Diagnosis of Chronic Endometritis in Endometrial Fluid Using Molecular Techniques to Improve the Outcomes of Assisted Reproductive Treatments
Diagnosis of Chronic Endometritis in Endometrial Fluid Using Molecular Techniques to Improve the Outcomes of Assisted Reproductive Treatments
Chronic endometritis (CE) is a persistent inflammation of the endometrial lining caused by
 the infection of the uterine cavity mainly by bacterial pathogens. It is known that CE can
 produce implantation failure, recurrent abortion and preterm labor. The study hypothesis is
 that patients who have repeated implantation failures (RIF) despite transferring good quality
 and normal embryos, diagnosed by preimplantation Genetic Diagnosis (PGD), into a receptive
 endometrium, diagnosed by Endometrial Receptivity Analysis (ERA), may present asymptomatic
 CE, being the altered endometrium the main cause of these implantation failures.

 The current diagnosis of CE is the microbiological culture of endometrial biopsy samples.
 Alternatively, examination of the uterine cavity by hysteroscopy is effective at 93.4%.
 However, both methods have limitations, such as the time needed to obtain the diagnosis, the
 economic cost of the tests, the possibility of detecting microorganisms in culture etc. For
 these reasons, the developing of a simple, fast, cheap and minimally invasive diagnostic tool
 for CE patients subjected to IVF treatments is expected. It would be very useful in order to
 establish a specific treatment and improve pregnancy rates in infertile patients. For this
 reason, the bacterial DNA present in the endometrial fluid samples will be obtained and the
 identification of the causative pathogens of CE will be done by sequencing (NGS) and/or
 quantitative PCR with specific oligonucleotides for the most common bacteria causing CE. The
 validation of this new method will be performed by comparison with the microbiological
 diagnosis of those same patients.
On day 5 of the Hormonal Replacement Therapy (after 5 days of progesterone administration),
 coinciding with the day of hysteroscopy indicated by the physician according to the routine
 clinical practice, samples of endometrial fluid (EF) and endometrial biopsy (EB) of patients
 with RIF will be obtained. These samples will be used for the diagnosis of CE by both classic
 and molecular methods. Specifically, EF sample will be analyzed by molecular techniques using
 Next Generation Sequencing (NGS) for the detection of infectious agents. EB sample will be
 divided into three parts, one of them will be used for molecular analysis (as control of the
 bacterial content present in the EF), another part will be subjected to microbiological
 analysis and the third part will be analyzed using histological techniques. In this way,
 comparisons will be made between the different diagnostic methods of CE in the two sample
 types (EF and EB) and the efficacy values of the new diagnostic test (sensitivity,
 specificity, positive and negative predictive values and Receiving Operating Characteristic
 curves) will be obtained using the microbiological diagnosis as a reference (gold standard
 method).

 Patients with a negative outcome for CE (approximately 40%) will continue with the expected
 assisted reproduction treatment (ART) according to the clinic's standard protocol and
 patients with a positive outcome for CE (approx. 60%) will receive antibiotic treatment
 according to usual clinical practice (time and type of antibiotic will be different according
 to the microbiological diagnosis). Once this treatment is completed, the improvement of these
 patients will be confirmed obtaining a second sample of EF and EB. The Assisted Reproductive
 Treatment (ART) will be performed once the specialist considers it appropriated.

 Finally, reproductive rates will be correlated with the diagnosis of CE. Therefore, an
 observational analytical study, in which a comparison of the ART results before and after its
 diagnosis of CE, will be carried out. The reproductive impact of an altered endometrial
 microbiota will be assessed analyzing the implantation rates, pregnancy and ongoing
 pregnancy.
Inclusion Criteria:

 -  Patients who provide their written informed consent after having been informed of the
 all study aspects.

 -  IVF or ICSI cycles with own oocytes, with blastocyst transfer (day 5 or 6 of stage
 development).

 -  Women with age comprised between 18 and 38 years (both included).

 -  Body mass index: 18.5 - 30 km/m2 (both included)

 -  Adequate ovarian reserve > 8 antral follicles (RFA) and/or Antimüllerian hormone
 (HAM)> 1 ng / mL.

 -  Concentration of spermatozoa > 2 million sperm/ml.

 Exclusion Criteria:

 -  Congenital or acquired uterine pathologies.

 -  Endometriosis.

 -  Patients with IUDs in the last 3 months.

 -  Patients who have taken prescribed antibiotic treatment in the last 3 months.

 -  Any disease or medical condition that could be unstable or could endanger the security
 of the patient and her compliance in the study.