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

Summary

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.

Abstract

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.

Introduction

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 "black box" of reproductive potential².

Pregnancy is established when a high-quality embryo successfully interacts with a receptive endometrium (uterine lining). The chances of successful preg....

Protocol

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

.......

Discussion

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 .......

Materials

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