Optimum endometrial environment for embryo implantation
Endometrial receptivity is the process undertaken by the uterine lining to prepare for the implantation of an embryo. While embryo development and endometrial preparation are concurrent yet independent, their synchronisation is critical to embryo apposition, adhesion, invasion, and further ongoing pregnancy. The limited period of optimal endometrial receptivity during which the endometrium is ready to receive an embryo, paired with an embryo’s readiness to implant, is commonly referred to as the “window of implantation” and is generally detected between days 20 and 24 of a normal 28-days menstrual cycle.
Pathophysiology of implantation
Many molecular pathways involve hormones, adhesion molecules, cytokines, and growth factors acting in concert to create a synchronous window of implantation. When synchrony is lost or receptivity is not achieved, the consequences is infertility as result of implantation failure.
Prior to implantation, the blastocyst shows evidence of polarity, assuming a particular orientation as it approaches the endometrium. Once the blastocyst is oriented correctly (apposition), the zona pellucida is shed. The blastocyst then comes in to contact with the epithelial layer and adheres to the endometrial surface (adhesion). Finally, the blastocyst penetrates the epithelial layer and invades the stroma (invasion).
A number of factors play an important role in all steps of implantation. For apposition, proper pinopods development requires leukemia inhibitory factor, progesterone, and beta-3 integrin, whereas adhesion involves various molecules, immune cells, and cytokines (namely beta-3 integrin and L selectin), and finally maternal immune tolerance is required including macrophages, dendritic cells (DCs), and uterine natural killer (uNK) cells to support immune regulation and facilitate the support of trophoblast invasion.
To improve implantation rates, it is important to find ways to pinpoint the window of implantation, ensure that the best embryo is selected and synchronise embryo transfer with the time of optimal endometrial receptivity. Importantly, ways of evaluating and enhancing endometrial performance and embryo quality without disrupting the delicate process of implantation itself must be identified.
Implantation failure can be caused by: 1) issues related to embryo development and/or aneuploidy, 2) reduced or absent endometrial receptivity, and 3) embryo-endometrial asynchrony. Factors that disturb receptivity include inflammatory events, thin endometria, fibroids, polyps, certain congenital uterine abnormalities, immunologically mediated disturbances, and endocrine causes. Endometrial receptivity is not an all-or-nothing phenomenon but rather exists on a spectrum. A mild defect in receptivity may cause placental abnormalities, which lead to issues like pre-eclampsia or low birth weight. More severe forms of receptivity aberrations may lead to early pregnancy loss and infertility.
Endometrial receptivity can be further impaired by an imbalance of steroid hormones by way of progesterone resistance and estrogen dominance. In both natural and artificial cycles, progesterone resistance can result from a pro-inflammatory state. Conditions that cause inflammation in the endometrium include endometriosis, endometritis, adenomyosis, and fluid within hydrosalpinges.
The role of the endometrium
The endometrium is a multilayered, dynamic organ overlaying the myometrium and comprises a functional layer and a basal layer. Each month, cells in the functional layer are separated from the basal layer during menstruation. The basal layer is attached to the myometrium and remains intact during menstruation, serving as a base for endometrial regeneration. The endometrium is composed of several different cell types, including luminal and glandular epithelial cells, stroma with stromal fibroblastic cells, immunocompetent cells and blood vessels. The numbers, activity, structure and function of these cells change throughout the menstrual cycle and change again during pregnancy.
Understanding the molecular processes responsible for coordinating the window of implantation and facilitating normal embryo implantation is critical to developing diagnostic tests and treatments for receptivity defects that contribute to recurrent pregnancy loss, implantation failure, and infertility.
What is the endometrial microbiota
It is the collection of trillions of micro-organisms (bacteria, fungi, viruses) that live in symbiosis in the endometrium, the inner lining of the uterine cavity. This community of micro-organisms plays a crucial role in female reproductive health. A “normal” composition of the endometrial microbiota has not yet been established. However, it has been observed that the endometrial microbiome is considered to be healthy when it is Lactobacillus-dominant (if the relative abundance of Lactobacillus exceeded 90%). Lactobacilli inhibit the adhesion of other bacteria to epithelial cells and produce lactic acid that kills or inhibits the growth of other bacteria, promoting homeostasis.
Endometrial microbiota testing, also known as endometrial microbiome analysis or endometrial microbial profiling, is a diagnostic procedure used to assess the microbial composition and diversity within the endometrium. It involves collecting a sample of endometrial tissue or fluid for analysis to identify and characterize the micro-organisms present in the endometrial environment.
What causes endometrial dysbiosis?
The normal microbiome of the uterine environment, like that of the vagina, is dominated by Lactobacillus. Other bacteria residing on the surface of the endometrium are: Acinetobacter (9.07%), Pseudomonas (9.09%), Sphingobium (5%) and Vagococcus (7.29%). The presence of the above microorganisms in the right proportions is extremely important for a woman’s health and fertility.
Endometrial dysbiosis is an imbalance of micro-organisms that can lead to negative outcomes for reproductive function. The composition of the endometrial microbiota is influenced by several factors, such as genetic predisposition, age, menstrual cycle, childbirth, vaginal microbiota, antibiotic use and sexual practices. Dysbiosis, is a disruption of the quantitative relations between microorganisms, often in favor of pathogenic bacteria.
The endometrial microbiome is tested by employing the double-sheathed catheters commonly used for embryo transfers to obtain endometrial specimens. The outer hollow catheter is placed with its distal part just above the internal os of the cervix. Thereafter, the inner catheter (with a smaller diameter) can easily pass through the first one, avoiding contacts with the vaginal and cervical mucosae. Once reached the endometrial cavity, endometrial collection of the endometrial fluid can be obtained by gentle traction on the syringe. Then, bacterial genomic DNA extraction from endometrial samples is performed and the bacterial 16S rRNA gene is identified by next-generation sequencing (NGS), thus performing metagenomics for the analysis.
The study of the endometrial microbiota can help the medical specialist to better understand the causes of infertility, endometriosis, recurrent miscarriage and endometritis. It can also help develop new diagnostic and treatment methods for these conditions and personalize medical care for women with fertility or reproductive health problems.
Inflammation of the endometrium
Endometritis is an acute or chronic inflammation of the endometrium caused by an abnormal endometrial microbiome. The symptoms can vary from a completely asymptomatic situation to causing chronic pelvic pain and dyspareunia. Nonetheless, when symptomatic the most common symptom is abnormal bleeding between periods and vaginal discharge. In its acute form, endometritis presents itself with high fever, severe pain in the abdomen and foul-smelling vaginal secretions.
The most prominent example of a pathology caused by an altered endometrial microbiota is chronic endometritis. In recent years, a growing interest in chronic endometritis has been seen, especially due to its putative role in infertility, recurrent pregnancy loss, and repeated IVF failures. Notably, in these conditions, chronic endometritis prevalence has been often reported to exceed 30%.
Different theories have been proposed for explaining chronic endometritis-related impaired endometrial receptivity, including the activation of local inflammatory processes with altered cytokine and chemokine secretion, abnormal leukocyte infiltration within the endometrium, altered uterine contractility, defective decidualisation, and defective endometrial vascularisation.
Based on a body of research, chronic endometritis significantly reduces ongoing pregnancy rates as well as live birth rates and clinical pregnancy rates in women undergoing IVF. Importantly, chronic endometritis resolution after antibiotic therapy may improve IVF outcome, leading to similar ongoing pregnancy rates, live birth rates and clinical pregnancy rates as compared to unaffected women.
Diagnosis of chronic endometritis
One of the crucial issues regarding chronic endometritis is the methodology used for its diagnosis. Methods to diagnose endometritis include:
- Hysteroscopy. There are certain endometrial features such as the presence of micro polyps and/or hyperemia that may be seen at hysteroscopy.
- Endometrial biopsy for plasma cells, commonly known as CD138 staining. The method is simple and objective, but it can often be negative and still the woman can suffer from endometritis. That is why the interpretation and guidance of each incident must be separate.
- Intrauterine tissue culture, which can also give false positive results.
- Alice and Emma Tests. Debatable is the clinical usefulness of novel tests such as Alice (analysis of infectious chronic endometritis) and Emma (endometrial microbiome metagenomic analysis). They detect the pathogens DNA.
The gold standard for the diagnosis of chronic endometritis is a diagnostic hysteroscopy with endometrial sample for the uterine microbiome.
Treatment
Many protocols of antibiotic treatment have been proposed. Basically, antibiotic choice may be guided based on the antibiogram (endometrial cultures required) or empirically chosen (standard protocol). The most commonly employed standard protocol consists of oral levofloxacin 500 mg and tinidazole 1,000 mg daily for 14 days. Alternatively, it has been proposed a first course with oral doxycycline 100 mg twice daily for 14 days. In case of persistence, a second treatment with oral levofloxacin 200 mg administered twice daily plus oral metronidazole 500 mg thrice daily for 14 days. If the endometrial culture is still positive, a third and last treatment course with oral levofloxacin 200 mg twice daily plus oral metronidazole 500 mg thrice daily for another 14 days. To this date, no studies demonstrated the superiority of one approach over the other.
Conclusions
Chronic endometritis can be found in up to 10% of women, however it is known that this percentage is much higher in women that have had recurrent embryo implantation failures and in those who have had recurrent miscarriages (up to 67%).
To conclude, chronic endometritis has an infectious origin. It causes a hostile uterine environment for the development and maintenance of pregnancy. Although there is a lack of randomised controlled trials looking at pregnancy outcomes in treated vs. untreated cohorts, the existing evidence suggests that chronic endometritis is highly prevalent among women with failed IVF outcome (up to 60%). It appears that it is possible to diagnose and treat chronic endometritis and thereby improve reproductive outcome. Hysteroscopy and endometrial biopsy are low-cost office procedures that are effective for diagnosing chronic endometritis and evaluating the effects of treatment. Based on this evidence we strongly suggest investigating the following for chronic endometritis before IVF treatment:
- Women with infertility of unknown cause
- Women with history of hydrosalpinx
- Women with endometriosis/adenomyosis
- Women with recurrent implantation failure and repeated miscarriage
- Women who have had scraping or other endometrial procedures in the past
- Women with known endometritis, or repeated incidences of vaginal infections
- Women with past coil insertion
How often should endometrial microbiome be tested?
Microbiome molecular analysis can be used to determine whether uterine microenvironment is normal or whether pre-treatment (antibiotics and probiotics) should be suggested for a specific period of time, in order to influence the microbiome towards a healthier population. This suggests a great benefit not only for women undergoing IVF, but also for every woman wishing to conceive. If there is any suspicion of a dysbiotic uterine environment (as in all cases described above) all women should be offered the investigation of their uterine microbiome.
There is though limited evidence on retesting the endometrial microbiome after therapy, thus, it is not recommended to waste time or money on repeated tests once treatment has been completed. The study of the endometrial microbiome continues to be a very active area of research. It is though our recommendation for a second look hysteroscopy with another microbiome sampling in cases or repeated implantation failures or first trimester miscarriages when a dysbiotic uterine environment has been diagnosed in the past.
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