Establishing a Mouse Model of Thin Endometrium

Thin endometrium has been widely recognized as the critical cause of infertility. Studies on the pathogenesis of thin endometrium rely on animal models, making the selection of an appropriate model crucial. Here, we describe a detailed protocol that produces a reliable and reproducible thin endometrial model in mice.

The operation is traumatic, as we need to dissect the mouse and expose the mouse uterine horn for manipulation. An improper operation may cause infection or even lead to the death of the mice. Indeed, careful training and experience are needed before the experimental operation.

We successfully developed an endometrial model with infusion time ranging from one to three minutes to replicate the varying degrees of thin endometrial symptoms observed in clinical cases. This allows us to create models with different levels of damage, depending on the specific needs of our research. The method used in our protocol offers the advantage of low cost, suitability, as well as experimental period.

It study the pathological mechanism and treatment in three initial as compared to other techniques. To begin, position the anesthetized mouse supine on the preheated operating table, and using medical tape, secure the limbs of the mouse. Employing sterile scissors and forceps, make a one centimeter incision in the abdomen, located about a centimeter from the urethral orifice, extending to the peritoneal cavity.

Gently move the entrails aside with sterile forceps to locate the uterus. Expose the right uterine horn and apply hemostatic clamps at the proximal end near the cervix and the distal end near the ovary. With a 25 gauge syringe, instill approximately 50 microliters of 95%ethanol into the uterine cavity, holding the needle parallel to the uterine horn.

Maintain the syringe for one to five minutes as appropriate. Then, withdraw the ethanol from the uterine cavity and flush the uterine cavity with sterile saline five times to remove any remaining ethanol. Now, remove the hemostatic clamps and return the uterus and entrails to their original position.

Using 5/0 absorbable surgical sutures, suture the peritoneum, followed by the epidermis. To begin, position a euthanized thin endometrium mouse model on the dissection table and cut the sutures using surgical scissors to expose the uterus. Then, using surgical forceps, extract the entire uterus carefully and detach the uterus from the surrounding entrails.

Immediately remove both uterine horns and divide each horn into three to four small segments. Place the uterine tissues in one milliliter of 4%paraformaldehyde taken in a 1.5 milliliter microcentrifuge tube. Place the formalin-fixed samples into tissue cassettes and transfer the tissue cassettes through a series of ethanol concentrations, followed by xylene and paraffin.

Fill a wax mold with molten paraffin wax heated to 55 degrees Celsius. Use heated forceps to quickly transfer the infiltrated tissue segments from the embedding cassette into the wax mold. Once the tissue segments are properly positioned, place the bottom of the embedding cassette on top of the histology mold.

Next, place the mold directly on a cooling plate set to four degrees Celsius for at least 20 minutes to let the paraffin solidify. Then, remove the sample from the mold and use a paraffin trimmer to trim any excess wax around it. The endometrial thickness and the number of endometrial glands significantly decreased in the one, two, and three-minute groups compared to the control group.

Severe uterine adhesion and an almost invisible uterine cavity were observed in the four and five minute groups, and no endometrial glands were found. Endometrial fibrosis increased with the duration of ethanol infusion, as shown by excessive staining in one to five minute groups compared to the control.