Testicular Thrombotic Microangiopathy: An Unrecognized Complication

Abstract

Thrombotic microangiopathy (TMA) causes global endothelial damage and multiorgan injury. No study has described reproductive organ involvement in TMA. Our study aimed to characterize testicular involvement in TMA. We reviewed autopsies from 4 patients who underwent hematopoietic cell transplant (HCT) complicated by TMA. Three patients had striking histologic evidence of TMA while the fourth had normal testicular histology. This suggests TMA injures the testicles and may adversely affect fertility. There is now an urgent need for a larger analysis of reproductive organ involvement in TMA.

Introduction:

Thrombotic microangiopathy (TMA) is a systemic vascular disease caused by multiple distinct pathologic processes. Subtypes of TMA include thrombotic thrombocytopenic purpura (TTP), complement-mediated TMA and Shiga toxin-mediated TMA, among others¹. TMAs classically cause endothelial injury and organ dysfunction in the kidneys, lungs, gastrointestinal tract and central nervous system. These tissues are affected at different frequencies depending on the causative subtype of TMA¹. Tissue damage from these entities occurs from a vicious cycle of endothelial injury, inflammation and thrombosis^{2, 3}. This cycle causes global endothelial damage and there are likely other organ systems injured by TMAs that result in currently unrecognized complications.

Complement-mediated TMA is common after hematopoietic cell transplant (HCT) and complicates between 10 and 35% of HCTs^2-4. Complement blocking therapies have increased survival in complement-mediated TMA and there is now a need to focus on long-term complication from TMA. No study has investigated reproductive system complications from TMAs. Infertility is an important complication of chemotherapy and HCT and approximately 70% of males who undergo HCT will develop permanent azoospermia ⁵. Sperm cryopreservation prior to HCT or chemotherapy is the current standard of care for fertility preservation, though several clinical trials are investigating cryopreservation of testicular tissue ⁶. Alkylators, platinum and radiation therapy are known risk factors for infertility, however there is no known association between TMA and infertility.

TMA is a systemic vascular disease and can theoretically affect any organ system. The effects of TMA on the reproductive system are unknown and fertility counseling is not routinely performed in patients diagnosed with TMA. The goal of our study was to investigate and characterize testicular involvement in male patients with TMA after HCT.

Methods:

We reviewed autopsy specimens from males who underwent HCT complicated by systemic, complement-mediated TMA at Cincinnati Children’s Hospital Medical Center between 2010 and 2015. Seven males who underwent autopsy during this timeframe were identified, and 4 of these patients had testicular tissue available for review in our study. Testicular tissue from a 17-year-old patient undergoing orchiectomy for evaluation of a testicular mass (benign) was used as a control. All specimens were reviewed with a senior pathologist at our institution. Previously recognized histologic findings used to diagnose TMA in other organ systems were used to evaluate testicular tissue⁷. These criteria included vascular lumen obliteration, intimal thickening and lamellated endothelial basement membranes, among others⁷. Chart reviews were performed for clinical details and patient demographics. All HCT patients at our institution are prospectively screened for TMA with weekly blood sC5b-9 levels and urine protein: creatinine ratios.

Results:

Patient demographics and HCT complications for all patients studied are shown in Table 1. The median age at time of HCT for patients with testicular TMA was 18 years old (range 14-29) and the HCT patient without testicular TMA (patient 4) was 23 years old at time of HCT. All HCT patients died from complications related to infection (n=4) and/or graft versus host disease (GvHD, n=3). The median time to death after HCT was 338 days (range, 164-828 days). Patient 4 was diagnosed with multi-organ TMA at death based on autopsy findings (i.e. did not have an existing TMA diagnosis prior to autopsy). The median time to TMA diagnosis after HCT for the remaining 3 patients was 39 days (range, 20-217 days). Two patients with testicular TMA received the C5 inhibitor, eculizumab, for TMA therapy. The remaining patient was diagnosed with TMA prior to routine eculizumab use for the treatment of TMA. The maximum sC5b-9 values for patients with testicular TMA were 628ng/mL (patient 1), 769 ng/mL (patient 2) and 385ng/mL (patient 3; normal range, < 244ng/mL). Blood sC5b-9 levels were not available for patient 4. A testosterone level was obtained prior to HCT in Patient 1 and was normal, but was not repeated prior to death. Fertility assessments were not obtained in any of the remaining patients with testicular TMA.

Table 1:

Patient Demographics and HCT Complications.

	Patient 1	Patient 2	Patient 3	Patient 4
Age at HCT (years)	18	29	14	23
Diagnosis	XLP-like	WAS	XIAP	AML
Conditioning	Alemtuzumab Fludarabine Melphalan	Busulfan Cyclophosphamide ATG	Alemtuzumab Fludarabine Melphalan	Fludarabine Melphalan
Donor, Match	Unrelated, 8/8	Sibling, 10/10	Sibling, 10/10	Unrelated, 10/10
Graft	PBSC	BM	BM	PBSC
Timing of TMA Diagnosis (days after HCT)	39	20	217	At Death
sC5b9 Max Value (ng/mL)	628	769	385	N/A
Eculizumab Therapy	No	Yes	Yes	No
Testicular TMA	Yes	Yes	Yes	No
GvHD	Acute	Acute	Acute, Chronic	Acute, Chronic
Timing of Death (days after HCT)	193	164	483	828
Cause of Death	Bacterial and Viral Sepsis, TMA	Disseminated Fungal Infection and GvHD	GvHD, Multiple Viremias	GvHD, Sepsis

Three patients had striking histologic evidence of testicular TMA. The overall severity of these findings varied but was most notable in patient 1 (Figure 1, Panel A). Testicular tissue had chronic damage to both the vasculature (small and moderate sized vessels) and surrounding stroma. Capillaries had abnormal wall thickening, lamellated basement membranes and thickened intima. The surrounding testicular stroma was expanded with significant hyalinization. These abnormal stromal findings are consistent with testicular hypoxia due to markedly increased capillary wall to lumen ratios and chronically diminished tissue perfusion. Patient 4 underwent HCT complicated by TMA but did not have histologic findings of TMA on testicular tissue examination (Figure 1, Panel B). Testicular tissue in this patient had normocellular stroma with preserved architecture. Vascular lumen sizes and basement membranes were normal, consistent with findings seen in the non-HCT control patient (Figure 1, Panel C).

Figure 1: Vascular Injury in Testicular Tissue Associated with TMA after HCT.

Blue arrows in Panel A show capillaries and small to medium sized vessels with evidence of chronic vascular changes associated with TMA. These rigid-appearing vessels have abnormally increased wall to lumen ratios, best seen at higher magnification (24x and 38.5x). Blood vessel basement membranes are thickened and lamellated, with occasional splintering. The collagenous stroma surrounding vessels shows hyalinizing fibrosis, suggesting chronic injury (blue asterisks), with an expansion evident even at low power (3x). Panel B shows overall normal vessels (black arrows), with no evidence of TMA-related vascular injury as shown in Panel A. The stroma is delicate and light, with normal cellularity and no fibrous expansion (black asterisks). Independently, the basement membrane of atrophic seminiferous tubules is thickened (green arc). Panel C shows normal testicular vasculature (black arrows) and stroma (black asterisks). The high power inset images highlight representative vessels present in each respective field. Images were obtained using a Leica’s Aperio AT2 whole slide scanner, and Leica’s eSlide Manager software platform. Images were captured at 40x magnification in eSlide Manager’s Web Viewer. Image Resolution (microns/pixel): 0.2517.

Discussion:

Testicular TMA has not been previously described in any patient population. Our study suggests testicular involvement in TMA is underdiagnosed, likely due to absence of symptoms. Patient 4 had essentially normal (mild tubular atrophy) testicular histology, which suggests testicular architecture can be preserved even after HCT, and that testicular involvement is not inevitable even in those with very active TMA. All patients in this study suffered from GvHD after HCT and the possibility of GvHD-related testicular injury was considered in this study. However, the abnormal histology observed in patients 1-3 was highly consistent with published TMA-related changes⁷ and not felt to be GvHD-related. Furthermore, there was a paucity of inflammatory infiltrate, apoptotic bodies and epithelial changes that would support a diagnosis of GvHD. The absence of inflammatory infiltrate also went against an infectious process.

The observed testicular stromal and vascular injury from TMA were extensive and the timing of testicular TMA onset is unknown. It is conceivable that chronic, smoldering TMA results in the observed testicular histology, however it is also possible that severe acute TMA-mediated vascular injury is capable of inflicting this damage. We therefore hypothesize that TMA-mediated vascular injury and tissue hypoxia irreversibly injures testicular tissue and can occur early or late in the disease course. Future studies are needed to identify the timing of testicular injury in TMA, however these studies will be limited by testicular tissue availability.

The clinical relevance of testicular TMA is also unknown, however further attention must now be paid to fertility after TMA diagnosis. The relationship between TMA and fertility is highly relevant to patients treated with minimal or even no preparative chemotherapy prior to HCT, and is perhaps most pertinent to patients with recurrent TMA syndromes, such as TTP and atypical hemolytic uremic syndrome, in whom infertility is not commonly considered. The fertility effects of vasculitic processes have been studied in patients with rheumatologic diseases but the majority of patients in these studies were treated with medications that are known to impair fertility (e.g. cyclophosphamide) and limited conclusions can be made regarding the direct association between vasculitis and fertility ^{8, 9}.

In conclusion, we have shown that testicular injury is a previously unrecognized complication of TMA. Future studies are needed to confirm when TMA-mediated testicular injury occurs and whether the observed tissue injury is associated with impaired fertility. Confirmation of a clinical impact of TMA on fertility would result in widespread clinical practice changes. Extension of these findings to investigate female reproductive organ involvement with TMA is also needed.

Abbreviations:

TMA

Thrombotic microangiopathy

HCT

Hematopoietic cell transplant

TTP

Thrombotic thrombocytopenic purpura

GvHD

Graft versus host disease