td>
			<td></td>
			<td></td>
			<td>For evaluating vaginal cytology smears and histological analysis.</td>
		</tr>
		<tr>
			<td>Steel scalpel blades #10</td>
			<td>B.Braun</td>
			<td>BB510</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>MEDICOM</td>
			<td>HMWS 077</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilized pyrex glass Petri dishes</td>
			<td>Corning</td>
			<td>70160-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermoregulated electric pad&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VICRYL RAPIDE (polyglactin 910) absorbable Suture</td>
			<td>Ethicon Inc.</td>
			<td>W9918</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine&#160;</td>
			<td>Alfasan International b.v</td>
			<td>2110333-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The successful establishment of OMA 
Of the 12 mice subjected to the transplantation protocol, 10 exhibited characteristic OMA lesions, both at a gross anatomical and histological level, translating to a success rate of 83%. Gross examination revealed fluid-filled lesions adherent to the ovaries, reminiscent of clinical OMA presentations (Figure 2). Histopathological scrutiny, as illustrated in Figure 3, confirmed the ectopic endometrial ti...

Read this Scientific Article Protocol about Establishment of an Experimental Mouse Model of Endometrioma to Study its Related Infertility at JoVE.com

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

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.