NORDFERTIL Research Lab – Jan-Bernd Stukenborg's research group

Background

Survival rates among childhood cancer patients have progressively increased over the past four decades, in particular as a result of the development of more effective cancer treatments. Three out of four children will be cured of their disease. However, success has come at a cost and some patients will suffer from adverse effects later on. One of these late effects may be treatment-induced infertility. Boys who have matured into puberty are offered sperm preservation before treatment starts. However, for boys before puberty there is as yet no procedure that may preserve fertility. Most childhood cancer treatments will have only a minor impact on future fertility, while some treatments are known to be very harmful as regards reproductive function. So far, boys facing such treatments have almost no chance to father their own biological children later in life.

To address the concerns of many patients and parents and to open a forum concentrating on infertility as a disease, or a treatment-related late effect in childhood cancer survivors, the “Nordic Network for Gonadal Preservation after Cancer Treatment in Children and Young Adults” was initiated in 2008 and resulted in recommendations concerning fertility preservation for girls/boys and young women/men with childhood cancer. Up to now, advances in females have been made, but a similar development is still missing for young boys. Therefore, the idea of establishing a scientific network in order to intensify and combine research efforts on fertility preservation for young boys was born. The network is named NORDFERTIL

The research of this network is centralized at the NORDFERTIL Research Lab Stockholm at Karolinska Institutet and includes research areas including pluripotent stem cell cultures as well as the development of novel in vitro systems for male gonadal cell development and differentiation.

In addition to the research performed, NORDFERTIL will focus on the establishment of protocols for clinical applications. Therefore, the results obtained by evaluation of testicular biopsy material and cell culture experiments will be used to generate and/or optimize already existing protocols for clinical use in cooperation with all units included in NORDFERTIL.

The following questions will be addressed:

1. Which are the best cryopreservation protocols as regards later differentiation of early male germ cells in vitro?
2. Which are the best strategies to monitor cancer cell contamination in vitro?
3. Which are the best strategies and culture conditions for human spermatogenesis in vitro?
4. Which clinical efficacy and safety measures should be monitored?

We would like to thank governmental and private funding bodies.

Swedish Childhood Cancer Foundation (Barncancerfonden)
Swedish Research Council (Vetenskapsrådet)
Stiftelsen Frimurare Barnhuset i Stockholm
Sällskapet Barnavård
Stiftelsen Samariten
HKH Kronprinsessan Lovisas förening för barnsjukvård/ Stiftelsen
Axel Tielmans Minnesfond
Jane and Dan Olssons Foundation
Karolinska Institutet
Magnus Bergvalls Foundation

Background

Boys who have matured into puberty are offered sperm preservation before treatment starts. However, for boys before puberty there is as yet no procedure that may preserve fertility. Most childhood cancer treatments will have only a minor impact on future fertility, while some treatments are known to be very harmful as regards reproductive function. So far, boys facing such treatments have almost no chance to father their own biological children later in life.

To address the concerns of many patients and parents and to open a forum concentrating on infertility as a disease, or a treatment-related late effect in childhood cancer survivors, the “Nordic Network for Gonadal Preservation after Cancer Treatment in Children and Young Adults” was initiated in 2008 and resulted in recommendations concerning fertility preservation for girls/boys and young women/men with childhood cancer. Up to now, advances in females have been made, but a similar development is still missing for young boys. Therefore, the idea of establishing a scientific network in order to intensify and combine research efforts on fertility preservation for young boys was born. The network is named NORDFERTIL.

The research of this network is centralized at the NORDFERTIL Research Lab Stockholm at Karolinska Institutet and includes research areas including pluripotent stem cell cultures as well as the development of novel in vitro systems for male gonadal cell development and differentiation.

In addition to the research performed, NORDFERTIL will focus on the establishment of protocols for clinical applications. Therefore, the results obtained by evaluation of testicular biopsy material and cell culture experiments will be used to generate and/or optimize already existing protocols for clinical use in cooperation with all units included in NORDFERTIL.

Research projects

Strategies to mature pluripotent stem and immature germ cells into mature gametes

Late side effects affecting future fertility in children undergoing gonadotoxic treatments do still exist. So far, no treatment can be offered to rescue fertility in those patients. In the past, major efforts have been put into optimizing in vivo and in vitro methods for maturation of immature germ cells, but these techniques still remain experimental. For the in vivo methods the immature testicular tissue or spermatogonial stem cells (SSCs) could be transplanted back to the patient after cancer treatment, however in such case there is a potential risk of introducing cancer cells back to the patient. Transplanting the tissue or the isolated SSCs to other animal species for maturation is also an option but then there is a potential risk of introducing the germ cells to xenogenic tissue hosting unknown viruses such as retroviruses. Therefore, such methodology would presently not be considered acceptable for clinical practice.

For in vitro methods, the SSCs could be differentiated in a monolayer on supporting feeder layer or on extracellular matrixes. Also it has been reported that pre-meiotic germ cells from mice can be differentiated in soft agar culture system (SACS) where the cells have 3D support. In addition to those 3D culture approaches, in 2011, Japanese researchers demonstrated for the first time viable offspring from in vitro generated murine sperm using an organ culture. This experiment demonstrates a big step towards the establishment of clinical tools to use in vitro differentiated gametes as potential fertility preservation for young cancer patients. More studies are required to meet efficiency/safety concerns and translate results to the situation in humans.

Jan-Bernd Stukenborg, Assoc Prof, PhD, Group leader

Hormonal effects in the female reproductive tract

The focus for my part of our projects is on hormonal effects and cross-talk between sex steroid hormone receptors and growth factors. The classical steroid hormone receptors are predominantly located in the nucleus, but in recent years there have been evidence also of membrane bound steroid hormone receptors e.g. GPER (G-protein bound estrogen receptor 1), PGRMC (progesterone receptor membrane component) 1 and PGRMC2. These membrane bound receptors could explain some of the very fast effects seen after hormone treatment, which cannot be mediated via the classical nuclear receptors since nuclear/genomic signaling requires longer time.

Lena Sahlin, Assoc Prof, PhD

PhD projects

Novel strategies to preserve future fertility in boys and men.

This PhD project aims to gain knowledge regarding risk potential of medical treatment causing infertility as well as to develop novel biotechnology approaches to encounter those with a focus on the somatic microenvironment present in the testis. Within the project we will combine a unique collection of human testicular material covering all important stages of human testis development from pre- and postnatal life until pubertal maturation with advanced in vitro models recently established in our lab.

Project 1: Definition of the human somatic cell compartments hosting SSCs in post-natal human testes.
Hypothesis – The Sertoli cells are a crucial component in the SSC niche in mammals. After birth final Sertoli cell maturation occurs in a two-step maturation process, the SSC niche in humans is established in a two-step maturation process, accordingly.

Project 2: Establishment of a protocol for functional gonadal somatic microenvironment to differentiate human gonadal cell in vitro.
Hypothesis - It is possible to create functional testicular somatic microenvironment using pre-pubertal human testicular tissue. Improved functional capacity of somatic environment will increase possibilities to differentiate human SSCs towards functional sperm.

Project 3: Definition of human SSC populations at different maturational age and protection of human SSC niche during medical treatments.
Hypothesis - The establishment of niche maintaining human SSCs is crucial to create environment for the two SSC populations, A_dark (silent SSC) and A_pale (proliferating SSC). The protection of fertility in males during gonadotoxic treatment is possible, by regulating SSC niche to prevent the silent A_dark SSCs to enter the proliferative state of A_pale SSCs. This protection can be reached by blocking specific pathways in the somatic microenvironment belonging to the SSC niche or structures in direct contact with it.

Yanhua Cui, MSc, PhD student

Effects of haematological and oncological diseases on spermatogonial stem cell populations in boys.

This PhD project aims to ensure a gain of knowledge about risk potential of different medical treatment causing infertility as well as novel biotechnology approaches focusing on the stem the population in the testes. This project will combine a unique collection of human testicular material covering all important stages of human testis development from prenatal to the postpubertal age with advanced in vitro models recently established in our lab. The planned experiments aim to establish a system for in vitro differentiation or maintenance of human male germ cells as well as to study effects of different diseases and medical treatments on the SSC populations in humans.

Project 1: –Definition of the human spermatogonial stem cell population in post-natal testes of boys.
Hypothesis – The quantity and expression profile of the spermatogonial stem cell population is directly affected by the primary disease and medical treatments used s in paediatric oncology and haematology units.

Project 2: Establishment of a system for human spermatogonial stem cell cultures and differentiation.
Hypothesis – In vitro differentiation and propagation protocols, used successfully for differentiation of rodent SSCs, can be used as a suitable platform to establish SSC differentiation in humans.

Project 3: Evaluation of protocols to protect human spermatogonial stem cells during medical treatments.
Hypothesis - The establishment of human SSCs and their niche is crucial to define the two SSC populations, A_dark (silent SSC) and A_pale (proliferating SSC). The protection of fertility in males during gonadotoxic treatment is possible, by preventing the silent Adark SSCs to enter the proliferative state of A_pale SSCs. This can be done by interfering with the somatic cellular compartment of the testis or via direct manipulation of the stem cell population with chemical compounds.

Hajar Baomar, MSc, PhD student

Human induced pluripotent stem cells differentiation towards the male germ cell lineage

Despite the research progress in the andrological area, detailed knowledge about the early development and the biology of male fertility is still inadequate. Studying the human germline development and maturation using fetal, prepubertal and adult testicular tissue as well as pluripotent stem cells can contribute to a better understanding of in vivo as well as in vitro gametogenesis processes.

Further we believe that this knowledge will help to understand infertility causing disorders and the poor outcomes in assisted reproduction.

Therefore my project focuses on one side in understanding the establishment and role of the tubular basement membrane and its function in germ cell specification, maintenance and differentiation.

On the other side I also focus on the X chromosome inactivation status of Klinefelter syndrome (47, XXY) derived iPSCs and the role of the additional X chromosome in germ cell specification, maintenance and differentiation.

Projects:

• Derivation of clinical grade hiPSC lines from healthy (46,XY) and Klinefelter syndrome (47,XXY) dermal fibroblasts
• Effect of laminin 521 on the pluripotency behavior of hESC
• Investigation of aspects the testicular stem cell niche
• Influence of differentiation techniques for differentiation of hiPSCs towards male germ cells

Magdalena Kurek, MSc, PhD student

Qualitative assessment of fresh and cryopreserved ovarian tissues from pediatric oncological patients

Cryopreservation of ovarian tissue is the only option to preserve fertility in childhood cancer patients facing highly toxic treatments. Chemo- and radiotherapy can damage the ovary, causing permanent infertility in 16% to 41% of girls.

Cryopreserved ovarian tissues can be transplanted back to the patient and are able to restore ovarian function. However, this option is not safe for certain malignancies at high risk of ovarian contamination (e.g. leukemia, Ewing sarcoma).

In vitro follicle growth and oocyte maturation has emerged as a safe alternative method but is still burdened by low efficacy rates, especially for younger patients. Age-related factors, cryostatic cell damage as well as chemotherapeutic agents to which patients have been exposed, have an impact on the oocyte viability in vitro. To date knowledge in this field remains limited, due to the difficulty of obtaining tissues for research from this population.

My research project includes 2 sets of tissues harvested from girls affected by cancer (age range 1-17 yrs):

1) Freshly formalin-fixed ovarian tissues collected after the initiation of chemotherapy (n=35). Expression of key factors involved in follicle activation, maturation and survival will be determined and related to individual patient's characteristics and chemotherapeutic regimens (methods: light microscopy).

2) Frozen ovarian tissues that have been cryopreserved for fertility preservation purposes after the initiation of chemotherapy (n=17), The effects of the cryopreservation process and the effect of chemotherapy on pathways regulating oocyte viability will be assessed in an in vitro culture system (methods: light microscopy, transmission electron microscopy, qPCR, Western Blot).

This project will provide new understandings on the impact of cancer treatments and tissue processing on the in vitro developmental potential of ovarian tissues harvested for fertility preservation purpose in pediatric cancer girls.

Valentina Pampanini, MD, PhD student

Group leader NORDFERTIL Research Lab Stockholm:

Forskare

Jan-Bernd Stukenborg

Telefon:	08-524 827 88
Enhet:	Institutionen för kvinnors och barns hälsa (KBH), K6
E-post:	jan-bernd.stukenborg@ki.se