Background

The noncanonical Wnt receptor and tyrosine kinase Ror2 has been associated with recessive Robinow syndrome (RRS) and dominant brachydactyly type B1. The phenotypes of mouse mutants implicate Ror2 in the development of the heart, lungs, bone, and craniofacial structures, which are affected in RRS. Following a recently identified role of Ror2 in the migration of mouse primordial germ cells, we extensively characterized its expression throughout the fetal internal reproductive system and the postnatal ductal system.

Results

We show that Ror2 gene products are present in the germ cells and somatic cells of the testis and the ovary of both the mouse and human fetus. In reproductive tract structures, we find that Ror2 is expressed in the mesonephros, developing Wolffian and Müllerian ducts, and later in their derivatives, the epididymal epithelium and uterine epithelium.

Conclusions

This study sets the stage to explore function for this tyrosine kinase receptor in novel regions of expression in the developing reproductive system in both mouse and human.

Objective

To investigate whether delaying the start of ovarian stimulation with GnRH antagonist improves ovarian response in poor responders.

Design

Retrospective study.

Setting

Academic medical center.

Patient(s)

Thirty patients, who responded poorly and did not get pregnant with conventional estrogen priming antagonist IVF protocol.

Intervention(s)

Delayed-start antagonist protocol (estrogen priming followed by early follicular-phase GnRH antagonist treatment for 7 days before ovarian stimulation).

Main outcome measure(s)

Number of dominant follicles and mature oocytes retrieved, mature oocyte yield, and fertilization rate.

Result(s)

The number of patients who met the criteria to proceed to oocyte retrieval was significantly higher in the delayed-start protocol [21/30 (70%)] compared with the previous conventional estrogen priming antagonist cycle [11/30 (36.7%)]. The number of dominant follicles was significantly higher in the delayed-start (4.2 ± 2.7) compared with conventional (2.4 ± 1.3) protocol. In patients who had oocyte retrieval after both protocols (n = 9), the delayed start resulted in shorter ovarian stimulation (9.4 ± 1.4 days vs. 11.1 ± 2.0 days), higher number of mature oocytes retrieved (4.9 ± 2.0 vs. 2.2 ± 1.1), and a trend toward increased fertilization rates with intracytoplasmic sperm injection (ICSI; 86 ± 17% vs. 69 ± 21%) compared with conventional protocol. After delayed start, the average number of embryos transferred was 2.8 ± 1.4 with implantation rate of 9.8% and clinical pregnancy rate of 23.8%.

Conclusion(s)

The delayed-start protocol improves ovarian response in poor responders by promoting and synchronizing follicle development without impairing oocyte developmental competence.

The embryonic stem cell cycle (ESCC) and let-7 families of miRNAs function antagonistically in the switch between mouse embryonic stem cell self-renewal and somatic differentiation. Here, we report that the human ESCC miRNA miR-372 and let-7 act antagonistically in germline differentiation from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). hESC and iPSC-derived primordial germ cell-like cells (PGCLCs) expressed high levels of miR-372 and conversely, somatic cells expressed high levels of let-7. Manipulation of miRNA levels by introduction of miRNA mimics or knockdown with miRNA sponges demonstrated that miR-372 promotes whereas let-7 antagonizes PGCLC differentiation. Knockdown of the individual miR-372 targets SMARCC1, MECP2, CDKN1, RBL2, RHOC, and TGFBR2 increased PGCLC production, whereas knockdown of the let-7 targets CMYC and NMYC suppressed PGCLC differentiation. These findings uncover a miR-372/let-7 axis regulating human primordial germ cell (PGC) specification. Stem Cells 2016;34:1985-1991.

Prepubertal boys treated with high-dose chemotherapy do not have an established means of fertility preservation because no established in vitro technique exists to expand and mature purified spermatogonial stem cells (SSCs) to functional sperm in humans. In this study, we define and characterize the unique testicular cellular niche required for SSC expansion using testicular tissues from men with normal spermatogenesis. Highly purified SSCs and testicular somatic cells were isolated by fluorescence-activated cell sorting using SSEA-4 and THY1 as markers of SSCs and somatic cells. Cells were cultured on various established niches to assess their role in SSC expansion in a defined somatic cellular niche. Of all the niches examined, cells in the SSEA-4 population exclusively bound to adult testicular stromal cells, established colonies, and expanded. Further characterization of these testicular stromal cells revealed distinct mesenchymal markers and the ability to undergo differentiation along the mesenchymal lineage, supporting a testicular multipotent stromal cell origin. In vitro human SSC expansion requires a unique niche provided exclusively by testicular multipotent stromal cells with mesenchymal properties. These findings provide an important foundation for developing methods of inducing SSC growth and maturation in prepubertal testicular tissue, essential to enabling fertility preservation for these boys.