HLA-Identical Embryos Selected after in Vitro Fertilization and Pre-Implantation Genetic Diagnosis Combined with HLA Typing: A Promising Option for Successful Allogeneic Hematopoietic Stem Cell Transplantation for Children with Genetic or Malignant Disorders Who Lack an HLA-Matched Related or Unrelated Donor
Authors: Goussetis Eugene , Tsirigotis Panagiotis , Peristeri Ioulia , Kitra Vassiliki , Avgerinou Georgia , Vessalas George , Paisiou Anna , Zisaki Kalliopi , Papassotiriou Ioannis , Kanavakis Emmanuel , Graphakos Stelios 
Published: October 2019

Abstract
Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative option for many children with various genetic or malignant diseases. However not all children in need for HSCT have a suitable donor available. For such cases in vitro fertilization (IVF) with pre-implantation genetic diagnosis (PGD) combined with human leukocyte antigen (HLA) tissue typing has been used to select an IVF healthy and HLA-matched embryo in order to give birth to a child who may serve as a stem cell donor. We report our experience with five children transplanted from HLA-matched siblings selected after IVF and PGD in our center.
Methods and Patients: The clinical protocol was approved by the center’s Institutional Research and Ethics Committee. Written informed consent was obtained from parents.
Hormonal stimulation for IVF, culture techniques, PGD and HLA-typing were performed according to standard protocols. Detection of the genetic disease mutation was performed by using mini-sequencing. Embryos genotyping was performed by using multiplex polymerase chain reaction (PCR) analysis of informative polymorphic short tandem repeat (STR) markers.
Healthy and HLA-identical embryos were transferred 6 days after fertilization to the uterus of the respective mothers. Confirmatory genetic testing and HLA-typing were performed during the first trimester of pregnancy by using chorionic villus sampling.
Patient’s characteristics are shown in Table 1. None of our patients had an available matched sibling donor. All patients underwent myeloablative conditioning consisting of busulfan, cyclophosphamide and antithymocytic globulin. GVHD prophylaxis consisted of CyA plus Methotrexate. Stem cell graft consisted of bone marrow plus cord blood in 4 out of 5 patients, while one patient received bone marrow only.
Results: All couples underwent a single cycle of IVF resulting into the generation of 78 embryos. Fourteen embryos were healthy and HLA-identical with their sibling and 10 of them were transferred to the uterus of the respective mother giving birth to a total of 6 healthy children. Results of IVF, PGD and HLA-typing are shown in more detail in Table 2.
The median age of the donor at the time of HSCT was 16 months. The median total nucleated cell (TNC) concentration of cord blood grafts was 2.3x107/kg, while the median CD34+ cell was 0.47x105/kg. The median TNC and CD34 cell concentration of bone marrow grafts was 1,97x108/kg and 5,09x106/kg respectively. All patients achieved sustained engraftment of donor cells. Mild acute GVHD of the skin was observed in one out of 5 patients. As of today, with a median follow up period of 4 years, all patients are alive with complete donor chimerism, without chronic GVHD and free of disease.
Conclusions: IVF and PGD/HLA typing methodology should be considered and discussed with parents in cases where no HLA-matched donor is available. The procedure can be applied only in cases requiring non-urgent HSCT, and in parents of reproductive age. Table 1:Patient’s characteristicsPatientSexAge (years)DiseasePrevious treatmentsDate of transplant1Male4,5Chronic granulomatous diseaseInterferon-gamma, antibiotics2/10/20072Male4Chronic granulomatous diseaseantibiotics1/7/20083Male11Chronic myeloid leukemiaHydroxyurea, imatinib21/7/20094Male4Thalassemia majorRBC transfusions4/5/20105Female4,5Diamond-Blackfan anemiaCorticosteroids, RBC transfusions30/4/2013
Table 2: Results of IVF, PGD and HLA-typing Patients IVF (cycles) Number of embryos after IVF Number of healthy and HLA-identical embryos Number of healthy and HLA-identical transferred embryos Number of healthy and HLA-identical borned children 1 1 37 6 2 1 2 1 11 2 2 1 3 1 10 2 2 1 4 1 10 1 1 1 5 1 10 3 3 2

Disclosures
No relevant conflicts of interest to declare.