Is Routine Pathology Evaluation of Tissue Removed for Fertility Preservation Necessary?

Structured Abstract

Background:

For all fertility preservation (FP) cases at our institution, a biopsy is performed for routine pathology from all gonadal tissue removed. This is not standard at all centers. We reviewed our experience with biopsy for pathological evaluation of ovarian and testicular specimens in FP cases to determine clinical utility.

Methods:

The medical records of individuals who underwent ovarian tissue cryopreservation (OTC) or testicular tissue cryopreservation (TTC) between 2011 and 2023 were retrospectively reviewed under an IRB-approved study at a free-standing tertiary care children’s hospital. Patient demographics, diagnosis, operative characteristics, and pathology results were collected.

Results:

One-hundred and eighty-three patients underwent OTC, and 134 patients underwent TTC. All patients had their gonadal tissue biopsied for routine pathology. Malignancy was identified in the biopsies of 4 OTC patients (2.2%) and 2 TTC patients (1.5%). Two OTC patients (1.1%) and 2 TTC patients (1.5%) did not have germ cells identified in their biopsy. All OTC and TTC patients and families elected to continue storing tissue for FP after discussion of pathology findings.

Conclusions:

Pathology results provide another data point to help inform patients and their families when making decisions on ovarian or testicular tissue storage and on how tissue may be utilized in the future to restore fertility and/or hormones. There is a low rate of identifying malignancy in gonadal tissue biopsies taken from FP specimens even in patients with known malignancy. However, when malignancy was identified, it could be unexpected and alter the diagnosis and treatment plan significantly for patients.

Introduction

Iatrogenic gonadal insufficiency in children and adolescents is a late effect of many treatments for pediatric cancers. Other therapies that use gonadotoxic treatments include conditioning regimens for stem cell transplants, and treatment of congenital or acquired rheumatologic conditions [1]. For prepubertal patients, ovarian tissue cryopreservation (OTC) and testicular tissue cryopreservation (TTC) are presently the only options for fertility preservation (FP) prior to the start of therapy. Postpubertal patients may opt for OTC and TTC for FP to not delay treatment or if they are not able to complete sperm banking or egg banking safely [2,3]. Ovarian tissue transplantation (OTT) after OTC is currently the only procedure for restoring ovarian hormone function and fertility using OTC tissue [4,5]. Testicular tissue transplantation has not been shown to restore fertility in humans to date, although there has been a successful live birth after testicular tissue cryopreservation and reimplantation in non-human primates [6].

In children who undergo a FP procedure prior to or during their treatment for a malignancy, there is a theoretical risk of reimplanting malignancy at the time of tissue transplantation after OTC or TTC [7–10]. In the Fertility & Hormone Preservation & Restoration Program at Ann & Robert H. Lurie Children’s Hospital of Chicago (Lurie Children’s), it is standard practice that a biopsy of ovarian or testicular tissue removed for FP is sent for routine pathologic examination. Biopsies are evaluated for the presence of germ cells, the presence of malignancy, and any other histopathologic abnormalities.

In this study, we examine our experience from January 2011 to March 2023 with routine pathologic examination of tissue removed for FP at Lurie Children’s to determine the rate of malignancy found in these biopsies as well as the presence and qualitative features of germ cells and surrounding tissue.

Methods

A retrospective study was conducted under an institutional review board approved protocol of all patients who were enrolled prospectively to undergo OTC or TTC for FP at Lurie Children’s between January 2011 and March 2023. Ovarian biopsies were performed on removed ovaries on the operating room back table using a 3–4mm dermatology punch biopsy (Figure 1). Ovarian biopsies were oriented by inking of the cut surface and were submitted in formalin for routine pathology. Testis wedge biopsies of 1–2mm were performed sharply and submitted in formalin for routine pathology (Figure 1). Slides of sectioned ovarian or testicular tissue were stained with hematoxylin and eosin prior to pathologist review (Figure 2). Slides then underwent selective immunostaining (such as PHOX 2B, TdT, PAX-5, and CD79a) for any possible malignant cells at the discretion of the pathologist and based on the patient’s diagnosis.

Figure 1. Ovary and Testicular Biopsy Removed for Fertility Preservation.

A small punch biopsy is taken from the ovary (A) and a small sharp biopsy is taken from the larger testis wedge biopsy (B). Blue arrows highlight biopsies sent to pathology for further evaluation.

Figure 2. Hematoxylin and eosin-stained photomicrographs of testicular and ovarian tissue.

Testicular tissue with numerous seminiferous tubules (A, blue stars) lined by a population of germ cells with a typical “fried egg” appearance or abundant, clear cytoplasm and uniform, round nuclei (A, inset, red arrows). These germ cells are supported by Sertoli cells which contain round nuclei and lightly eosinophilic cytoplasm (A, inset, yellow arrows). Ovarian tissue demonstrates ovarian-type stroma composed of numerous spindle cells with storiform architecture (B, green stars). Embedded within the stroma are numerous follicles (B, inset, red arrows) composed of large, round oocytes surrounded by low-cuboidal to flattened follicular cells. Scale bars in each frame are 50μm.

A retrospective chart review was conducted and data including patient demographics, principal diagnosis, planned future treatment, and specimen pathology findings were collected for review. Analysis was conducted using nonparametric Mann-Whitney U tests for continuous data and Chi-Square tests for categorical data performed R Statistical Software (v4.2.3; R Core Team 2022).

Results

A total of 317 patients’ data were reviewed who had either an OTC or TTC at Lurie Children’s between 2011 and 2023. Of those, 183 underwent OTC and 134 underwent TTC. Most OTC and TTC patients were white and did not identify as Hispanic (Table 1). Approximately half of patients, 103 OTC patients (56.3%) and 64 TTC patients (47.8%), underwent some alkylating chemotherapy or radiation treatment prior to their FP procedure. Three patients were excluded from our analysis: 1 OTC and 1 TTC patient did not have a biopsy sent for unknown reasons; 1 OTC biopsy specimen revealed only fibromuscular tissue and no ovarian parenchyma after histopathologic evaluation.

Table 1.

Demographics, Diagnoses, and Type of Treatment Prior to Fertility Preservation of Patients Included in this Study.

	OTC (n=183)	TTC (n=134)
Race (%)
White	117 (63.9)	86 (64.2)
Other	26 (14.2)	30 (22.4)
Black	15 (8.2)	9 (6.7)
Asian	10 (5.5)	4 (3.0)
Declined to respond	15 (8.2)	5 (3.7)
Ethnicity (%)
Hispanic	29 (15.8)	30 (22.4)
Not Hispanic	138 (75.4)	97 (72.4)
Declined to respond	16 (8.7)	7 (5.2)
Diagnosis (%)
Sarcoma	64 (35.0)	30 (22.4)
Other solid tumor	46 (25.1)	21 (15.7)
Leukemia/Lymphoma	32 (17.5)	34 (25.4)
Benign hematologic condition	19 (10.4)	15 (11.2)
Brain/Spine tumor	17 (9.3)	23 (17.2)
Rheumatologic condition	2 (1.1)	4 (3.0)
Other	4 (2.2)	7 (5.2)
Treatment Prior to FP (%)
Radiation therapy	16 (8.7)	6 (4.5)
Alkylating chemotherapy	101 (55.2)	62 (46.3)

The diagnoses of patients who underwent FP procedures are listed in Table 1. Most patients, 155 OTC (87.4%) and 106 TTC (79.1%) patients, had malignant neoplasm. The remainder had a benign hematologic or rheumatologic condition (Table 1). After unilateral oophorectomy for FP, four OTC patients (2.2%) were found to have malignant neoplasm within their ovary upon routine pathologic evaluation of a biopsy from the procured ovary (Table 2, patients 1–4; Figure 3). Two TTC patients (1.5%) were found to have malignancy upon routine pathology of the testis biopsy taken at time of TTC (Table 2, patients 7–8; Figure 3). No patients with benign diagnoses had evidence of a malignant neoplasm within their gonadal biopsy.

Table 2.

Characteristics of Patients with Abnormalities on FP Specimen Biopsy.

Patient	Age in years (Tanner stage)	Procedure	Diagnosis prior to FP	CED of treatment prior to FP (mg/m2)	Risk to fertility of prior treatment	Malignancy present in gonadal biopsy	Germ cells present	Continued long term storage
1	14 (4)	OTC	Immature teratoma	0	0	Mixed germ cell tumor	Yes	Yes
2	15 (5)	OTC	Desmoplastic Small Round Cell Tumor	3396	Minimally increased risk	Desmoplastic Round Cell Tumor	Yes	Yes
3	13 (5)	OTC	Rhabdomyosarcoma	0	0	Rhabdomyosarcoma	Yes	Yes
4	4 (1)	OTC	High-Risk Neuroblastoma	4000	Minimally increased risk	B Cell Lymphoblastic Leukemia	Yes	Yes
5	2 (1)	OTC	Neuroblastoma	2000	Minimally increased risk	None	No	Yes
6	10 (1)	OTC	B Cell Lymphoblastic Leukemia	3000	Minimally increased risk	None	No	Yes
7	2 (1)	TTC	B Cell Lymphoblastic Leukemia	7000	Significantly increased risk	B Cell Lymphoblastic Leukemia	Yes	Yes
8	2 (1)	TTC	B Cell Lymphoblastic Leukemia	0	0	B Cell Lymphoblastic Leukemia	Yes	Yes
9	10 (1)	TTC	Hypergonadotropic Hypogonadism	0	0	None	No	Yes
10	4 (1)	TTC	Aplastic Anemia	0	0	None	No	Yes

Patients who either were found to have malignant cells or an absence of germ cells on biopsy of FP tissue are included in this table. Fertility preservation (FP); Cyclophosphamide equivalency dose (CED); Ovarian tissue cryopreservation (OTC); Testicular tissue cryopreservation (TTC).

Figure 3. Hematoxylin and eosin-stained photomicrographs of gonadal tissue demonstrating involvement by various malignant neoplasms.

Biopsy specimen from patient 1 contains malignant mixed germ cell tumor composed of immature teratoma with immature neuroepithelium (A, black arrow) and a closely associated yolk sac component (A, blue arrow) is shown within the ovarian tissue (A, inset) with adjacent viable follicles (A, inset, red arrows). The biopsy from patient 2 contains tongue-like projections of desmoplastic small round cell tumor (B, blue arrows) invading adjacent ovarian tissue (B, green star) contain viable follicles (B, inset, red arrows). Biopsy from patient 3 shows malignant small round blue cell tumor (C, blue star) invading into ovarian tissue (C, green star) that contains viable follicles (C, top inset, red arrow). The malignancy shows architecture resembling sac-like alveoli of the lung (C, bottom inset), consistent with alveolar rhabdomyosarcoma. Biopsy specimen from patient 4 contains ovarian tissue with viable follicles (D, red arrows) infiltrated by sheets of B-lymphoblasts, consistent with B-cell acute lymphoblastic leukemia/lymphoma (D, blue star). Scale bars in each frame are 50μm.

Two of the patients with malignant neoplasm identified in their ovarian tissue had known or suspected ovarian involvement by their disease. Patient 1 (Figure 3A) was found to have a mixed germ cell tumor after undergoing bilateral oophorectomy for bilateral ovarian masses. The mixed germ cell tumor involved primarily the medulla of the ovary, while also abutting close to cortical tissue; however, no intimate association or destructive invasion of the ovarian cortex was seen, and viable germ cells were present. The mass on the contralateral ovary was found to be an immature teratoma. Cortical tissue on the periphery of the masses was cryopreserved with the understanding that reimplantation of this tissue in its native form may not be recommended in the future. Patient 2 (Figure 3B) was diagnosed with an intraabdominal desmoplastic small round cell tumor that required cytoreductive surgery. The ovary procured for cryopreservation had visible deposits of tumor that were grossly removed prior to being processed for cryopreservation. Microscopically, the tumor was primarily situated within the medulla/hilum, but showed a widely infiltrating, “tongue-like” pattern of invasion with tumor seen within the cortex and close to viable germ cells (less than 0.1 cm away). Two additional patients whose ovarian biopsy contained a malignant neoplasm were not suspected to have ovarian involvement of their disease prior to OTC. Metastatic alveolar rhabdomyosarcoma was identified in the ovarian biopsy from Patient 3. Microscopically, the tumor was centered in the medulla with focal extension into the cortex, with invasive cells seen close to (less than 0.1 cm away) viable germ cells (Figure 3C). The primary tumor was in this patient’s left hand, and metastatic disease had been identified prior to the oophorectomy for OTC, with sites of metastasis including the upper extremity and axillary lymph nodes, the left pleura, and numerous osseous lesions of the axial skeleton and femurs, but no known intraabdominal or ovarian metastases had been identified. Patient 4 (Figure 3D), who was initially diagnosed with high-risk neuroblastoma, was noted to have infiltrating sheets of small round blue cells intimately associated with and surrounding cortical germ cells. This pattern of invasion in the ovary prompted further evaluation of the patient’s ovarian biopsy as well as reevaluation of the prior biopsy of her flank mass. Additional immunohistochemical staining with Pax5, CD20, CD79, CD10, Bc12, and PGP9.5 confirmed a revised diagnosis of B-cell acute lymphoblastic leukemia. The patient had already received 2 cycles of chemotherapy ANBL12P1 at the time of revision of her diagnosis [11]. She was then transitioned to AALL1131 [12].

Both patients who had malignancy present within the biopsy of their testicular tissue were suspected of having leukemia within their testis prior to TTC (Table 2). Patient 7 had known involvement of the left testis with B-cell lymphoblastic leukemia and biopsy for cryopreservation was performed on the right testis. This biopsy confirmed leukemic involvement of both testes in this patient. Patient 8 had bilateral testicular enlargement and had a tissue biopsy removed for TTC at the same time that an open biopsy was performed to confirm leukemia within the testis. This biopsy confirmed the presence of leukemia within the testis. In both cases, these testicular biopsy findings contributed to the patients’ cancer staging which ultimately altered their treatment plans to include testicular radiation.

The presence or absence of germ cells within the gonadal tissue biopsy is also noted during pathology evaluation. Germ cells were present in biopsy tissue from 179 OTC patients (98.9%) and 131 TTC patients (98.5%). Germ cells were not identified in the ovarian biopsy taken for pathology of two OTC patients (1.1%, Table 2). Patient 5, who was 2 years old at the time of OTC, had received 2g/m² of cyclophosphamide, and Patient 6, who was 10 years old had received 3g/m² of cyclophosphamide. According to risk stratification data, these doses only minimally increase the risk of premature ovarian failure [13]. Germ cells were not identified in the testicular pathology biopsy tissue of two TTC patients (1.5%, Table 2). Neither of the TTC patients had received preoperative radiation or chemotherapy. Patient 9 had his TTC performed during orchiectomy for hypergonadotropic hypogonadism. Patient 10 was scheduled to undergo conditioning for stem cell transplant for aplastic anemia and had not received any gonadotoxic therapy prior to TTC.

Discussion

An important concern in pediatric FP is the risk of reimplanting malignant cells at the time of tissue autotransplantation for fertility restoration as an adult [7–10]. At our institution, biopsies of any tissue removed from the body must undergo evaluation by a pathologist. In this study, we demonstrate that there is a low rate of identifying malignancy in gonadal tissue biopsies taken from FP specimens even in patients with known malignancy. However, when malignancy was identified, it could alter the diagnosis and treatment plan significantly for patients.

All patients who undergo OTC are counselled that there is no guarantee that OTT will produce biological children in the future. For the two patients with known or suspected ovarian involvement of their disease (Table 2, patient 1–2) the patients and their families were counselled preoperatively that autotransplantation of the native ovarian tissue would not be safe and were educated on other potential future options that are currently undergoing investigation, including in vitro growth and maturation of oocytes from the cryopreserved tissue to support future pregnancy, and methods to separate germ cells from malignant tissue for reimplantation within a bioprosthetic ovary [14–19]. For patients 3 and 4, the findings of metastatic disease within their ovaries were unexpected. These pathological results were promptly disclosed to the families. They received similar counselling regarding potential future use of their ovarian tissue. Given this information, all families agreed to continue storage of the cryopreserved tissue in hopes that advancements in research would allow for future options in obtaining a biological offspring.

It is important to note that patients 3 and 4 would not have undergone routine biopsy of their ovaries based on what was known about their disease prior to OTC. Patient 3 had known metastatic rhabdomyosarcoma. Her primary tumor was within her left hand and had metastasized to regional lymph nodes, the left pleura, left flank, and the axial skeleton. However, there was no evidence of intraperitoneal disease prior to OTC. Ultimately, this finding did not change her diagnosis or treatment as her disease was already known to be widely metastatic to other areas. Patient 4 was diagnosed with stage IV high risk neuroblastoma prior to OTC. Her diagnosis was subsequently changed to B-cell acute lymphoblastic leukemia, and she was transitioned from ANBL12P1 to AALL1131 [11,12]. This patient has been in remission from her leukemia for over 5 years and has transitioned into survivorship. The long-term course of this patient’s treatment without OTC is unknown, however it is possible that the patient would have continued treatment per ANBL12P1 and proceeded with autologous stem cell transplant. The information recovered through the ovarian biopsy examined as part of her OTC contributed to transitioning this patient to the most appropriate treatment plan for her cancer and prevented further exposure to high doses of cyclophosphamide and radiation therapy [11,12].

Presently, there have been no live births in humans with reimplanted testicular tissue although there was an infant Rhesus macaque born using sperm from a testicular transplant of TTC tissue [6]. All families of patients who underwent TTC procedures were counselled that there is no guarantee that testicular tissue reimplantation will be successful but that the success in non-human primates is encouraging that the technology will progress to successful live births in humans. Patients 6 and 7 were specifically counselled that reimplantation of their native tissue is not safe due to the risk of reintroduction of malignancy and that utilizing their tissue would require methods to mature the sperm outside their bodies which are still being developed [20–22]. Methods of maturing spermatogonia that do not require reimplantation of the testicular tissue are presently under development and include the use of testicular organoids and xenografting patient testicular tissue to promote spermatogenesis [20,21,23–25].

The absence of germ cells was observed in 2 patients who underwent OTC and 2 patients who underwent TTC (Table 2). Patient 5 underwent OTC and subsequently did not have germ cells present on ovarian tissue biopsy. in both patients as the level of exposure to alkylating agent chemotherapy would only be assessed as a minimally increased risk for gonadal failure or infertility [13,26]. Variability in the density of follicles within ovarian tissue may play a role in this finding and may be a consequence of sampling than a true absence of germ cells [27,28]. In the patient who underwent TTC for hypergonadotropic hypogonadism did not have evidence of testicular tissue in his biopsy. Both testes were noted to be atrophic at the time of the procedure and the absence of germ cells is likely secondary to the maldevelopment of his testes [29]. Patient 10 also did not have germ cells in his testicular biopsy which was unexpected as he had not begun any form of gonadotoxic treatment. The patient was scheduled to undergo conditioning for stem cell transplant for aplastic anemia. His pathology report noted “fragments of seminiferous tubules without any defined germ cells.” The absence of germ cells in this case may be more reflective of the biopsy sample that was sent rather than a true absence of germ cells from the testicular tissue. Alternatively, sickle cell disease has been previously shown to affect germ cell development, including instances where testicular tissue in sickle cell patients are devoid of germ cells [30].

It has been previously shown that malignancy can be detected within tissue cryopreserved for FP even if it is not detected during routine pathological examination [10]. Novel molecular and imaging methods to evaluate samples for metastatic disease and for evaluating overall tissue health are still being developed [10,31]. These techniques will be important adjuncts to the histopathologic examination performed at the time of tissue cryopreservation, as the presence of metastatic disease, particularly in leukemia patients, cannot be fully excluded by this pathological evaluation of a small portion of the gonad.

We acknowledge the limitations of our study and the practice of performing small biopsies on tissue cryopreserved for FP. While patients were enrolled prospectively for FP under an IRB-approved research protocol, this data was collected in a retrospective fashion and relied on the medical record for patient data and pathology reports. Sampling of the tissue by surgeons and evaluation of the tissue by pathologists was not standardized. Additionally, this study was conducted at a single tertiary care children’s hospital with a robust experience in FP for pediatric patients, which may limit the generalizability of these results to other centers. Perhaps most importantly, the biopsy of the gonad evaluated by the pathologist is small in comparison to the specimen that is cryopreserved for FP. This biopsy may not be representative of all the tissue preserved and does not eliminate the risk of occult malignancy in the gonadal tissue and patients need be fully informed of the risk of potentially reimplanting malignancy. There have been no reported recurrences of cancer after tissue reimplantation after OTC in the adult population, even in large series, which supports that the overall risk of malignancy reimplantation is low [32–34]. Additional caution needs to also be taken in patients who have not begun gonadotoxic therapy at the time of FP or who have a history of hematologic malignancies or neuroblastoma as these groups are considered the highest risk for reimplantation of malignancy with the reimplantation of gonadal tissue [34,35].

Overall, the percentage of biopsy specimens obtained during FP that contained malignant cells by histological observation was low. However, in the small number of patients who did have malignancy present in their biopsies, this information impacted overall diagnosis, staging, and/or treatment for their cancer diagnosis. Routine pathologic examination of tissue removed for FP also provides important information for counselling patients and their families regarding the utility of continued cryopreservation and may inform future options for restoring fertility and/or hormones from this tissue. Future education efforts for surgeons who perform FP may include highlighting importance of sending a routine ovarian or testis biopsy and working with pathology to standardize the reporting of potential malignant cells and germs cells from biopsy specimens.

Conclusion

Pathology results from gonadal tissue biopsy during fertility preservation provide another data point to help inform patients and their families when making decisions on ovarian or testicular tissue storage and on how tissue may be utilized in the future to restore fertility and/or hormones. Ongoing developments in techniques to separate germ cells from tissue containing malignancy and to mature germ cells outside the body will be important in offering patients who cannot have their gonadal tissue reimplanted after cryopreservation possible options for future biological children.

Highlights:

• Ovarian and testicular tissue removed for fertility preservation can contain cancer cells.

• There is a low rate of identifying malignancy in gonadal tissue biopsies taken from FP specimen. Biopsies provided information about patient disease and provided vital information for future decisions about tissue reimplantation.

Abbreviations:

OTC

ovarian tissue cryopreservation

TTC

testicular tissue cryopreservation

FP

fertility preservation