Objectives: Parathyroid allotransplantation has been a curative option for patients with severe permanent hypoparathyroidism for more than 50 years. Despite advancements, parathyroid allotransplant outcomes still depend on reliable parameters, including HLA-allele matching criteria. Here, we report 2 cases of parathyroid allotransplant between ABO/Rh-compatible donors and recipients with HLA-DR and HLA-DQ allele match.
Materials and Methods: The recipients, aged 39 and 43 years, had been on the parathyroid allotransplant wait list for 5 years. Both experienced persistent hypocalcemia-related symptoms despite daily supplementation with intensive supplementation therapy. Ineffectiveness and side effects of increasing supplementation doses resulted in parathyroid allotransplant. The living unrelated donors, both with chronic kidney disease, underwent subtotal parathyroidectomy. Half of the retrieved parathyroid tissue was cryopreserved. After histopathological approval, parathyroid allotransplant was performed laparoscopically by transplanting parathyroid cells over the omentum. Preoperative assessments were negative, including panel reactive antibody and T-cell/B-cell crossmatch tests. Postoperatively, recipients received 10 mg/day methylprednisolone for 1 month. Outcomes were monitored for 5 years.
Results: After parathyroid allotransplant, recipient 1 showed reduced symptoms and medication requirements, decreasing to 600 mg calcium and 400 IU cholecalciferol daily, which were maintained for 4 years. However, by year 5, oral supplementation returned to initial levels. Recipient 2 exhibited similar improvements, with symptoms relieved and oral supplementation halved to half doses. Serum calcium levels at year 5 were 8.6 mg/dL and 9.0 mg/dL for recipients 1 and 2, respectively.
Conclusions: Our cases, to our knowledge, are the first to document panel-reactive antibody and T-cell and B-cell crossmatch-negative parathyroid allotransplant with HLA class II allele matching. HLA matching may enhance survival rates posttransplant. Only parathyroid cells were transplanted in both cases, and recipients received short-term immunosuppression with steroids. Further studies with larger cohorts are needed to establish the relationship between HLA allele matching and posttransplant survival rates.

Key words : Composite tissue, Graft function, HLA, Hypoparathyroidism, PRA

Introduction
Permanent hypoparathyroidism is characterized by chronic hypocalcemia, hyperphosphatemia, and reduced quality of life.^1,2 Parathyroid allotransplantation (PaTx) has emerged as a promising approach; however, its clinical use remains limited by variable graft survival and immunological uncertainty. Despite its relatively low immunogenicity,³ HLA class II-mediated alloimmune responses play a key role in graft survival.^4-6 HLA class II molecules (particularly HLA-DR and HLA-DQ) are central regulators of alloantigen presentation and T-cell activation. Multiple transplant studies have demonstrated that mismatches at class II loci are more strongly associated with graft rejection and long-term failure than mismatches at class I loci.^7-9 Moreover, donor-specific antibodies (DSAs) targeting HLA class II antigens correlate with antibody-mediated rejection and inferior graft outcomes, especially when mean fluorescence intensity levels are elevated.^8,10 Emerging molecular-matching approaches further support the concept that immunogenic load at the DRB1 and DQB1 loci predicts alloimmune risk more precisely than traditional serologic typing.^11-13 Despite substantial evidence in kidney, liver, and islet transplant, parathyroid transplant remains underexplored in this context. Reports in PaTx are limited, often involving small cohorts and heterogeneous immunosuppressive protocols.^1,2,14 Some studies have emphasized the role of HLA class I matching,^14-16 whereas others suggest that HLA class II compatibility may be more relevant for tolerance and graft persistence.¹⁷ Consequently, the actual clinical necessity of HLA class II matching in PaTx remains unclear. Clinical improvement may occur despite limited PTH recovery, suggesting partial graft activity or paracrine effects.^18,19 Therefore, this study evaluated long-term clinical and biochemical outcomes of HLA class II–matched PaTx with negative crossmatch status.

Materials and Methods
Parathyroid allotransplant has been conducted with the permission of the Turkish Ministry of Health, National Scientific Board for Transplantation. Since 2013, our PaTx unit is the first officially permitted center to conduct PaTx.

Patient selection
Two patients with severe permanent hypoparathyroidism were included in this study. The recipients were aged 39 years (recipient 1) and 43 years (recipient 2) and had been on the PaTx wait list for approximately 5 years. Both patients exhibited persistent hypocalcemia-related symptoms, including numbness and muscle weakness, despite intensive and optimized oral supplementation. At the time of transplant, both recipients were receiving 2000 mg of elemental calcium daily, 1600 IU of cholecalciferol, and 150 μg of alfacalcidol. Further dose escalation was not feasible because of limited clinical benefit and the occurrence of supplementation-related side effects. Given the refractory nature of their symptoms and biochemical instability, PaTx was indicated as a therapeutic intervention.

Donor characteristics
Parathyroid tissue was obtained from living unrelated donors who presented with parathyroid hyperplasia secondary to chronic kidney failure. All donors were under routine follow-up in the nephrology clinic and were referred to the general surgery department for surgical management. Before intervention, written informed consent was obtained from all donors in accordance with institutional and ethical guidelines. As part of the preoperative evaluation, comprehensive infectious disease screening was performed to ensure donor and recipient safety. Serological assessments included anti-HIV, anti-hepatitis C virus, hepatitis B virus markers (anti-HBc, anti-HBs, anti-HBe antibodies, and HBs antigen), cytomegalovirus immunoglobulin G and immunoglobulin M, and Epstein-Barr virus immunoglobulin G and immunoglobulin M antibodies. In addition, venereal disease research laboratory testing was conducted. Only donors with negative or clinically acceptable serological profiles proceeded to surgery. All donors underwent subtotal parathyroidectomy under standard surgical protocols. Parathyroid glands were carefully retrieved, and histopathological evaluation was performed to confirm the diagnosis of parathyroid hyperplasia and to verify suitability for transplant. Remaining parathyroid tissue was snap frozen via liquid nitrogen until transplant. The postoperative course was uneventful in all donors, and they were discharged on postoperative day 3 without any complications.

Preoperative immunological assessment
Comprehensive immunological compatibility assessments were performed for both donor-recipient pairs. Panel reactive antibody (PRA) screening was conducted and confirmed to be negative in both recipients. In addition, T-cell and B-cell crossmatch tests were performed preoperatively and yielded negative results, indicating the absence of preformed DSAs. HLA typing was performed between donors and recipients. Data were obtained from the HLA Typing Laboratory at Istinye University as a procured service.

Transplantation
Transplant procedures were conducted within our parathyroid transplantation unit. After immunological evaluation and histopathological validation of the donor tissue, we proceeded with PaTx. The technique for the isolation of parathyroid tissue cells was conducted by using our proprietary method (patent number 201815244), as previously described in the literature.¹⁴ Approximately 262 × 106 (recipient 1) to 419 × 106 (recipient 2) parathyroid suspension cells were administered to each recipient, derived from their respective donors. Each recipient was administered 500 mg intravenous methylprednisolone 1 day before the procedure and an additional 250 mg intravenous methylprednisolone 1 hour before transplant. Under general anesthesia, parathyroid cells were transplanted via a laparoscopic technique as previously described.²⁰ Cells were placed directly over the omentum. Recipients were discharged after 2 days of postoperative care without complications. Recipients were subjected to >5-year follow-up assessments.

Results
Both recipients had previously undergone total thyroidectomy at other medical facilities and were diagnosed with permanent hypoparathyroidism during the early postoperative period. Parathyroid allotransplant was performed approximately 9 years after the initial diagnosis of hypoparathyroidism. Notably, patients had prolonged time on the PaTx wait list, reflecting the severity and chronicity of their condition despite optimized medical management. The preoperative evaluation and the donor-recipient matching process followed a stepwise immunological assessment. Initial compatibility screening included ABO blood group matching and T- and B-cell crossmatch testing, all of which yielded negative results. After confirmation of negative crossmatch findings, PRA levels were negative in both recipients, allowing progression to HLA class II allele matching and subsequent transplant planning. An overview of the study timeline, including key assessment points and follow-up phases, is provided in Figure 1.

Clinical and biochemical outcomes after parathyroid allotransplant
Both recipients demonstrated clinically meaningful improvements following PaTx, with symptom relief and reduced medication requirements observed within the first 6 months posttransplant (Figure 2). Early postoperative follow-up revealed a marked decrease in oral calcium and cholecalciferol supplementation, accompanied by stabilization of serum calcium levels within or near the normal reference range. At 6 months, recipient 1 exhibited a substantial reduction in daily calcium supplementation from 2000 mg to 600 mg and cholecalciferol from 1600 IU to 400 IU. These reduced doses were consistently maintained for up to 4 years. During this period, hypocalcemia-related symptoms, including numbness and muscle weakness, were significantly alleviated, and no hospitalization related to hypocalcemia was required. However, at year 5 of follow-up, a gradual increase in oral supplementation was necessary to return to pretransplant doses, despite maintained serum calcium levels. Recipient 2 showed a comparable early response, and oral calcium and vitamin D supplementation were reduced by approximately 50%. Unlike recipient 1, this reduction was sustained throughout the 5-year follow-up period. At the final evaluation, recipient 2 continued on half-dose oral supplementation with stable biochemical parameters and minimal residual symptoms.

Sustained clinical benefit during mid-term follow-up
Between 1 and 4 years posttransplant, clinical benefits were maintained. Both recipients demonstrated stable serum calcium levels within or near the lower normal range while continuing reduced-dose oral supplementation. During this period, no hospitalizations related to hypocalcemia or acute neuromuscular symptoms were recorded. Quality-of-life assessments revealed sustained improvement in daily functioning, symptom control, and overall patient satisfaction. Importantly, no episodes of graft-related adverse events or complications associated with short-term immunosuppressive therapy were observed.

Long-term outcomes at 5-year follow-up
At the 5-year follow-up, divergent clinical trajectories were observed between the 2 recipients. Recipient 1 experienced a gradual increase in oral calcium and vitamin D requirements, ultimately returning to pretransplant supplementation levels. Despite this, serum calcium concentrations remained stable, and no severe hypocalcemia-related hospitalizations were reported. In contrast, recipient 2 maintained reduced supplementation at approximately half of the pretransplant doses, with continued symptom control and stable biochemical parameters. Serum calcium concentrations at 5 years posttransplant were 8.6 mg/dL for recipient 1 and 9.0 mg/dL for recipient 2, indicating persistent biochemical benefit despite differences in long-term supplementation requirements. Biochemical parameters, including serum calcium, phosphate, and intact PTH levels, remained stable throughout follow-up (Table 1).

Immunological compatibility and transplant performance
Immunological screening for both donor-recipient pairs was negative. High-resolution HLA typing demonstrated compatibility at the HLA class II level, with 1 shared HLA-DRB1 and 1 shared HLA-DQB1 allele identified in each donor-recipient pair (Table 2). In contrast, no complete allele matching was observed across HLA class I loci (HLA-A, HLA-B, HLA-C). The observed early and sustained clinical improvements suggest that immunological compatibility, particularly at the HLA class II level, may contribute to prolonged graft functionality and symptom control. These findings support the concept that HLA-matched PaTx can achieve long-term clinical benefit with minimal immunosuppressive exposure.

Discussion
Across solid-organ transplants, mismatches at HLA class II loci, particularly DR and DQ, have consistently shown stronger associations with rejection and long-term graft failure than class I mismatches.^4-6 Molecular and clinical data have indicated that class II disparities drive T-cell-mediated alloimmune activation and DSA development, both of which are central to chronic graft injury.^8,21,22 Although PaTx has historically been considered of relatively low immunogenic risk, especially when performed with cell suspensions rather than whole glands, this assumption has not eliminated graft loss or variable long-term function. Our 2 cases, which both had shared HLA-DRB1 and DQB1 alleles but no class I matching, showed early clinical improvement and prolonged functional benefit, suggesting that class II compatibility alone may be sufficient to modulate alloimmune risk in parathyroid cell grafts.^17,18,23 This observation aligned with the broader transplant literature, which has emphasized DR/DQ matching as a dominant immunogenetic factor and supported extending these principles to endocrine cell transplantation.^4,9 Our findings demonstrated that partial HLA-DR and DQB1 compatibility, in the setting of negative PRA and crossmatch, is associated with sustained clinical and biochemical benefit, even without full PTH recovery. The absence of preformed DSAs, together with class II compatibility,^8,10,21 likely contributed to the lack of overt rejection and allowed short-term corticosteroid immunosuppression. A key finding is that sustained clinical stabilization occurred despite low or fluctuating PTH levels. Both of our cases had reduced supplementation and normocalcemia maintenance, yet circulating intact PTH did not fully normalize. This dissociation challenges the long-standing paradigm that graft success must be defined with future studies. Interindividual variability likely reflected factors beyond HLA matching, including graft mass, cell viability at implantation, microvascularization, recipient metabolic differences, or subclinical immune activity not captured by routine testing. Such divergence has been also shown in kidney and islet transplant despite optimal matching, underscoring that HLA compatibility reduces but does not eliminate long-term functional decline.^4,21 Emerging tools, such as eplet mismatch analysis and PIRCHE-II models, have shown superior predictive performance for immunogenic load at class II loci in kidney transplant.^11-13 Although not applied in our study, our results suggested that PaTx could benefit from similar molecular-level matching in the future. Given the small tissue volume and minimal immunosuppression used in PaTx, even subtle immunogenic differences might influence long-term persistence. Integrating high-resolution molecular matching could therefore enhance donor selection in endocrine cell transplant.^12,24,25 The main limitation of our study was the small sample size, which is inherent to the rarity of PaTx. The absence of longitudinal DSA monitoring and intermittent sampling during the COVID-19 pandemic restricted mechanistic interpretation; however, the 5-year follow-up still provides valuable long-term insight.

Conclusions
Our findings indicated that immunogenetic compatibility was associated with prolonged clinical benefit in PaTx with minimal immunosuppression. Importantly, clinical stabilization did not require full systemic PTH recovery, suggesting that graft success in PaTx should not be defined solely by systemic PTH normalization. Our results support incorporating HLA class II matching in PaTx donor selection strategies. Future studies with molecular compatibility tools are needed to optimize endocrine cell transplant outcomes.