Encapsulated Allografts Preclude Host Sensitization and Promote Ovarian Endocrine Function in Ovariectomized Young Rhesus Monkeys and Sensitized Mice.

Transplantation of allogeneic donor ovarian tissue holds great potential for female cancer survivors who often experience premature ovarian insufficiency. To avoid complications associated with immune suppression and to protect transplanted ovarian allografts from immune-mediated injury, we have developed an immunoisolating hydrogel-based capsule that supports the function of ovarian allografts without triggering an immune response. Encapsulated ovarian allografts implanted in naïve ovariectomized BALB/c mice responded to the circulating gonadotropins and maintained function for 4 months, as evident by regular estrous cycles and the presence of antral follicles in the retrieved grafts. In contrast to non-encapsulated controls, repeated implantations of encapsulated mouse ovarian allografts did not sensitize naïve BALB/c mice, which was confirmed with undetectable levels of alloantibodies. Further, encapsulated allografts implanted in hosts previously sensitized by the implantation of non-encapsulated allografts restored estrous cycles similarly to our results in naïve recipients. Next, we tested the translational potential and efficiency of the immune-isolating capsule in a rhesus monkey model by implanting encapsulated ovarian auto- and allografts in young ovariectomized animals. The encapsulated ovarian grafts survived and restored basal levels of urinary estrone conjugate and pregnanediol 3-glucuronide during the 4- and 5-month observation periods. We demonstrate, for the first time, that encapsulated ovarian allografts functioned for months in young rhesus monkeys and sensitized mice, while the immunoisolating capsule prevented sensitization and protected the allograft from rejection.

iPSCs derived from esophageal atresia patients reveal SOX2 dysregulation at the anterior foregut stage.

A series of well-regulated cellular and molecular events result in the compartmentalization of the anterior foregut into the esophagus and trachea. Disruption of the compartmentalization process leads to esophageal atresia/tracheoesophageal fistula (EA/TEF). The cause of EA/TEF remains largely unknown. Therefore, to mimic the early development of the esophagus and trachea, we differentiated induced pluripotent stem cells (iPSCs) from EA/TEF patients, and iPSCs and embryonic stem cells from healthy individuals into mature three-dimensional esophageal organoids. CXCR4, SOX17 and GATA4 expression was similar in both patient-derived and healthy endodermal cells. The expression of the key transcription factor SOX2 was significantly lower in the patient-derived anterior foregut. We also observed an abnormal expression of NKX2.1 (or NKX2-1) in the patient-derived mature esophageal organoids. At the anterior foregut stage, RNA sequencing revealed the critical genes GSTM1 and RAB37 to be significantly lower in the patient-derived anterior foregut. We therefore hypothesize that a transient dysregulation of SOX2 and the abnormal expression of NKX2.1 in patient-derived cells could be responsible for the abnormal foregut compartmentalization.

Generation of three induced pluripotent stem cells lines from patients with esophageal atresia/tracheoesophageal fistula type C.

Esophageal atresia/tracheoesophageal fistula (EA/TEF) is the most common congenital anomaly of the upper gastrointestinal tract affecting 1 in 3,000 which could stem from a developmental anomaly of the foregut. The cause is not fully understood. We generated three iPSC cell lines using peripheral blood mononuclear cells (PBMCs) from EA/TEF type C patients. Pluripotency and trilineage differentiation capacity of these three iPSC cell lines were confirmed by gene and protein expression profiles and the differentiation ability into the three germ layers. The generated disease-specific cell lines could serve as a tool to investigate the mechanisms of EA/TEF and acquired associated diseases.

Genetic Mouse Models and Induced Pluripotent Stem Cells for Studying Tracheal-Esophageal Separation and Esophageal Development.

Esophagus and trachea arise from a common origin, the anterior foregut tube. The compartmentalization process of the foregut into the esophagus and trachea is still poorly understood. Esophageal atresia/tracheoesophageal fistula (EA/TEF) is one of the most common gastrointestinal congenital defects with an incidence rate of 1 in 2,500 births. EA/TEF is linked to the disruption of the compartmentalization process of the foregut tube. In EA/TEF patients, other organ anomalies and disorders have also been reported. Over the last two decades, animal models have shown the involvement of multiple signaling pathways and transcription factors in the development of the esophagus and trachea. Use of induced pluripotent stem cells (iPSCs) to understand organogenesis has been a valuable tool for mimicking gastrointestinal and respiratory organs. This review focuses on the signaling mechanisms involved in esophageal development and the use of iPSCs to model and understand it.

Encapsulation of ovarian allograft precludes immune rejection and promotes restoration of endocrine function in immune-competent ovariectomized mice.

Premature ovarian insufficiency (POI) is a significant complication of cytotoxic treatments due to extreme ovarian sensitivity to chemotherapy and radiation. POI is particularly devastating for young girls reaching puberty, because it irreversibly affects their physical and cognitive development. Changes occurring during puberty determine their height, bone health, insulin responsiveness, lipid metabolism, cardiovascular health and cognition. The only available treatment for POI during puberty is hormone replacement therapy (HRT), which delivers non-physiological levels of estrogen, lacks other ovarian hormones and pulsatility, and is not responsive to feedback regulation. Here we report that ovarian allografts encapsulated in a hydrogel-based capsule and implanted in ovariectomized mice restore ovarian endocrine function in immune competent mice. Ovarian tissue from BALB/c mice was encapsulated in poly(ethylene-glycol) (PEG) hydrogels, with a proteolytically degradable core and a non-degradable shell. The dual capsules were implanted subcutaneously in immune competent ovariectomized C57BL/6 mice for a period of 60 days. As expected, non-encapsulated ovarian allografts implanted in a control group sensitized the recipients as confirmed with donor-specific IgG in the serum, which increased 26-fold in the 3 weeks following transplantation (p = 0.02) and infiltration of the graft with CD8 T cells consistent with allo-immunity. In contrast, encapsulation in the Dual PEG capsules prevented sensitization to the allograft in all the recipients with no evidence of lymphocytic infiltration. In summary, the approach of hydrogel-based immunoisolation presents a minimally invasive and robust cell-therapy to restore hormonal balance in ovarian insufficiency. This report is the first to demonstrate the application of a tunable PEG-based hydrogel as an immunoisolator of allogeneic ovarian tissue to restore endocrine function in ovariectomized mice and prevent cell-mediated immune rejection in immune competent mice.

Immuno-Isolating Dual Poly(ethylene glycol) Capsule Prevents Cancer Cells from Spreading Following Mouse Ovarian Tissue Auto-Transplantation.

For female cancer survivors, premature ovarian insufficiency (POI) is a common complication of anticancer treatments. Ovarian tissue cryopreservation before treatment, followed by auto-transplantation after remission is a promising option to restore fertility and ovarian endocrine function. However, auto-transplantation is associated with the risk of re-introducing malignant cells harbored in the stroma of the ovarian autograft. To mitigate this risk, we investigated in this pilot study whether an immuno-isolating dual-layered poly(ethylene glycol)(PEG) capsule can retain cancer cells, while supporting folliculogenesis. The dual PEG capsule loaded with 1000 4T1 cancer cells retained 100% of the encapsulated cells in vitro for 21 days of culture. However, a greater cell load of 10,000 cells/capsule led to capsule failure and cells' release. To assess the ability of the capsule to retain cancer cells, prevent metastasis, and support folliculogenesis in vivo we co-encapsulated cancer cells with ovarian tissue in the dual PEG capsule and implanted subcutaneously in mice. Control mice implanted with 2000 non-encapsulated cancer cells had tumors formed within 14 days and metastasis to the lungs. In contrast, no tumor mass formation or metastasis to the lungs was observed in mice with the same number of cancer cells encapsulated in the capsule. Our findings suggest that the immuno-isolating capsule may prevent the escape of the malignant cells potentially harbored in ovarian allografts and, in the future, improve the safety of ovarian tissue auto-transplantation in female cancer survivors.

Rapid Mouse Follicle Stimulating Hormone Quantification and Estrus Cycle Analysis Using an Automated Microfluidic Chemiluminescent ELISA System.

Follicle stimulating hormone (FSH) plays a critical role in female reproductive development and homeostasis. The blood/serum concentration of FSH is an important marker for reporting multiple endocrinal functions. The standardized method for mouse FSH (mFSH) quantification based on radioimmunoassay (RIA) suffers from long assay time (∼2 days), relatively low sensitivity, larger sample volume (60 µL), and small dynamic range (2-60 ng/mL); thus, it is insufficient for monitoring fast developing events with relatively small mFSH fluctuations (e.g., estrous cycles of mammals). Here, we developed an automated microfluidic chemiluminescent ELISA device along with the disposal sensor array and the corresponding detection protocol for rapid and quantitative analysis of mFSH from mouse tail serum samples. With this technology, highly sensitive quantification of mFSH can be accomplished within 30 min using only 8 µL of the serum sample. It is further shown that our technique is able to generate results comparable to RIA but has a significantly improved dynamic range that covers 0.5-250 ng/mL. The performance of this technology was evaluated with blood samples collected from ovariectomized animals and animals with reimplanted ovarian tissues, which restored ovarian endocrine function and correlated with estrus cycle analysis study.

Immunoisolating poly(ethylene glycol) based capsules support ovarian tissue survival to restore endocrine function.

A common irreversible adverse effect of life-saving anticancer treatments is loss of gonadal endocrine function and fertility, calling for a need to focus on post-treatment quality of life. Here, we investigated the use of poly(ethylene glycol)-vinyl sulfone (PEG-VS) based capsules to support syngeneic donor ovarian tissue for restoration of endocrine function in mice. We designed a dual immunoisolating capsule (PEG-Dual) by tuning the physical properties of the PEG hydrogels and combining proteolytically degradable and nondegradable layers to meet the numerous requirements for encapsulation and immunoisolation of ovarian tissue, such as nutrient diffusion and tissue expansion. Tuning the components of the PEG-Dual capsule to have similar physical properties allowed for concentric encapsulation. Upon implantation, the PEG-based capsules supported ovarian tissue survival and led to a significant decrease in follicle stimulating hormone levels 60 days postimplantation. Mice that received the implants resumed regular estrous cycle activity and follicle development in the implanted grafts. The PEG-Dual capsule provided an environment conducive for tissue survival, while providing a barrier to the host environment. This study demonstrated for the first time that immunoisolating PEG-VS capsules can support ovarian follicular development resulting in the restoration of ovarian endocrine function and can be applied to future allogeneic studies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1381-1389, 2018.

The impact of functional groups of poly(ethylene glycol) macromers on the physical properties of photo-polymerized hydrogels and the local inflammatory response in the host.

Poly(ethylene glycol) (PEG) can be functionalized and modified with various moieties allowing for a multitude of cross-linking chemistries. Here, we investigate how vinyl sulfone, acrylate, and maleimide functional end groups affect hydrogel formation, physical properties, viability of encapsulated cells, post-polymerization modification, and inflammatory response of the host. We have shown that PEG-VS hydrogels, in the presence of a co-monomer, N-vinyl-2-pyrrolidone (NVP), form more efficiently than PEG-Ac and PEG-Mal hydrogels, resulting in superior physical properties after 6 min of ultraviolet light exposure. PEG-VS hydrogels exhibited hydrolytic stability and non-fouling characteristics, as well as the ability to be modified with biological motifs, such as RGD, after polymerization. Additionally, unmodified PEG-VS hydrogels resulted in lesser inflammatory response, cellular infiltration, and macrophage recruitment after implantation for 28 days in mice. These findings show that altering the end group chemistry of PEG macromer impacts characteristics of the photo-polymerized network. We have developed a tunable non-degradable PEG system that is conducive for cell or tissue encapsulation and evokes a minimal inflammatory response, which could be utilized for future immunoisolation applications. STATEMENT OF SIGNIFICANCE: The objective of this study was to develop a tunable non-degradable PEG system that is conducive for encapsulation and evokes a minimal inflammatory response, which could be utilized for immunoisolation applications. This study has demonstrated that reactive functional groups of the PEG macromers impact free radical mediated network formation. Here, we show PEG-VS hydrogels meet the design criteria for an immunoisolating device as PEG-VS hydrogels form efficiently via photo-polymerization, impacting bulk properties, was stable in physiological conditions, and elicited a minimal inflammatory response. Further, NVP can be added to the precursor solution to expedite the cross-linking process without impacting cellular response upon encapsulation. These findings present an additional approach/chemistry to encapsulate cells or tissue for immunoisolation applications.

Fertility Preservation in 2016: Where Are We?

This review focuses on advances in the field of fertility preservation achieved over the past few decades. The main purpose of fertility preservation is to help young cancer survivors retain their fertility by restoring either their ability to procreate or simply their reproductive endocrine function. Although different options are available in this regard, successful fertility preservation is yet to reach its full potential. Herein, we review the most significant work conducted over the past five decades and present new methodologies.

Restoring Ovarian Endocrine Function with Encapsulated Ovarian Allograft in Immune Competent Mice.

Premature ovarian insufficiency (POI) is a major complication of cytotoxic treatments due to extreme ovarian sensitivity to chemotherapy and radiation. In pediatric cancer patients modern therapy has improved the long-term survival to over 80% in the United States. However, these cancer survivors face long-term health problems related to treatment toxicity. In female cancer survivors POI leads to sterility, along with the consequences of estrogen deficiency such as premature osteopenia, muscle wasting, accelerated cardiovascular diseases and a vast array of other health and developmental problems. These long-lasting effects are particularly significant for young girls reaching puberty. As such, restoring ovarian endocrine function is paramount in this population. In the present study, we evaluated the feasibility of restoring ovarian endocrine function in ovariectomized mice by transplanting syngeneic and allogeneic ovarian tissue encapsulated in alginate capsules or TheraCyte®. Histological analysis of the implants retrieved after 7 and 30 days' post implantation showed follicular development up to the secondary and antral stages in both syngeneic and allogeneic implants. Implantation of syngeneic and allogeneic ovarian grafts encapsulated in TheraCyte devices restored ovarian endocrine function, which was confirmed by decreased serum FSH levels from 60 to 70 ng/mL in ovariectomized mice to 30-40 ng/mL 30 days after implantation. Absence of allo-MHC-specific IgG and IgM antibodies in the sera of implanted mice with allogeneic ovarian tissue encapsulated in TheraCyte indicate that the implants did not evoke an allo-immune response, while the allogeneic controls were rejected 21 days after implantation. Our results show that TheraCyte effectively isolates the graft from immune recognition but also supports follicular growth.

Immunoisolation to prevent tissue graft rejection: Current knowledge and future use.

This review focuses on the concept of immunoisolation and how this method has evolved over the last few decades. The concept of immunoisolation came out of the need to protect allogeneic transplant tissue from the host immune system and avoid systemic side effects of immunosuppression. The latter remains a significant hurdle in clinical translation of using tissue transplants for restoring endocrine function in diabetes, growth hormone deficiency, and other conditions. Herein, we review the most significant works studying the use of hydrogels, specifically alginate and poly (ethylene glycol), and membranes for immunoisolation and discuss how this approach can be applied in reproductive biology.

Synthetic hydrogel supports the function and regeneration of artificial ovarian tissue in mice.

Many prepubertal girls and young women suffer from premature ovarian insufficiency induced by chemotherapy given for treatment of cancer and autoimmune diseases. Auto-transplantation of cryopreserved ovarian tissue could restore the lost ovarian endocrine function and fertility. Unfortunately, tissue ischemia, inconsistent graft quality and the risk of re-introducing malignant cells may stand in the way of the clinical translation of this approach. To address these risks and limitations, we engineered an artificial ovary from immature follicles using a synthetic hydrogel, poly(ethylene glycol) vinyl-sulfone (PEG-VS), as a supportive matrix. Enzymatically-isolated follicles from 6 - 7 day old mice ovaries were encapsulated in 7% PEG-VS hydrogels modified with 0.5mM RGD and crosslinked with a tri-functional matrix metalloproteinase (MMP)-sensitive peptide. PEG hydrogels with the encapsulated follicles were orthotopically implanted into ovariectomized mice to investigate if PEG hydrogel supports folliculogenesis and steroidogenesis in vivo. After 30 days, grafts revealed multiple fully developed antral follicles and corpora lutea, which corresponded with regular ovulation cycles and follicle-stimulating hormone (FSH) levels. The elevated levels of FSH, caused by bilateral ovariectomy, were reversed by the implanted follicles and maintained at physiological levels for 60 days. Importantly, primordial and primary follicles still represented 60% of the follicular pool, demonstrating selective recruitment of primordial follicles into the growing pool. Functioning blood vessels in the grafts 30 and 60 days after implantation proved the capability of PEG hydrogels to undergo graft remodeling and revascularization. Our results demonstrate that PEG hydrogels with encapsulated immature ovarian follicles successfully functioned as an artificial ovarian tissue for 60 days in vivo.

Vitrification and xenografting of human ovarian tissue.

OBJECTIVE: To assess the efficiency of two vitrification protocols to cryopreserve human preantral follicles with the use of a xenografting model. DESIGN: Pilot study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Ovarian biopsies were obtained from seven women aged 30-41 years. INTERVENTION(S): Ovarian tissue fragments were subjected to one of three cryopreservation protocols (slow freezing, vitrification protocol 1, and vitrification protocol 2) and xenografted for 1 week to nude mice. MAIN OUTCOME MEASURE(S): The number of morphologically normal follicles after cryopreservation and grafting and fibrotic surface area were determined by histologic analysis. Apoptosis was assessed by the TUNEL method. Morphometric analysis of TUNEL-positive surface area also was performed. Follicle proliferation was evaluated by immunohistochemistry. RESULT(S): After xenografting, a difference was observed between the cryopreservation procedures applied. According to TUNEL analysis, both vitrification protocols showed better preservation of preantral follicles than the conventional freezing method. Moreover, histologic evaluation showed a significantly higher proportion of primordial follicles in vitrified (protocol 2)-warmed ovarian tissue than in frozen-thawed tissue. The proportion of growing follicles and fibrotic surface area was similar in all groups. CONCLUSION(S): Vitrification procedures appeared to preserve not only the morphology and survival of preantral follicles after 1 week of xenografting, but also their ability to resume folliculogenesis. In addition, vitrification protocol 2 had a positive impact on the quiescent state of primordial follicles after xenografting.

Effect of cryopreservation and transplantation on the expression of kit ligand and anti-Mullerian hormone in human ovarian tissue.

BACKGROUND: Although cryopreservation and transplantation of ovarian tissue represent a promising alternative to safeguard fertility in cancer patients, low recovery rates of oocytes aspirated from antral follicles and a significant number of empty follicles have been observed in women with transplanted frozen-thawed ovarian tissue. In order to understand how freezing and/or grafting may affect follicular development, the follicular expression of kit ligand (KL) and anti-Müllerian hormone (AMH), two key factors activating and inhibiting follicle growth, were assessed after long-term grafting in severe combined immunodeficient (SCID) mice. METHODS: Ovarian biopsies from eight patients were used for fresh and frozen-thawed tissue xenografting in 13 SCID mice for a period of 28 weeks, including 2 weeks of gonadotrophin stimulation. KL, AMH and proliferating cell nuclear antigen (PCNA) immunostaining were quantified before and after grafting in the two treatment groups (fresh and frozen-thawed grafted ovarian tissue). RESULTS: Lower expression of KL was found in primordial and primary follicles after grafting of both fresh and frozen-thawed tissue. Consistent expression of AMH was found in most growing follicles at a similar rate in both graft types. In fresh and frozen-thawed grafts, 13-14% of primordial follicles were PCNA-positive, indicating a similar maintenance of quiescent follicles despite follicle activation. CONCLUSIONS: Grafting and/or gonadotrophin stimulation appear to affect the follicular expression of KL, which may alter oocyte quality. AMH expression in growing follicles after ovarian tissue transplantation may be one of the factors contributing to the preservation of resting follicles in 28-week-old grafts.

Impact of freezing and thawing of human ovarian tissue on follicular growth after long-term xenotransplantation.

PURPOSE: To assess follicular growth after xenografting in order to understand how freezing and/or grafting may affect follicular development. METHODS: Human ovarian biopsies were used for fresh and frozen-thawed xenografting to SCID mice. After xenotransplantation, follicular morphology and proportion, oocyte and follicle diameter, and quantitative and qualitative parameters of antral follicles were analyzed. RESULTS: The proportion of growing follicles was significantly higher in grafted than non-grafted ovarian tissue. Follicular growth to the antral stage was observed and there was no significant difference in oocyte or follicle diameter in fresh or frozen-thawed grafts. Although no significant difference was observed in antral area or zona pellucida thickness, the theca layer in antral follicles from frozen-thawed grafted tissue was found to be significantly thinner than in fresh grafts. CONCLUSION: Antral follicles obtained after grafting of frozen-thawed human ovarian tissue showed a thinner theca cell layer compared to those from fresh grafts, which could affect follicular development and function. Further studies are nevertheless warranted to confirm the identity of theca cells and assess if they retain the ability to respond to luteinizing hormone and produce androgens.

Vitrification of human ovarian tissue: effect of different solutions and procedures.

OBJECTIVE: To test the effect of different vitrification solutions and procedures on the morphology of human preantral follicles. DESIGN: Pilot study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Ovarian biopsies were obtained from nine women aged 22-35 years. INTERVENTION(S): Ovarian tissue fragments were subjected to [1] different vitrification solutions to test their toxicity or [2] different vitrification methods using plastic straws, medium droplets, or solid-surface vitrification before in vitro culture. MAIN OUTCOME MEASURE(S): Number of morphologically normal follicles after toxicity testing or vitrification with the different treatments determined by histologic analysis. RESULT(S): In the toxicity tests, only VS3 showed similar results to fresh tissue before and after in vitro culture (fresh controls 1 and 2). In addition, this was the only solution able to completely vitrify. In all vitrification procedures, the percentage of normal follicles was lower than in controls. However, of the three protocols, the droplet method yielded a significantly higher proportion of normal follicles. CONCLUSION(S): Our experiments showed VS3 to have no deleterious effect on follicular morphology and to be able to completely vitrify, although vitrification procedures were found to affect human follicles. Nevertheless, the droplet method resulted in a higher percentage of morphologically normal follicles.

Immunohistochemical localization of growth factors after cryopreservation and 3 weeks' xenotransplantation of human ovarian tissue.

OBJECTIVE: To compare early follicular growth after fresh and frozen-thawed human ovarian tissue xenografting and to investigate whether expression of c-kit, kit ligand (KL), and growth differentiation factor-9 (GDF-9) is maintained after freezing and xenografting of human ovarian tissue. DESIGN: Prospective experimental study. SETTING: Gynecology research unit in a university hospital. ANIMAL(S): Ten nude (Swiss nu/nu) 6-week-old female mice. INTERVENTION(S): The ovarian biopsy samples were obtained from six women, aged 20 to 30 years. Fresh and frozen-thawed ovarian fragments intraperitoneally grafted into nude mice for 3 weeks. MAIN OUTCOME MEASURE(S): Histologic analysis and immunohistochemical evaluation of c-kit, KL, and GDF-9 expression before and after grafting. RESULT(S): The integrity and proportion of growing follicles increased similarly in both fresh (64%) and frozen-thawed (59%) xenografts compared with non-grafted tissue (fresh: 40%; frozen-thawed: 21%). Both C-kit and KL staining were detected in the oocytes and granulosa cells of preantral follicles, and GDF-9 expression was observed in the oocytes of preantral follicles in all groups. CONCLUSION(S): Freezing does not appear to have a major impact on early follicular growth after transplantation. This study shows, for the first time, expression of c-kit, KL, and GDF-9 in human preantral follicles after ovarian tissue cryopreservation and xenotransplantation.

Survival of human pre-antral follicles after cryopreservation of ovarian tissue, follicular isolation and in vitro culture in a calcium alginate matrix.

BACKGROUND: Ovarian tissue cryopreservation is a promising technique to safeguard fertility in cancer patients. However, in some types of cancer, there is a risk of transmitting malignant cells present in the cryopreserved tissue. To avoid such a risk, pre-antral follicles could be isolated from ovarian tissue and grown in vitro. On the basis of this assumption, the aim of our study was to investigate in vitro survival and growth of pre-antral follicles after cryopreservation of ovarian tissue and follicular isolation, followed by encapsulation in alginate beads. METHODS: Ovarian biopsies from four patients were frozen and thawed. Pre-antral follicles were then isolated and embedded in an alginate matrix before in vitro culture for 7 days. RESULTS: Small pre-antral follicles (42.98 +/- 9.06 microm) from frozen-thawed tissue can survive and develop after enzymatic isolation and in vitro culture. A total of 159 follicles were incubated in a three-dimensional system (alginate hydrogel) and, after 7 days, all of them showed an increase in size (final size 56.73 +/- 13.10 microm). The survival rate of the follicles was 90% (oocyte and all granulosa cells viable). CONCLUSION: Our preliminary results indicate that alginate hydrogels may be a suitable system for in vitro culture of isolated human pre-antral follicles. However, more studies are required to establish whether follicular morphology and functionality can be maintained using this matrix.