A healthy HLA-matched baby born by using a combination of aCGH and Karyomapping: the first latin american case

PGD for HLA typing is a procedure that can be performed when an affected child requires a transplant to treat a non-hereditary disorder related to the hematopoietic and/or immune system. Hematopoietic stem cell transplantation from an HLA-identical donor provides the best treatment option. Three conventional ovarian stimulation procedures for IVF were performed in a couple with a 10-year-old child diagnosed with T-cell acute lymphoblastic leukemia of high risk. Trophectoderm biopsy and aCGH examination were performed on 15 blastocysts, three on the first IVF procedure, four on the second cycle, and eight on the third. Three euploid blastocysts HLA-compatible with the genome of the affected child were identified. One euploid blastocyst HLA-compatible with the affected child was warmed and transferred, resulting in an HLA-matched live birth. In conclusion, combined aCGH for aneuploidy screening and Karyomapping may be performed in a single biopsy procedure.


INTRODUCTION
T-cell acute lymphoblastic leukemia is an immature lymphoid tumor characterized by diffuse infiltration of the bone marrow by malignant hematopoietic cells expressing immature T-cell markers. Clinically, patients with T-cell acute lymphoblastic leukemia have elevated white blood cell counts and hematopoietic insufficiency, neutropenia, anemia, and thrombocytopenia. In addition, they often show thymic mediastinal masses and meningeal infiltration of the central nervous system at the time of diagnosis. Immature T-cell tumors presenting as thymic masses with limited bone marrow infiltration are diagnosed instead as T-lymphoblastic lymphomas (Belver & Ferrando, 2016).
Preimplantation genetic diagnosis (PGD) has been applied as a selection tool to avoid inheritable diseases (Ren et al., 2016). However, the primary limitation of PGD for single gene disorders is the small amount of DNA. Whole genome amplification is required to detect the mutation. DNA amplification may produce a preferential amplification of one parental allele, which can cause heterozygous loci to appear homozygous. This process, known as allele dropout (ADO), may lead to misdiagnosis. In addition, the extreme sensitivity of amplification and detection methods makes them more susceptible to contamination. To avoid these potential causes of misdiagnosis, standard practice includes targeted haplotyping of the genetic locus by multiplex amplification of one or more closely related or intragenic polymorphic markers with or without direct detection of mutations (Natesan et al., 2014).
With Karyomapping, the entire parental genome can be haplotyped using the high-density single nucleotide polymorphism (SNP) method. Karyomapping provides an integral diagnostic method based on the ligation of singlegene defects. Genotyping parents and close relatives with known diseases eliminates the need for custom test development, and since Karyomapping defines four sets of SNP markers for each of the parental chromosomes, it allows the simultaneous execution of molecular cytogenetic analysis (Thornhill et al., 2015).
PGD for HLA typing is a procedure that can be performed as a single indication when the affected child requires a transplant to treat a non-hereditary disorder related to the hematopoietic and/or immune system, such as certain types of leukemia, or simultaneously with PGD to rule out a disease linked to a single family gene (E.g. β thalassemia) and allow pregnancy to develop with an unaffected HLAcompatible embryo (Kakourou et al., 2016).
Hematopoietic stem cell transplantation from an HLAidentical donor is the best treatment option for bone marrow transplantion, as it reduces the incidence of graft-versus-host disease (Soni et al., 2014). This case report describes what was, to our knowledge, the first PGD live birth of an HLAmatched child in Latin America using aCGH for chromosome screening and Karyomapping for HLA matching.

CASE REPORT
In November of 2014, a couple came to our assisted reproduction clinic with plans of having a child. They had been living together for 15 years and had two living children, a boy and girl. Recently, however, the couple had a miscarriage (2014).
Their 10-year-old boy had been diagnosed with T-cell acute lymphoblastic leukemia, but tests for bone marrow transplantation performed at the National Institute for Neoplastic Diseases in Peru revealed he and his sister were not histocompatible. After having searched the Bone Marrow Donors Worldwide database, they were offered the possibility of undergoing IVF with PGD. They were informed of the scope and limitations of PGD in HLA typing.
Following our clinical protocol, the 36-year-old woman was instructed to perform a hormone profile test and undergo pelvic ultrasound examination. The tests revealed she had polycystic ovaries, and treatment with Metformin was prescribed (Metformin -1500 mg/day). Though her hormone profile was normal, prolactin was found to be elevated (39 ng/mL) and she was treated with Cabergoline (Dostinex -Pfizer).
The 42-year-old man was instructed to undergo conventional sperm analysis. According to the WHO 2010 manual (World Health Organization, 2010), he was normozoospermic. The couple was also advised to undergo psychological counseling. Considering their clinical history and the evident determination of the couple, it was decided they would undergo IVF cycles in combination with PGD for HLA matching.

IVF protocol
Three controlled ovarian stimulation (COS) cycles were performed. The patients were properly advised about the procedures and signed informed consent terms.
The last COS cycle was performed in April of 2015. Patient was administrated 150 IU of FSH/LH (Bravelle -Ferring) and 75 IU of hp-HMG (Menopur-Ferring) from day 2 to day 11 of the cycle. Ant-GnRH (Cetrotide-Merck Serono) with follicle ≥14 (d 8, 9, 10, and 11) and ovulation was synchronized with Ana-GnRH (Gonapeptil-Ferring). In this cycle, 33 oocytes were aspirated; 26 were mature, 18 were fertilized and eight blastocysts were biopsied on days 5/6. On the same day, all blastocyst generated in previous cycles were warmed and biopsied. All blastocysts were vitrified for genetics analysis. (Table 1).
Trophectoderm biopsy and aCGH examination were performed on 15 blastocysts, three on the first IVF procedure, four on the second cycle, and eight on the third. PGS with aCGH results showed that eight blastocysts were euploid ( Table 2).
The same DNA isolated and amplified for aCGH was used to perform PGD with Karyomapping. In this case, the HLA region has 128 informative SNPs in the mother and 78 in the father. SNPs were classified as compatible or noncompatible with respect to the analysis of the same SNPs in the affected child's cells.
The SNPs reported as compatible were grouped as P1 (paternal origin) and M1 (maternal origin) (Table 3). In order for an embryo to be diagnosed as compatible, it had to present the M1/P1 pattern (Table 3). Three euploid blastocysts HLA-compatible with the affected child genome were identified ( Table 4).

Endometrium preparation for Embryo Transfer
In the middle of the luteal phase (day 21) of the previous menstrual cycle the patient was administered Leuprolide Acetate (Lupron Depot ® -Abbott), a GnRH agonist, 3.75 mg intra muscular. On the first day of subsequent menses, transvaginal ultrasound was performed and hormone therapy with Estradiol Valerate (Progynova ® -Shering) was initiated in a variable dose protocol, with an oral dose of 4 mg/day from day 1 to day 7, and 6 mg/day from day 8 until an endometrial thickness of 10 mm and trilaminar pattern was reached, on day 18. At that time, Micronized Progesterone 600 mg vaginal per day was initiated and the dose of Estradiol Valerate was increased to 8 mg/day. Deferred embryo transfer was performed in July of 2015. After 5.5 days of progesterone, a single HLA-matched embryo was warmed and cultured. Two hours later, the embryo re-expanded and was transferred with the aid of ultrasound guidance. Two weeks after embryo transfer, a first β-HCG score of 4,420 mIU/ml was obtained; and two days later, the value was 10,494 mIU/ml.
The patient went regularly to her prenatal checkups at Clínica Concebir San Isidro -Lima -Peru, and on March 28 th , 2016, a cesarean section was performed, resulting in the birth of a male newborn weighing 3.350 kg with an Apgar score of 9/9. At birth, umbilical cord stem cells were collected and cryopreserved.
At the time this report was being written, the parents performed a first post-natal HLA-compatibility test and found that both children had matching HLA, which made it possible for bone marrow transplantation to occur.

DISCUSSION
Fertility treatments may be combined with genetic tests to prevent the occurrence of chromosomal alterations (aCGH) or genetic diseases (Karyomapping). Verlinsky et al. (2001) described the first case of PGD + HLA-typing for Fanconi anemia. To our knowledge, the present case report describes the first live birth of an HLA-matched baby tested with PGD in Latin America by using a combination of aCGH and Karyomapping.
This case report described two clinical challenges. The first was the PCO-like ovaries. An antagonist GnRH protocol was used and modified after the first cycle, because of the low quality of oocytes determined by low blastocyst formation. In the following cycles the dose of hp-HMG was decreased on account of the polycystic ovaries. A GnRH agonist was also used as a final trigger for oocyte maturation to minimize the risk of ovarian hyperstimulation syndrome (Kol, 2004). In women with polycystic ovary syndrome or polycystic ovaries, Metformin before or during assisted reproductive technology cycles increases clinical pregnancy rates and decreases the risk of ovarian hyperstimulation syndrome. However, there is no conclusive evidence of benefits in life birth rates (Tso et al., 2015).   Table 3. Individuals reported as compatible in Karyomapping were grouped as P1 (paternal origin) and M1 (maternal origin). The second challenge was the combination of PGD techniques (aCGH and Karyomapping). There are few reports describing this type of protocol. One of the studies was performed between 2007 and 2014 and enrolled 12 couples interested in taking part in a PGD-HLA-typing program. All couples had children affected by either a genetic or an acquired disease affecting their hematopoietic and/or immune system. Seven couples were treated in 26 cycles and two healthy HLA-matched babies were born, leading to a live birth rate of 28.6% per transfer and 7.7% per initiated cycle (Fernández et al., 2014). Nevertheless, PGD was not combined with chromosome screening for aneuploidies.

HLA -RESULTS INTERPRETATION
Three IVF cycles were performed, since a large number of embryos was required to obtain a chromosomally euploid HLA-matched embryo. The multidisciplinary team, which included gynecologists, biologists and geneticists, considered the conditions of these types of diagnoses were very demanding. In the three IVF cycles performed, trophectoderm biopsies were performed on 15 blastocysts, of which eight (53.3%) were euploid on aCGH; and three (20%) were HLA-compatible with the affected child. Similar results have been reported in a French study, in which 19% (3 out of 16) of the embryos were unaffected and HLAcompatible (Lamazou et al., 2011).
IVF combined with HLA-typing has become another option to find HLA-matched donors. The literature indicates that the chances of finding an HLA-identical donor within a family or in Hematopoietic stem cell banks are low (Fernández et al., 2014). In pathological conditions, PGD can be used to select unaffected HLA-matched embryos for transfer. After birth, umbilical cord and blood stem cells may be collected for transplantation into an affected child, a proven strategy to cure these children (Natesan et al., 2014).
Hematopoietic stem cell (HSC) transplantation from an HLA-identical donor is the best therapeutic option for genetic diseases affecting the hematopoietic and/ or immune system in children, and may be an option for individuals with acquired diseases as well (Gaziev et al., 2000;Hellani et al., 2012).
Regarding legislation, there is no regulation for ART procedures in Peru. All clinical and laboratory practices were performed following the highest ethical standards and in close compliance with European legislation (Van de Velde et al., 2009). Nevertheless, there are ethical concerns when this type of procedure is carried out (Pennings et al., 2002).
In conclusion, combined aCGH for aneuploidy screening and Karyomapping was performed in a single biopsy procedure, resulting in an HLA-matched live birth. This type of procedure may be extended to other single gene disorders.