Podocalyxin molecular characteristics and endometrial expression: high conservation between humans and macaques but divergence in mice†.

Podocalyxin (PODXL) is a newly identified key negative regulator of human endometrial receptivity, specifically down-regulated in the luminal epithelium at receptivity to permit embryo implantation. Here, we bioinformatically compared the molecular characteristics of PODXL among the human, rhesus macaque, and mouse, determined by immunohistochemistry and in situ hybridization (mouse tissues) whether endometrial PODXL expression is conserved across the three species and examined if PODXL inhibits mouse embryo attachment in vitro. The PODXL gene, mRNA, and protein sequences showed greater similarities between humans and macaques than with mice. In all species, PODXL was expressed in endometrial luminal/glandular epithelia and endothelia. In macaques (n = 9), luminal PODXL was significantly down-regulated when receptivity is developed, consistent with the pattern found in women. At receptivity, PODXL was also reduced in shallow glands, whereas endothelial expression was unchanged across the menstrual cycle. In mice, endometrial PODXL did not vary considerably across the estrous cycle (n = 16); however, around embryo attachment on d4.5 of pregnancy (n = 4), luminal PODXL was greatly reduced especially near the site of embryo attachment. Mouse embryos failed to attach or thrive when co-cultured on a monolayer of Ishikawa cells overexpressing PODXL. Thus, endometrial luminal PODXL expression is down-regulated for embryo implantation in all species examined, and PODXL inhibits mouse embryo implantation. Rhesus macaques share greater conservations with humans than mice in PODXL molecular characteristics and regulation, thus represent a better animal model for functional studies of endometrial PODXL for treatment of human fertility.

A human-based assisted reproduction protocol for the menstruating spiny mouse, Acomys cahirinus.

The Egyptian or Common spiny mouse (A. cahirinus) is the first rodent species to show human-like menstruation and spontaneous decidualisation. We consider from these, and its other, human-like characteristics that this species will be a more useful and appropriate small animal model for human reproductive studies. Based on this, there is a need to develop specific laboratory-based assisted reproduction protocols including superovulation, in-vitro fertilisation, embryo cryopreservation and transfer to expand and make this model more relevant. Because standard rodent superovulation has not been successful in the spiny mouse, we have selected to test a human protocol. Female spiny mice will receive a subcutaneous GnRH agonist implant and be allowed to recover. Menstrual cycle lengths will then be allowed to stabilize prior to ovarian stimulation. After recovery, females will be injected IP once a day for 4 days with a FSH analogue, to induce follicular growth, and on day 5 will be injected IP with a hCG analogue to trigger ovulation. Females will either be culled 36hrs after trigger to collect oocytes or immediately paired with a stud male and two cell embryos collected 48hrs later. Mature oocytes will be inseminated using fresh spiny mouse spermatozoa and all in-vitro grown and in-vivo collected two cell embryos will be cryopreserved using methods developed in a close spiny mouse relative, the Mongolian gerbil. For embryo transfer, vitrified embryos will be rapidly warmed and non-surgically transferred to surrogate mice. Surrogates will be monitored until pregnancy is apparent (roughly 30 days) and then left undisturbed until birth, 38-40 days after transfer. By successfully developing robust assisted reproduction protocols in A. cahirinus we will be able to use this rodent as a more effective model for human reproduction.

Assessment of potential biomarkers of pre-receptive and receptive endometrium in uterine fluid and a functional evaluation of the potential role of CSF3 in fertility.

The endometrium lines a women's uterus becoming receptive, and allowing embryo implantation to occur, for just a few days during the post-ovulatory mid-secretory phase of each menstrual cycle. We investigated whether concentrations of proposed receptivity biomarkers (VEGF, IL8, FGF2, CSF3 sFlt-1, sGP130 and PlGF) secreted by the endometrium into the uterine cavity and forming the microenvironment for embryo implantation is altered among a population of age-matched women with unexplained (idiopathic) infertility compared to fertile women during the receptive mid-secretory phase (n = 16 fertile, 18 infertile) and the prior pre-receptive early secretory phase (n = 19 fertile, 18 infertile) of their cycle. In the mid-secretory cohort significantly elevated concentrations of five biomarkers; PlGF (p = 0.001), IL8 (p = 0.004), sGP130 (p = 0.009), sFlt-1 (p = 0.021), and CSF3 (p = 0.029) was present in uterine fluid of infertile women during the mid-secretory phase, but only CSF3 was significantly elevated in the pre-receptive early secretory phase (p = 0.006). In vitro studies of glycosylated and non-glycosylated forms of CSF3 at representative fertile (20 ng/mL) and infertile (70 ng/mL) effects on endometrium and embryo behaviour were performed. Non-glycosylated CSF3 at fertile concentrations significantly (p < 0.001) elevated endometrial epithelial cell proliferation however chronic treatment or elevated (infertile) concentrations of CSF3 in glycosylated form abrogated the positive effects. Both forms of CSF3 increased trophoblast cell invasion (p < 0.001) regardless of concentration. Mouse embryo outgrowth was significantly (p < 0.01) increased at fertile but not at infertile concentrations. The study confirmed potential utility of five biomarkers of endometrial receptivity for future application in the mid-secretory phase while highlighting CSF3 is elevated in the earlier pre-receptive phase. Our data provides evidence that CSF3 acts on both human endometrium and embryo in a manner that is concentration and glycosylation dependent.

Successful in vitro culture of pre-antral follicles derived from vitrified murine ovarian tissue: oocyte maturation, fertilization, and live births.

Cryopreservation of ovarian tissue is an important option for preserving the fertility of cancer patients undergoing chemotherapy and radiotherapy. In this study, we examined the viability and function of oocytes derived in vitro from pre-antral follicles as an alternative method for restoring fertility. Pre-antral follicles (specified as secondary follicle with a diameter around 100-130 µm) were mechanically isolated from vitrified-warmed and fresh adult mouse ovarian tissues and cultured for 12 days followed by an ovulation induction protocol at the end of this period to initiate oocyte maturation. Oocytes were then released from these follicles, fertilized in vitro, and cultured to the blastocyst stage and vitrified. After storage in liquid nitrogen for 2 weeks, groups of vitrified blastocysts were warmed and transferred into pseudo-pregnant recipient females. Although most of the isolated mouse pre-antral follicles from fresh (79.4%) and vitrified (75.0%) ovarian tissues survived the 12-day in vitro culture period, significantly fewer mature oocytes developed from vitrified-warmed pre-antral follicles than from the fresh controls (62.2 vs 86.4%, P<0.05). No difference was observed in embryo cleavage rates between these two groups, but the proportion of embryos that developed into blastocysts in the vitrification group was only half that of the controls (24.2 vs 47.2%, P<0.05). Nevertheless, live births of healthy normal pups were achieved after transfer of vitrified blastocysts derived from both experimental groups. This study shows that successful production of healthy offspring using an in vitro follicle culture system is feasible, and suggests that this procedure could be used in cancer patients who wish to preserve their fertility using ovarian tissue cryopreservation.

Live offspring from vitrified blastocysts derived from fresh and cryopreserved ovarian tissue grafts of adult mice.

Ovarian tissue cryopreservation and transplantation can be used to preserve fertility for cancer patients. In this study, we assessed the viability and function of ovarian tissue from adult mice that was cryopreserved by solid surface vitrification or traditional slow-cooling using various in vitro and in vivo techniques, including allotransplantation, in vitro oocyte maturation, embryo culture in vitro, blastocyst cryopreservation, embryo transfer, and development. The importance of cumulus cells for oocyte maturation, fertilization, and embryo development was investigated. Graft recovery, follicle survival, and oocyte retrieval was similar in control, vitrified, and slow-cooled groups. High rates of oocyte maturation, cleavage, and blastocyst formation were achieved, with no significant differences between the control, vitrified or slow-cooled ovarian tissue grafts. The presence of cumulus cells was important for oocyte maturation, fertilization, and subsequent development. Cumulus-oocyte complexes with no surrounding cumulus cells (N-COCs) or with an incomplete layer (P-COCs) had significantly lower rates of oocyte maturation and blastocyst formation than cumulus-oocyte complexes with at least one complete layer of cumulus cells (F-COCs; maturation rate: 63, 78 vs 94%; blastocyst rate: 29, 49 vs 80%). Live births were achieved using vitrified blastocysts derived from oocytes taken from vitrified and slow-cooled ovarian tissue heterotypic allografts. Successful production of healthy offspring from these vitrified blastocysts suggests that this technique should be considered as a useful stage to pause in the assisted reproduction pathway. This provides an alternative protocol for restoring fertility and offering cancer patients a better indication of their chances of pregnancy and live birth.