Peripheral blood mononuclear cells composition for the autoimmune infertility treatment in IVF practice A.M. Feskov, I.A. Feskova, I.S. Zhylkova, N.N. Sotnik, V.A. Feskov Clinic of Professor Feskov A.M., Ukraine and S. V. Zhilkov MEZADATA Medical LLC, USA Presented in Prague, Czech Republic, December 2014

Embryo implantation: who is responsible? 1. Embryo quality VS 2. Endometrium quality

Scheme of the best result: Immune system endometrium Endocrine system High quality embryo Gametes quality

Role of immune system in embryo implantation Maternal immune system recognizes certain presence of the developing and implanting embryo in the Fallopian tube and uterus cavity due to embryo- and species-specific signals such as degraded products of zona pellucida glycoprotein and/or HCG. Effector immune cells move to the ovary and the endometrium via blood circulation to regulate corpus luteum (CL) function and induce endometrial differentiation. Local immune cells at the implantation site also contribute to induction of embryo invasion, secreting chemoattractants by HCG stimulation. To achieve smooth transformation into CL of pregnancy, it is reasonable to expect that immune cells transmit information from the developing embryo to CL before embryo implantation. In support of this hypothesis, the HLA-DR and leukocyte functional antigen (LFA)-3/CD58, which could mediate the direct interaction with T lymphocytes, were expressed on human CL during corpus luteum formation.

Immune system and peripheral blood mononuclear cells: what has been already known? Feskov, A., et al. US 61/629,651, 11/23/2011 TITLE OF THE INVENTION: METHOD OF IN VITRO FERTILIZATION WITH DELAY OF EMBRYO TRANSFER AND USE OF PERIPHERAL BLOOD MONONUCLEAR CELLS … The in vitro fertilization method of the invention provides the use of the PBMCs obtained by the above culturing technique in order to improve pregnancy inception… Okitsu O, Kiyokawa M, Oda T, Miyake K, Sato Y, Fujiwara H. J Reprod Immunol. 2011 Dec;92(1-2):82-7. doi: 10.1016/j.jri.2011.07.001. Epub 2011 Oct 27 Intrauterine administration of autologous peripheral blood mononuclear cells increases clinical pregnancy rates in frozen/thawed embryo transfer cycles of patients with repeated implantation failure …intrauterine administration of autologous PBMC freshly isolated from patients, effectively improves embryo implantation in patients with three or more IVF failures… Feskov O.M., Feskova I.A., Zhylkova I.S., Bezpechna I.M., Blazhko O.V. World of Medicine and Biology, №3, pp. 132-134, 2011. The influence of intrauterine administration of peripheral blood mononuclear cells on implantation rates in “fresh” and “cryo” IVF programs …This study showed that intrauterine application of PBMC definitely (р <0,05) increased the implantation rates and clinical pregnancy rates both after embryo transfer in fresh IVF cycles and after the thawed embryos transfer… Yu N1, Yang J, Guo Y, Fang J, Yin T, Luo J, Li X, Li W, Zhao Q, Zou Y, Xu W. Am J Reprod Immunol. 2014 Jan;71(1):24-33. doi: 10.1111/aji.12150. Epub 2013 Aug 1. Intrauterine administration of peripheral blood mononuclear cells (PBMCs) improves endometrial receptivity in mice with embryonic implantation dysfunction …Embryo implantation dysfunction model group showed a significant decrease in pregnancy rate, implantation sites and the expression of both the endometrial LIF and VEGF during the implantation window. EID with PBMCs group showed a higher pregnancy rate and endometrial LIF and VEGF expression compared to EID group…

Aim of present investigation: Study question: The influence of the intrauterine peripheral blood mononuclear cells (PBMCs) application on the embryo implantation rates was studied in patients with repeated implantation failures (RIF) in IVF cycles. Study design: The study population consists of 180 infertile female patients, who already had two or more unsuccessful IVF attempts with transfer of the high quality embryos in the past.

Participants: Group 1: 90 patients with the median age of 34.2±4.3 years old. Intrauterine PBMCs were applied in group 1 consequentially: First in “fresh” IVF cycle: PBMCs were applied for 90 patients. 28 clinical pregnancies were achieved after 90 ET of “fresh” embryos. The rest 62 patients moved to the cryo-IVF attempt. For these 62 patients, PBMCs were again applied prior transfer of the vitrified embryos that were under cryo-preservation after first IVF attempt above. 26 clinical pregnancies were achieved after 62 ET of thawed embryos.

Participants: Group 2: 90 patients with the median age of 35.2±5.6 years old. Intrauterine PBMCs were not applied in group 2: First in “fresh” IVF cycle 20 clinical pregnancies were achieved after 90 ET of “fresh” embryos. The rest 70 patients moved to the cryo-IVF attempt. For these 70 patients, the transfer of the vitrified embryos was done. 15 clinical pregnancies were achieved after 70 ET of thawed embryos.

IVF procedure COS (controlled ovary stimulation): protocol with a-GnRH was used for patients of both groups. The period of ovary stimulation for every patient was no less then 10 days. To the moment of transvaginal puncture, the average size of follicles was about 18 mm. To maintain the luteal phase, the medicines with progesterone were used for patients in both groups. Embryo culture: After oocytes were retrieved, they were cultured in Universal IVF Medium (Medicult) in 6.0 % CO2 and 37.0ºC. The embryos were cultured in Global Total Medium (LifeGlobal Group).

Preparation and intrauterine application of PBMC 1. PBMCs were isolated by Ficoll-Hypaque centrifugation. 2. After extraction, PBMCs were cultured in vitro. The conditions of culture were carbon dioxide atmosphere and 37 ºC. 3. Fresh PBMCs were obtained from the same patient few days after the first portion of PBMCs culture. Freshly isolated and cultured PBMCs were mixed and transferred to uterus cavity.

Results: Fresh-IVF Cryo- IVF Total Clinical Pregnancy Rate   Clinical Pregnancy Rate Group 1 (with PBMCs) Group 2 (without PBMCs) Fresh-IVF 31.1% (28 pregnancies after 90 ET) 22.2% (20 pregnancies after 90 ET) Cryo- IVF 41.9% (26 pregnancies after 62 ET) 21.4% (15 pregnancies after 70 ET) Total 60.0% (54 pregnancies for 90 patients) 38.9% (35 pregnancies for 90 patients)

Results: Cryo- IVF Clinical Pregnancy Rate 21.4% 41.9% Special attention should be addressed to the clinical pregnancy rate after the transfer of thawed embryos in group with PBMCs application. It is further demonstrated that the combining of delayed or Cryo-IVF technique (cryopreservation IVF technique or “Cryo-IVF”) with enhanced preparation of the woman’s uterus by the administration of her own PBMCs prior embryo transfer does result in a significant increase in the rate of pregnancy of infertile women as compared to previously-known IVF procedures; possibly it is a synergistic effect both of allowing the female patient’s autoimmune system to regain hormonal balance after COS and allowing the inception process to be realized in uterine cavity of improved quality.   Clinical Pregnancy Rate Group 1 (with PBMCs) Group 2 (without PBMCs) Cryo- IVF 41.9% (26 pregnancies after 62 ET) 21.4% (15 pregnancies after 70 ET)

Conclusions: Intrauterine application of PMBC increases rates of the embryo implantation and can be used for patients with repeated implantation failures (RIF). For patients with RIF, the clinical pregnancy rate after applying of the PBMC is significantly higher compare with patients without PBMC use (p<0.05); this is observed for embryo transfers both in the “fresh” and “cryo” IVF cycles. PBMC method might be also effective in infertility treatment of older female patients, because among this age group the RIF patients constitute significant majority.

Thank you for your attention ! Contact: Zhilkova@Feskov.com.ua