Alison Y. Ting

In vitro development of secondary follicles from cryopreserved rhesus macaque ovarian tissue after slow-rate freeze or vitrification

BACKGROUND Ovarian tissue cryopreservation is the only option for preserving fertility in prepubertal girls and cancer patients requiring immediate treatment. Following ovarian tissue cryopreservation, fertility can be restored after tissue transplant or in vitro follicle maturation. METHODS Macaque (n= 4) ovarian cortex was cryopreserved using slow-rate freezing (slow freezing) or vitrification. Tissues were fixed for histology or phosphohistone H3 (PPH3) analysis, cultured with bromodeoxyuridine (BrdU) or used for three-dimensional secondary follicle culture. Follicular diameter and steroid hormones were measured weekly. RESULTS Slow freezing induced frequent cryo-injuries while vitrification consistently maintained morphology of the stroma and secondary follicles. PPH3 was similar in fresh and vitrified, but sparse in slow-frozen tissues. BrdU uptake appeared diminished following both methods compared with that in fresh follicles. In vitro follicle survival and growth were greater in fresh than in cryopreserved follicles. Antrum formation appeared similar after vitrification compared with the fresh, but was reduced following slow freezing. Steroid production was delayed or diminished following both methods compared with fresh samples. CONCLUSIONS Secondary follicle morphology was improved after vitrification relative to slow freezing. Following vitrification, stroma was consistently more compact with intact cells typical to that of fresh tissue. BrdU uptake demonstrated follicle viability post-thaw/warming. For the first time, although not to the extent of fresh follicles, macaque follicles from cryopreserved tissue can survive, grow, form an antrum and produce steroid hormones, indicating some functional preservation. The combination of successful ovarian tissue cryopreservation with in vitro maturation of follicles will offer a major advancement to the field of fertility preservation.

Estrogen Regulates Vaginal Sensory and Autonomic Nerve Density in the Rat

Abstract Vaginal function is strongly influenced by reproductive hormone status. Vaginal dysfunction during menopause is generally assumed to occur because of diminished estrogen-mediated trophic support of vaginal target cells. However, peripheral neurons possess estrogen receptors and are potentially responsive to gonadal steroid hormones. In the present study, we investigated whether sensory and autonomic innervation of the vagina varies among rats during the estrus phase of the estrous cycle, following chronic ovariectomy, and after sustained estrogen replacement. Relative to rats in estrus, ovariectomized rats showed a 59% elevation in nerve density, as determined using the panneuronal marker PGP 9.5. This increase persisted even after correcting for differences in vaginal tissue size, indicating true axonal proliferation after ovariectomy rather than changes secondary to altered volume. Increased total innervation after ovariectomy was attributable to increased densities of sympathetic nerves immunostained for tyrosine hydroxylase (70%), cholinergic parasympathetic nerves immunoreactive for vesicular acetylcholine transporter (93%), and calcitonin gene-related peptide-immunoreactive sensory nociceptor nerves (84%). Myelinated primary sensory innervation revealed by RT-97 immunoreactivity did not appear to be affected. Sustained 17β-estradiol administration reduced innervation density to an extent comparable to that of estrus, implying that estrogen is the hormone mediating vaginal neuroplasticity. These findings indicate that some aspects of vaginal dysfunction during menopause may be attributable to changes in innervation. Increased sympathetic innervation may augment vasoconstriction and promote vaginal dryness, while sensory nociceptor axon proliferation may contribute to symptoms of pain, burning, and itching associated with menopause and some forms of vulvodynia.

Fibrin promotes development and function of macaque primary follicles during encapsulated three-dimensional culture

STUDY QUESTION Does fibrin introduced into the extracellular matrix affect the growth and maturation of individual primate follicles during encapsulated three-dimensional (3D) culture? SUMMARY ANSWER While not altering follicle survival, fibrin-alginate (FIBRIN) improves macaque primary, but not secondary, follicle development during encapsulated 3D culture in terms of growth, steroidogenesis, anti-Müllerian hormone (AMH)/vascular endothelial growth factor (VEGF) production and oocyte maturation. WHAT IS KNOWN ALREADY Efforts to grow non-human primate ovarian follicles from the secondary to the antral stage during encapsulated 3D culture have been successful. However, the growth and maturation of primary follicles in vitro has not been reported in primates, especially in chemically defined conditions. STUDY DESIGN, SIZE, DURATION In vitro follicle maturation was investigated using the rhesus macaque (Macaca mulatta). Ovaries (n = 7 pairs) were obtained during the early follicular phase of the menstrual cycle (cycle day 1-4). Primary (80-120 µm diameter) and secondary (125-225 µm diameter) follicles were isolated mechanically, randomly assigned to experimental groups, encapsulated into alginate (0.25% w/v) or FIBRIN (25 mg/ml fibrinogen-0.25% alginate) and cultured for 13 and 5 weeks, respectively. MATERIALS, SETTING, METHODS Individual follicles were cultured in alpha minimum essential medium supplemented with FSH. Follicle survival and growth were assessed by microscopy. Follicles that reached the antral stage were treated with recombinant hCG. Metaphase II (MII) oocytes were inseminated via ICSI. Follicle morphology was evaluated by hematoxylin and eosin (H&E) staining. Immunohistochemistry was performed for cytochrome P450 family 17 subfamily A polypeptide 1 (CYP17A1) and 19 subfamily A polypeptide 1 (CYP19A1). Culture medium was analyzed for estradiol (E2) and progesterone by chemiluminescence, androstenedione (A4) by radioimmunoassay, as well as anti-Müllerian hormone (AMH) and vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE A total of 105 primary and 133 secondary follicles were collected. The presence of fibrin in the alginate matrix had no effect on either primary or secondary follicle survival. Growing primary and secondary follicles formed an antrum at Weeks 9 and 3, respectively. The percentage of growing follicles was higher (P < 0.05) for primary follicles cultured in FIBRIN than alginate at Week 13. The diameters were larger for the growing secondary follicles cultured in alginate than FIBRIN at Week 5 (P < 0.05). H&E staining revealed the typical morphology for small antral follicles. CPY17A1 immunostaining was detected in theca cells, while CYP19A1 was observed in granulosa cells. E2 increased (P < 0.05) during antrum formation in growing follicles at Week 9 for primary and Week 3 for secondary follicles. AMH levels in medium from growing primary follicles increased (P < 0.05) after Week 4 with peak levels at Weeks 9-11. AMH increased (P < 0.05) in growing secondary follicles at Weeks 3-5. VEGF levels in medium were elevated (P < 0.05) in growing primary follicles at Week 9. VEGF increased (P < 0.05) in medium from growing secondary follicles at Weeks 3-5. E2, AMH and VEGF production was higher (P < 0.05) in primary follicle culture with FIBRIN than alginate alone. One primary follicle cultured in FIBRIN (1 of 5 follicles harvested) and a secondary follicle cultured in alginate alone (1 of 15 follicles harvested) yielded an MII oocyte. The fertilized oocyte from primary follicle culture arrested without cell division after fertilization, while the oocyte from secondary follicle culture cleaved and reached the morula stage. LIMITATIONS, REASONS FOR CAUTION The study reports on in vitro development and function of individual macaque follicles, that is limited to the interval from the primary and secondary stage to the small antral stage. The findings await translation to human ovarian follicles. WIDER IMPLICATIONS OF THE FINDINGS The 3D model for primate follicle development offers a unique opportunity to investigate the growth and regulation of primate primary, as well as secondary follicles, and their enclosed oocytes, as they grow to the antral stage by monitoring and manipulating factors or signaling pathways in vitro. Since primate primary follicles, in addition to secondary follicles, can be cultured to the antral stage to provide mature oocytes, they represent an additional source of pre-antral follicles for in vitro follicle maturation with the potential to provide gametes for assisted reproductive technology as an option for fertility preservation in women, including patients with cancer. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by The Oncofertility Consortium (NIH U54 RR024347-HD058294, PL1-EB008542), NIH U54-HD18185 (Eunice Kennedy Shriver Specialized Cooperative Centers Program in Reproduction and Infertility Research), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), Oregon National Primate Research Center 8P51OD011092. There are no conflicts of interest.

Use of hormonal protection for chemotherapy-induced gonadotoxicity

It is still controversial that GnRH agonist (GnRHa) protects ovarian function from chemotherapy-induced gonadotoxicity. Indeed, the results of many studies related to this issue are neither consistent nor convincing because of the weak study design and the inadequate sample size. We identified 11 prospective controlled studies (8 nonrandomized and 3 randomized) for the systemic review and meta-analysis. The meta-analysis showed that GnRHa cotreatment during chemotherapy can protect ovarian function. However, it is worthy to note that the result of this meta-analysis is influenced by nonrandomized studies. The protective effect of GnRHa will remain elusive until the currently ongoing large, prospective, randomized studies are completed. In addition, tamoxifen, a selective estrogen receptor modulator, may have the protective effect against loss of follicles and ovarian function, which was caused by chemotherapy.

Ovarian Endocrine Disruption Underlies Premature Reproductive Senescence Following Environmentally Relevant Chronic Exposure to the Aryl Hydrocarbon Receptor Agonist 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

Abstract The aryl hydrocarbon receptor (AHR) mediates the effects of many endocrine disruptors and contributes to the loss of fertility in polluted environments. While previous work has focused on mechanisms of short-term endocrine disruption and ovotoxicity in response to AHR ligands, we have shown recently that chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces premature reproductive senescence in female rats without depletion of ovarian follicular reserves. In the current study, premature reproductive senescence was induced using a range of low-dose exposure to TCDD (0, 1, 5, 50, and 200 ng kg−1 wk−1) beginning in utero and continuing until the transition to reproductive senescence. Doses of 50 and 200 ng TCDD kg−1 wk−1 delayed the age at vaginal opening and accelerated the loss of normal reproductive cyclicity with age without depletion of follicular reserves. Serum estradiol concentrations were decreased in a dose-dependent fashion (≥5 ng kg−1 wk−1) across the estrous cycle in perisenescent rats still displaying normal cyclic vaginal cytology. Serum FSH, LH, and progesterone profiles were unchanged by TCDD. The loss of reproductive cyclicity following chronic exposure to TCDD was not accompanied by decreased responsiveness to GnRH. Ovarian endocrine disruption is the predominant functional change preceding the premature reproductive senescence induced by chronic exposure to low doses of the AHR-specific ligand TCDD.

Morphological and functional preservation of pre-antral follicles after vitrification of macaque ovarian tissue in a closed system

STUDY QUESTION What are the appropriate conditions to vitrify the macaque ovarian cortex in a large-volume, closed system that will preserve functional pre-antral follicles? SUMMARY ANSWER The combination of glycerol, ethylene glycol (EG) and polymers with cooling in liquid nitrogen (LN2) vapor and a two-step warming procedure was able to preserve tissue and follicle morphology as well as function of a small population of secondary follicles in the macaque ovarian cortex following vitrification in a closed system. WHAT IS KNOWN ALREADY For prepubertal cancer patients or those who require immediate cancer therapy, ovarian tissue cryopreservation offers the only hope for future fertility. However, the efficacy of live birth from the transplantation of cryopreserved ovarian tissue is still unclear. In addition, live birth from cryopreserved ovarian tissue has only been demonstrated after tissue autotransplantation, which poses the risk of transmitting metastatic cancer cells back to the cancer survivor in certain cancers. STUDY DESIGN, SIZE, DURATION Non-human primate model, n = 4, randomized, control versus treatment. End-points were collected from tissue histology, tissue culture (48 h) and isolated secondary follicle culture (6 weeks). PARTICIPANTS/MATERIALS, SETTING, METHODS Two vitrification solutions (VSs) containing EG + glycerol (VEG) and EG + dimethylsulfoxide (VED) were examined for vitrification, devitrification and thermodynamic properties. Once the optimal VS was determined, macaque ovarian cortical pieces (3 × 3 × 0.5 mm(3)) were divided into fresh and two vitrified groups (VEG and VED). For the vitrification groups, tissues were exposed to 1/4, 1/2 and 1× VS for 5 min/step as well as 1× VS + polymers for 1 min at 37°C, loaded into high-security straws with 1 ml of VS + polymers, heat sealed and cooled in LN2 vapor. Samples were warmed in a 40°C water bath and cryoprotective agents were diluted with 1, 0.5, 0.25 and 0 M sucrose. Tissues were fixed for histological analysis and cultured with bromodeoxyuridine (BrdU). Secondary follicles from VEG tissues were encapsulated and cultured (n = 24/treatment/animal). Follicle health, diameter and steroid [progesterone, androstenedione (A4), estradiol (E2)] production were analyzed weekly. MAIN RESULTS AND THE ROLE OF CHANCE Dense stroma and intact pre-antral follicles were observed using VS containing 27% glycerol, 27% EG and 0.8% polymers with cooling in LN2 vapor and a two-step warming. Higher cooling and warming rates led to fracturing. BrdU uptake was evident in granulosa cells of growing follicles in fresh and vitrified tissues. Secondary follicles from fresh tissues (70 ± 12%) and tissues vitrified with VEG (52 ± 2%) showed similar survival rates (all data: mean ± SEM; P > 0.05). For both groups, the initial follicle diameter was similar and increased (P < 0.05) by Week 3, but diameters in vitrified follicles were smaller (P < 0.05) by Week 6 (566 ± 27 µm) than those of the fresh follicles (757 ± 26 µm). Antrum formation rates were lower (P < 0.05) for vitrified (37 ± 6%) relative to fresh (64 ± 8%) follicles. There was no significant change in levels in culture media of E2, P4 and A4 between fresh and VEG groups at any time point during culture. LIMITATIONS, REASONS FOR CAUTION Only in vitro studies are reported. Future in vivo tissue transplantation studies will be needed to confirm long-term function and fertility potential of vitrified ovarian tissues. WIDER IMPLICATIONS OF THE FINDINGS This is the first demonstration of antral follicle development during 3D culture following ovarian tissue vitrification in a closed system using primate ovarian tissue. While diminished antrum formation and slower growth in vitro reflect residual cryodamage, continued development of ovarian tissue vitrification based on cryobiology principles using a non-human primate model will identify safe, practical and efficient protocols for eventual clinical use. Tissue function following heterotopic transplantation is currently being examined. STUDY FUNDING/COMPETING INTEREST(S) National Institutes of Health (NIH) Oncofertility Consortium UL1 RR024926 (1RL1-HD058293, HD058295, PL1 EB008542), the Eunice Kennedy Shriver NICHD/NIH (U54 HD018185) and ONPRC 8P51OD011092-53. G.M.F. works for the company that makes the polymers used in the current study.

Amphiregulin promotes the maturation of oocytes isolated from the small antral follicles of the rhesus macaque

BACKGROUND In non-primates, the epidermal growth factor (EGF) and EGF-related ligands such as amphiregulin (AREG) serve as critical intermediates between the theca/mural cells and the cumulus-oocyte-complex (COC) following the mid-cycle LH surge. Studies were designed in primates (1) to analyze AREG levels in follicular fluid (follicular fluid) obtained from pre-ovulatory follicles, as well as (2) to assess dose-dependent effects of AREG on oocytes from small antral follicles (SAFs) during culture, including meiotic and cytoplasmic maturation. METHODS Controlled ovulation protocols were performed on rhesus monkeys (n=12) to determine AREG content within the single, naturally selected dominant follicle after an ovulatory stimulus. Using healthy COCs (n=271) obtained from SAFs during spontaneous cycles (n=27), in vitro maturation (IVM) was performed in the absence or presence of physiological concentrations of AREG (10 or 100 ng/ml) with or without gonadotrophins (FSH, 75 mIU/ml; LH, 75 mIU/ml). At the end of the culture period, oocyte meiotic maturation was evaluated and ICSI was performed (n=111), from which fertilization and early embryo development was followed in vitro. RESULTS AREG levels in follicular fluid from pre-ovulatory follicles increased (P<0.05) following an ovulatory bolus of hCG at 12, 24 and 36 h post-treatment. At 12 h post-hCG, AREG levels in follicular fluid ranged from 4.8 to 121.4 ng/ml. Rhesus macaque COCs incubated with 10 ng/ml AREG in the presence of gonadotrophins displayed an increased percentage of oocytes that progressed to the metaphase II (MII) stage of meiosis (82 versus 56%, P<0.05) and a decreased percentage of metaphase I (MI) oocytes (2 versus 23%, P<0.05) relative to controls, respectively. The percentage of either MI or MII oocytes at the end of the culture period was not different between oocytes cultured with 100 ng/ml AREG or in media alone. Fertilization and first cleavage rates obtained by ICSI of all IVM MII oocytes were 93 and 98%, respectively, and did not vary among treatment groups. Of the MII oocytes that fertilized (n=103), 37 were randomly selected and maintained in culture to assess developmental potential. A total of 13 early blastocysts were obtained, with four embryos developing to expanded blastocysts. CONCLUSIONS These data indicate that AREG levels increase in rhesus macaque pre-ovulatory follicles after an ovulatory stimulus, and a specific concentration of AREG (10 ng/ml) enhances rhesus macaque oocyte nuclear maturation but not cytoplasmic maturation from SAFs obtained during the natural menstrual cycle. However, owing to the small number of samples in some treatment groups, further studies are now required.

Synthetic polymers improve vitrification outcomes of macaque ovarian tissue as assessed by histological integrity and the in vitro development of secondary follicles

Ovarian tissue cryopreservation is the only proven option for fertility preservation in female cancer patients who are prepubertal or require immediate treatment. However it remains unclear which cryopreservation protocol is best in cases where the tissue may contain cancerous cells, as these should be matured in vitro rather than autografted. This study evaluated different cryoprotectant exposure times and whether the addition of synthetic polymers (Supercool X-1000, Z-1000 and polyvinylpyrrolidone [PVP K-12]) to the vitrification solution is beneficial to tissue morphology, cellular proliferation and subsequent in vitro function of secondary follicles. Pieces of macaque (n=4) ovarian cortex were exposed to vitrification solution containing glycerol (25%, v/v) and ethylene glycol (25%, v/v) for 3 or 8 min, without (V3, V8) or with (VP3, VP8) polymers (0.2% [v/v] X-1000, 0.4% Z-1000 and 0.2% PVP). Fresh and vitrified tissues were fixed for histology and phosphohistone H3 (PPH3) analysis, or used for secondary follicle isolation followed by encapsulated 3D culture. Five-week follicle survival and growth, as well as steroid hormones (estradiol [E(2)], progesterone, androstenedione) were measured weekly. Morphology of the stroma and preantral follicles as well as PPH3 expression, was preserved in all vitrified tissues. Vitrification with polymers and shorter incubation time (VP3) increased in vitro follicle survival and E(2) production compared to other vitrified groups. Thus, a short exposure of macaque ovarian tissue to a vitrification solution containing synthetic polymers preserves morphology and improves in vitro function of secondary follicles.

Effect of chronic exposure to the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin in female rats on ovarian gene expression

The aryl hydrocarbon receptor (AHR) mediates the effects of many endocrine disruptors and contributes to the loss of fertility in polluted environments. Female rats exposed chronically to environmentally relevant doses of the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) across their lifespan experience accelerated reproductive senescence preceded by ovarian endocrine disruption. The purpose of this study was to determine the changes in ovarian gene expression that accompany the loss of ovarian function caused by chronic exposure to TCDD. Beginning in utero, female Sprague-Dawley rats received TCDD (1, 5, 50, or 200 ng/kg-week; n=4 per group) or vehicle weekly throughout their lifespan, and were sacrificed on diestrus just prior to loss of reproductive cyclicity at 11 months of age. Microarray analysis was used to determine differences in ovarian gene expression between control and TCDD-treated (200 ng/kg-week) animals. To confirm microarray results, real-time PCR was used to assess changes in gene expression among treatment groups. TCDD treatment decreased (p<0.05) proestrus serum estradiol concentrations with no effect on serum progesterone. In ovaries from rats treated with 200 ng/kg-week TCDD compared to controls, 19 genes of known function were found to be up-regulated, while 31 ovarian genes were found to be down-regulated >or=1.5-fold (p<or=0.05). Gene expression of 17 alpha-hydroxylase decreased following chronic TCDD treatment, suggesting the decrease in estradiol biosynthesis may be a consequence of decreased substrate. Taken together with past studies indicating a lack of effect on hypothalamus or pituitary function, the apparent regulation of key ovarian genes support the hypothesis that chronic TCDD exposure directly affects ovarian function.

Differential effects of estrogen and progesterone on development of primate secondary follicles in a steroid-depleted milieu in vitro

STUDY QUESTION What are the direct effects of progesterone (P4) and estradiol (E2) on the development and function of primate follicles in vitro from the pre-antral to early antral stage? SUMMARY ANSWER In a steroid-depleted milieu, E2 improved follicle survival, growth, antrum formation and oocyte health, whereas P4 exerted minimal beneficial effects on follicle survival and reduced oocyte health. WHAT IS KNOWN ALREADY Effects of P4 and E2 on follicle development have been studied primarily in large antral and pre-ovulatory follicles. Chronic P4 exposure suppresses antral follicle growth, but acute P4 exposure promotes oocyte maturation in pre-ovulatory follicles. Effects of E2 can be stimulatory or inhibitory depending upon species, dose and duration of exposure. STUDY DESIGN, SIZE, DURATION Non-human primate model, randomized, control versus treatment. Macaque (n = 6) secondary follicles (n = 24 per animal per treatment group) were cultured for 5 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS Adult rhesus macaque secondary follicles were encapsulated in 0.25% alginate and cultured individually in media containing follicle stimulating hormone plus (i) vehicle, (ii) a steroid-synthesis inhibitor, trilostane (TRL, 250 ng/ml), (iii) TRL + low E2 (100 pg/ml) or progestin (P, 10 ng/ml R5020) and (iv) TRL + high E2 (1 ng/ml E2) or P (100 ng/ml R5020). Follicles reaching the antral stage (≥750 µm) were treated with human chorionic gonadotrophin for 34 h. End-points included follicle survival, antrum formation, growth pattern, plus oocyte health and maturation status, as well as media concentrations of P4, E2 and anti-Müllerian hormone (AMH). MAIN RESULTS AND THE ROLE OF CHANCE In a steroid-depleted milieu, low dose, but not high dose, P improved (P < 0.05) follicle survival, but had no effect (P > 0.05) on antrum formation and AMH production. Low-dose P increased (P < 0.05) P4 production in fast-grow follicles, and both doses of P elevated (P < 0.05) E2 production in slow-grow follicles. Additionally, low-dose P increased (P < 0.05) the percentage of no-grow follicles, and high-dose P promoted oocyte degeneration. In contrast, E2, in a steroid-depleted milieu, improved (P < 0.05) follicle survival, growth, antrum formation and oocyte health. E2 had no effect on P4 or E2 production. Follicles exposed to E2 yielded mature oocytes capable of fertilization and early cleavage, at a rate similar to untreated control follicles. LIMITATIONS, REASONS FOR CAUTION This study is limited to in vitro effects of P and E2 during the interval from the secondary to small antral stage of macaque follicles. WIDER IMPLICATIONS OF THE FINDINGS This study provides novel information on the direct actions of P4 and E2 on primate pre-antral follicle development. Combined with our previous report on the actions of androgens, our findings suggest that androgens appear to be a survival factor but hinder antral follicle differentiation, E2 appears to be a survival and growth factor at the pre-antral and early antral stage, whereas P4 may not be essential during early folliculogenesis in primates. STUDY FUNDING/COMPETING INTERESTS NIH P50 HD071836 (NCTRI), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), ONPRC 8P51OD011092. There are no conflicts of interest.