Monica M. Laronda

A bioprosthetic ovary created using 3D printed microporous scaffolds restores ovarian function in sterilized mice

Emerging additive manufacturing techniques enable investigation of the effects of pore geometry on cell behavior and function. Here, we 3D print microporous hydrogel scaffolds to test how varying pore geometry, accomplished by manipulating the advancing angle between printed layers, affects the survival of ovarian follicles. 30° and 60° scaffolds provide corners that surround follicles on multiple sides while 90° scaffolds have an open porosity that limits follicle–scaffold interaction. As the amount of scaffold interaction increases, follicle spreading is limited and survival increases. Follicle-seeded scaffolds become highly vascularized and ovarian function is fully restored when implanted in surgically sterilized mice. Moreover, pups are born through natural mating and thrive through maternal lactation. These findings present an in vivo functional ovarian implant designed with 3D printing, and indicate that scaffold pore architecture is a critical variable in additively manufactured scaffold design for functional tissue engineering.

A microfluidic culture model of the human reproductive tract and 28-day menstrual cycle

The endocrine system dynamically controls tissue differentiation and homeostasis, but has not been studied using dynamic tissue culture paradigms. Here we show that a microfluidic system supports murine ovarian follicles to produce the human 28-day menstrual cycle hormone profile, which controls human female reproductive tract and peripheral tissue dynamics in single, dual and multiple unit microfluidic platforms (Solo-MFP, Duet-MFP and Quintet-MPF, respectively). These systems simulate the in vivo female reproductive tract and the endocrine loops between organ modules for the ovary, fallopian tube, uterus, cervix and liver, with a sustained circulating flow between all tissues. The reproductive tract tissues and peripheral organs integrated into a microfluidic platform, termed EVATAR, represents a powerful new in vitro tool that allows organ–organ integration of hormonal signalling as a phenocopy of menstrual cycle and pregnancy-like endocrine loops and has great potential to be used in drug discovery and toxicology studies.

The Development of Cervical and Vaginal Adenosis as a Result of Diethylstilbestrol Exposure In Utero

Exposure to exogenous hormones during development can result in permanent health problems. In utero exposure to diethylstilbestrol (DES) is probably the most well documented case in human history. DES, an orally active synthetic estrogen, was believed to prevent adverse pregnancy outcome and thus was routinely given to selected pregnant women from the 1940s to the 1960s. It has been estimated that 5 million pregnant women worldwide were prescribed DES during this period. In the early 1970s, vaginal clear cell adenocarcinomas (CCACs) were diagnosed in daughters whose mother took DES during pregnancy (known as DES daughters). Follow-up studies demonstrated that exposure to DES in utero causes a spectrum of congenital anomalies in female reproductive tracts and CCACs. Among those, cervical and vaginal adenoses are most commonly found, which are believed to be the precursors of CCACs. Transformation related protein 63 (TRP63/p63) marks the cell fate decision of Müllerian duct epithelium (MDE) to become squamous epithelium in the cervix and vagina. DES disrupts the TRP63 expression in mice and induces adenosis lesions in the cervix and vagina. This review describes mouse models that can be used to study the development of DES-induced anomalies, focusing on cervical and vaginal adenoses, and discusses their molecular pathogenesis.

Estrogen Actions in the Male Reproductive System Involve Estrogen Response Element-Independent Pathways

The estrogen receptor-alpha (ERalpha) acts through multiple pathways, including estrogen response element (ERE)-dependent (classical) and ERE-independent (nonclassical) mechanisms. We previously created a mouse model harboring a two-amino-acid mutation of the DNA-binding domain (E207A, G208A) that precludes direct binding of ERalpha to an ERE. After crossing heterozygous mutant mice with an ERalpha knockout (ERKO) line, it was possible to assess the degree of physiological rescue by the isolated ERalpha nonclassical allele (-/AA; AA) when compared with ERKO mice (-/-) and to wild type (+/+; WT). In male ERKO mice up to 8 months of age, testosterone levels were high, although LH levels were similar to WT. Testosterone was normal in the AA mice, indicating that the AA allele rescues the enhanced testosterone biosynthesis in ERKO mice. Male ERKO mice exhibited distention of the seminiferous tubules as early as 2-3 months of age as a consequence of decreased water resorption in the efferent ducts. By 3-4 months of age, ERKO mice had impaired spermatogenesis in approximately 40% of their tubules, and sperm counts and motility declined in association with the histological changes. In the AA mice, histological defects were greatly reduced or absent, and sperm counts and motility were rescued. Levels of aquaporins 1 and 9, which contribute to water uptake in the efferent ducts, were reduced in ERKO mice and partially or fully rescued in AA mice, whereas another water transporter, sodium-hydrogen exchanger-3, was decreased in both ERKO and AA mice. We conclude that non-ERE-dependent estrogen pathways are sufficient to rescue the defective spermatogenesis observed in ERKO mice and play a prominent role in ERalpha action in the testis, including pathways that regulate water resorption and androgen biosynthesis.

Sox3 Functions in a Cell-Autonomous Manner to Regulate Spermatogonial Differentiation in Mice

The X-linked Sox3 gene encodes a member of the Sry high-mobility group box proteins, which play a role in many developmental processes including neurogenesis and testis development. This study further examined the role of Sox3 in spermatogenesis. Males without Sox3 expression exhibited a similar number of germ cell nuclear antigen-positive germ cells at 1, 5, and 10 d postpartum (dpp) compared to their wild-type littermates, but there was significant germ cell depletion by 20 dpp. However, spermatogenesis later resumed and postmeiotic germ cells were observed by 56 dpp. The VasaCre transgene was used to generate a germ cell-specific deletion of Sox3. The phenotype of the germ cell-specific Sox3 knockout was similar to the ubiquitous knockout, indicating an intrinsic role for Sox3 in germ cells. The residual germ cells in 20 dpp Sox3(-/Y) males were spermatogonia as indicated by their expression of neurogenin3 but not synaptonemal complex protein 3, which is expressed within cells undergoing meiosis. RNA expression analyses corroborated the histological analyses and revealed a gradual transition from relatively increased expression of spermatogonia genes at 20 dpp to near normal expression of genes characteristic of undifferentiated and meiotic germ cells by 84 dpp. Fluorescent-activated cell sorting of undifferentiated (ret tyrosine kinase receptor positive) and differentiated (kit receptor tyrosine kinase-positive) spermatogonia revealed depletion of differentiated spermatogonia in Sox3(-/Y) tubules. These results indicate that Sox3 functions in an intrinsic manner to promote differentiation of spermatogonia in prepubertal mice but it is not required for ongoing spermatogenesis in adults. The Sox3(-/Y) males provide a unique model for studying the mechanism of germ cell differentiation in prepubertal testes.

Foxl2, a Forkhead Transcription Factor, Modulates Nonclassical Activity of the Estrogen Receptor-α

Foxl2 is a forkhead transcription factor required for ovary development and ovarian follicle maturation. In this report, we identified and characterized a functional relationship between Foxl2 expression and estrogen receptor (ER)-alpha signaling. We show that Foxl2 has no effect on classical ERalpha-mediated transcription, which occurs through canonical estrogen response elements. However, Foxl2 suppresses ERalpha signaling through nonclassical tethered transcriptional pathways. Specifically, the selective ER modulator tamoxifen stimulates activator protein-1 (AP1)-dependent transcription via the ERalpha, and this enhancement is blocked by Foxl2. Two lines of evidence suggest that Foxl2 suppression is mediated by physical interactions with ERalpha rather than direct action at AP1 binding sites. First, ERalpha is coimmunoprecipitated with Foxl2. Second, activation of a upstream activating sequence (UAS) reporter by Gal4-cJun in the presence of ERalpha and tamoxifen was blocked by Foxl2, demonstrating suppression in the absence of an AP1 site. Cyclooxygenase-2 (COX2), which is required for ovulation, was identified through expression profiling as a candidate physiological target for nonclassical ERalpha signaling and thus modulation by ERalpha/Foxl2 interactions. This possibility was confirmed by two sets of experiments. COX2 protein levels were induced by ERalpha in the presence of tamoxifen, and protein expression was suppressed by Foxl2. In addition, ERalpha stimulation of the COX2 promoter was repressed by Foxl2. We conclude that ERalpha and Foxl2 interact and that Foxl2 selectively suppresses ERalpha-mediated transcription of AP1-regulated genes. These data provide a potential point of convergence for ERalpha and Foxl2 to regulate ovarian development and function.

Recreating the female reproductive tract in vitro using iPSC technology in a linked microfluidics environment

The female reproductive tract produces hormones for reproductive function and cardiovascular, bone and sexual health; the tract supplies a finite number of gametes, and it supports fetal development. Diseases that affect each of the female reproductive tract organs, along with treatments that have direct, deleterious effects on the reproductive tract (for example, chemotherapeutics), are understudied due to the lack of model systems that phenocopy in vivo function. This review describes a path toward developing female reproductive tract mimics. The models use isolated primary support cells cultured onto a biological scaffold and within a microfluidic system to create a niche and support the desired differentiation of epithelia, germ and somatic cells from patient-derived induced pluripotent stem cells. Improving our fund of knowledge about reproductive tract biology and creating reproductive organs for patients who have lost gonadal, uterine or vaginal/ cervical function is a major step forward in womens health and an important advancement in personalized medicine.

A review of reported surgical techniques in fertility preservation for prepubertal and adolescent females facing a fertility threatening diagnosis or treatment

BACKGROUND Ovarian tissue cryopreservation is increasing as a preferred option for fertility preservation for prepubertal and young adolescent females facing a fertility threatening diagnosis or treatment. DATA SOURCES Ovid MEDLINE and PubMed searches for terms related to ovarian tissue removal for fertility preservation revealed there is no current consensus on operative technique for surgical ovarian cortical tissue removal in adult females. Additionally, there are limited published reports of surgical approach and outcomes in the pediatric population. In total, 22 publications were reviewed for their operative approach, ovarian tissue harvesting techniques, complications and outcomes. CONCLUSIONS Reported operative approaches and techniques for ovarian tissue cryopreservation for pediatric and adolescent patients are variable. Further investigations into operative technique and tissue harvesting that maintains healthy ovarian follicles for transplant will help establish standard technical principles for surgery in pediatric and adolescent females undergoing fertility preservation.

Good manufacturing practice requirements for the production of tissue vitrification and warming and recovery kits for clinical research

Products that are manufactured for use in a clinical trial, with the intent of gaining US Food and Drug Administration (FDA) approval for clinical use, must be produced under an FDA approved investigational new drug (IND) application. We describe work done toward generating reliable methodology and materials for preserving ovarian cortical tissue through a vitrification kit and reviving this tissue through a warming and recovery kit. We have described the critical steps, procedures, and environments for manufacturing products with the intent of submitting an IND. The main objective was to establish an easy-to-use kit that would ensure standardized procedures for quality tissue preservation and recovery across the 117 Oncofertility Consortium sites around the globe. These kits were developed by breaking down the components and steps of a research protocol and recombining them in a way that considers component stability and use in a clinical setting. The kits were manufactured utilizing current good manufacturing practice (cGMP) requirements and environment, along with current good laboratory practices (cGLP) techniques. Components of the kit were tested for sterility and endotoxicity, and morphological endpoint release criteria were established. We worked with the intended down-stream users of these kits for development of the kit instructions. Our intention is to test these initial kits, developed and manufactured here, for submission of an IND and to begin clinical testing for preserving the ovarian tissue that may be used for future restoration of fertility and/or hormone function in women who have gonadal dysgenesis from gonadotoxic treatment regimens or disease.

Laparoscopic unilateral oophorectomy for ovarian tissue cryopreservation in children

BACKGROUND/PURPOSE Many survivors of childhood cancer will experience premature gonadal insufficiency or infertility as a consequence of their medical treatments. Ovarian tissue cryopreservation (OTC) remains an experimental means of fertility preservation with few reports focused on the surgical technique and postoperative outcomes for OTC in children. METHODS This is a single institution, retrospective review of OTC cases from January 2011 to December 2017. Children were eligible for OTC if they had a greater than 80% risk of premature ovarian insufficiency or infertility owing to their anticipated gonadotoxic medical treatment. RESULTS OTC was performed in 64 patients. Median age was 12 years old (range: 5 months-23 years). Nearly half (48%) of the patients were premenarchal. Laparoscopic unilateral oophorectomy was performed in 84% of patients. There were no surgical complications. In 76% of patients, OTC was performed in conjunction with an ancillary procedure. The majority (96%) of patients were discharged within 24 hours. Median time from operation to medical therapy was six days, with no unanticipated treatments delays attributable to OTC. CONCLUSIONS Laparoscopic unilateral oophorectomy for OTC can be performed safely, in combination with other ancillary procedures, as an outpatient procedure without delaying medical therapy for children facing a fertility-threatening diagnosis or treatment. LEVEL OF EVIDENCE IV.