Testicular tissue biopsies contain stem cells with the potential of generating sperm from biopsied tissue

Histological sections from testicular biopsies from boys of various ages with normal testicular function (left panel) and from boys with nonmosaic Klinefelter syndrome (right panel). From fetal life until puberty the testicular architecture seems similar except that the number of germ cells is markedly reduced in the sample from the prepubertal Klinefelter boy. During and after puberty gross morphological changes with widespread degeneration and hyalinization of the seminiferous tubules in the Klinefelter subject are noted. In the adult most of the biopsy consists of clumps of Leydig cells. Bars represent 100 μm. Gonocytes are indicated by arrows (reprinted from Aksglaede L, Skakkebaek NE, Almstrup K & Juul A. Clinical and biological parameters in 166 boys, adolescents and adults with nonmosaic Klinefelter syndrome: a Copenhagen experience. Acta Paediatrica 2011 100 793–806, with permission).

Testicular tissue samples obtained from 189 males who were facing procedures that could imperil fertility were cryopreserved at one university, proving the feasibility of centralized processing and freezing of testicular tissue obtained from academic medical centers, including Children’s National, scattered around the world.

“It’s not surprising that the University of Pittsburgh would record the highest number of samples over the eight-year period (51 patients), given its role as the central processing facility for our recruiting network of academic medical centers,” says Michael Hsieh, Ph.D., director of transitional urology at Children’s National.

“Children’s National recruited the third-highest number of patients, which really speaks to the level of collaboration I have with Jeff Dome’s team and their commitment to thinking about the whole patient and longer-term issues like fertility.”

An estimated 2,000 U.S. boys and young men each year receive treatments or have cancers or blood disorders that place them at risk for infertility.

While older youths who have undergone puberty can bank their sperm prior to undergoing sterilizing doses of chemotherapy or radiation, there have been scant fertility preservation options for younger boys.

However, some older adolescents and young men are too sick or stressed to bank sperm.

Advances in developmental and cell biology have allowed for extension of previous knowledge and experiences to more specialized areas of medicine, including reproduction.1,2 

As has been described first in mouse models experimentally, spermatogonial stem cells (SSCs) have been used to generate sperm either in vivo 3 or in vitro.4 

SSC transplantation has been tried successfully in different species 5 including nonhuman primates, 6 but not yet in humans.

Progress in achieving successful and efficient in vitro spermatogenesis is less advanced than SSC transplantation but is promising as newer approaches, ie, the use of three-dimensional (3D) culture systems, are developing and becoming more refined.79

Various groups of patients such as cancer survivors, those with idiopathic non-obstructive azoospermia, and those with Klinefelter syndrome (KS) are examples of patients who may benefit from regenerative treatments using SSC technology either in vivo or in vitro when available in the future (Figure 1).

Cancer survivors are the group that may benefit the most from this technology.

In the past three decades, the survival rate of patients suffering from cancer has increased significantly.10,11 

However, one of the most common long-term complications of cancer treatments is the subsequent difficulty to conceive a child.12 

Semen cryopreservation is often offered to adult patients before the start of chemotherapy or radiation.

Unfortunately, this option is not available for pediatric patients prior to the onset of puberty.

Investigators have postulated that cryopreservation of testicular tissue prior to the start of gonadotoxic treatments could open the door for future fertility treatments. Many groups have tried different methods to cryopreserve human testicular samples to maintain morphology, viability, and functionality of the cells after freezing and thawing.

This article aimed to systematically reviewing the evolution of human testicular tissue cryopreservation and its effect on subsequent SSC prop

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Figure 1
Potential future clinical applications using stored testicular tissue from high-risk patients. I. isolation and in vitro propagation of SSCs to have adequate number of SSCs for transplantation. Patients will try for natural conception or IVF/ICSI. II. Isolation of testicular cells and in vitro differentiation of cells into sperms and use in IVF/ICSI. III. Ex vivo culturing of testicular tissue to differentiate SSCs to sperms and use in IVF/ICSI. IV. Xenografting of cryopreserved tissue under the skin at the back of mouse until they differentiate to sperms and are used for ICSI/IVF. These options are all experimental and not clinically available.
Abbreviations: ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; SSCs, spermatogonial stem cells.

For patients with no sperm to bank or who are too sick or stressed to bank sperm, the experimental procedure of freezing testicular tissue in anticipation that future cell- or tissue-based therapies can generate sperm is the only option.

Recent research in experimental models indicates that such testicular tissue biopsies contain stem cells, blank slate cells, hinting at the potential of generating sperm from biopsied tissue.

“This study demonstrates that undifferentiated stem and progenitor spermatogonia may be recovered from the testicular tissues of patients who are in the early stages of their treatment and have not yet received an ablative dose of therapy.

The function of these spermatogonia was not tested,” writes lead author Hanna Valli-Pulaski, Ph.D., research assistant professor at the University of Pittsburgh, and colleagues in a study published online May 21, 2019, in Human Reproduction.

Right now, hematologists and oncologists discuss future treatment options with patients and families, as well as possible long-term side effects, including infertility.

At Children’s National, they also mention the ongoing fertility preservation study and encourage families to speak with Dr. Hsieh.

He meets with families, explains the study goals – which include determining better ways to freeze and thaw tissue and separating malignant cells from normal cells – what’s known about experimental fertility preservation and what remains unknown. Roughly half of patients decide to enroll.

“This study is unique in that there is definitely a potential direct patient benefit,” Dr. Hsieh adds.

“One of the reasons the study is compelling is that it presents a message of hope to the families.

It’s a message of survivorship: We’re optimistic we can help your child get through this and think about long-term issues, like having their own families.”

In this phase of the study, testicular tissue was collected from centers in the U.S. and Israel from January 2011 to November 2018 and cryopreserved.

Patients designated 25% of the tissue sample to be used for the research study; 75 percent remains stored in liquid nitrogen at temperatures close to absolute zero for the patient’s future use.

The fertility preservation patients ranged from 5 months old to 34 years old, with an average age of 7.9 years.

Thirty-nine percent of patients had started medical treatment prior requesting fertility preservation.

Sixteen percent received non-alkylating chemotherapy while 23% received alkylating chemotherapy, which directly damages the DNA of cancer cells.

The research team found that the number of undifferentiated spermatogonia per seminiferous tubule increase steadily with age until about age 11, then rise sharply.

“We recommend that all patients be counseled and referred for fertility preservation before beginning medical treatments known to cause infertility.

Because the decision to participate may be delayed, it is encouraging that we were able to recover undifferentiated spermatogonia from the testes of patients already in the early stages of chemotherapy treatments,” Dr. Hsieh says.

More information: H Valli-Pulaski et al, Testicular tissue cryopreservation: 8 years of experience from a coordinated network of academic centers, Human Reproduction (2019). DOI: 10.1093/humrep/dez043

Journal information: Human Reproduction
Provided by Children’s National Medical Center


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