Validation of Addition of Uterine Fluid to Human Embryo Culture Medium

Infertility or infertility affects about 15% of couples of reproductive age. It is estimated that 80 million people around the world suffer from this problem. Assisted reproduction techniques (ART) use culture media (during in vitro fertilization or early embryo development) with protein sources that are very different from natural sources. This media could produce an added stress to the gametes and embryos that could cause epigenetic alterations and health effects during adult life.

Our working hypothesis is based on studies in animal models (pig and cow), in which it was observed that the culture media with reproductive fluids used as additives instead of conventional sources of proteins (such as serum albumin) , produce embryos with an epigenetic profile closer to that of embryos generated in the maternal oviduct. Moreover, with these fluids, blastocysts obtained have a greater number of cells and hatchability than those produced with serum albumin alone.

Therefore, the University of Murcia, with an extensive experience in this area, and the IVI Murcia (Valencian Infertility Institute) research team have come together to launch this research project in order to determine the advantages of the use of human reproductive fluids as additives in embryonic culture media. To do this, 2 specific objectives are proposed,:

1. Creation of the first collection of human uterine fluid samples for Assisted Reproduction use.
2. To evaluate the use of uterine fluid as a media supplement in the culture media for assisted reproduction techniques, by evaluating embryo quality cultured with autologous fluid from voluntary patients (autologous culture)

This achievement would allow us the development of protocols in the nearest ART physiological conditions which represent not only a technical challenge but a biomedical responsibility that must be addressed to prevent future diseases of the offspring.

1. Creation of the first collection of human uterine fluid samples for Assisted Reproduction use. Female reproductive system will be collected at the Virgen de la Arrixaca University Clinical Hospital from surgical pieces of patients undergoing surgical procedures. IVI Murcia Clinics uterine fluids from oocyte donors will be collected. These fluids will be store in the Arrixaca Hospital Biobank for future researches.
2. Validation of the addition of uterine fluid to human embryonic culture media:

Couples undergoing assited Reproduction Treatment in IVI Murcia will be asked for their inclusion in this study. Uterine fluid will be take 48 hours after peak of LH (luteinizing hormone), in natural cycle. Oocytes from these patients, will be divided into two groups: Half of the zygotes of each patient, control group, will be grown in the conditions usually used in the clinic, and the other half, experimental group, will add 1-5% of uterine fluid from the patient (v / v)

On day 3 and on day 5 of culture the quality of the embryos in both groups will be assessed according to the usual criteria of the clinic and the best quality and highest probability of implantation will be transferred, following the usual clinical practice. The samples of uterine fluid, after the initial processing in the IVI-Murcia clinic will be transferred to the Department of Physiology of the University of Murcia where they will be fractionated to perform a quality control according to the following specifications:

Uterine fluid pH 7.0-7.6 Osmolarity (mOsm / kg) 260-320 Endotoxin (EU / mL) \<0.15 Sterility No growth

Only samples that meet these criteria will continue to be part of the study

Eligibility Criteria:
Inclusion Criteria:

* Couples in icsi treatment

Woman:

BMI between 17 and 30.Under controlled ovarian stimulation and at least 8 metaphase II oocytes collected

Man:

Sperm concentration higher than 5 million sperm / ml

Exclusion Criteria:

* Woman:

Diagnosis of endometriosis Uterine pathology that compromises embryonic development Patients undergoing treatment for repeat abortion or implantation failures Abnormal karyotype Patients included in the Pre-Implantation Genetic Diagnosis (DPI) program

Male:

Men whit abnormal karyotype, High values of DNA fragmentation Abnormal FISH (Fluorescence in situ Hybridization) Sperm from epididymal aspirate or testicular biopsy