Significance: All current reversible hormonal contraceptives rely on modulating gonadotropins or sex steroids by acting on the hypothalamic–pituitary–gonadal axis. Primordial follicle activation, the first step of folliculogenesis, is independent of gonadotropins or steroids. In this study we show that Müllerian inhibiting substance (MIS) can completely block primordial follicle activation, representing a unique mechanism of contraception that spares the pool of quiescent primordial follicles (ovarian reserve). Chemotherapy is thought to cause the over-recruitment of primordial follicles. Here we show that treatment with MIS during cycles of carboplatin, doxorubicin, or cyclophosphamide can significantly protect the ovarian reserve in mice. Thus, MIS may provide a paradigm of a reversible contraceptive that could mitigate damage to the ovarian reserve associated with gonadotoxic chemotherapeutics.

Significance: This study improves our understanding of the role of Müllerian inhibiting substance (MIS/AMH) in antagonizing primordial follicle activation. By applying scRNA-seq methods to neonatal mouse ovaries, we revealed transcriptional signatures associated with MIS treatment in ovarian cell types present during the first wave of folliculogenesis and follicle development. We showed that MIS inhibits pregranulosa cell differentiation and the proliferation of ovarian surface epithelium and stromal cells. MIS also uncoupled granulosa and germ cell maturation, leading to abnormal development of activated follicles. These findings identify markers and pathways related to primordial follicle quiescence that could be targeted in contraception, preservation of ovarian reserve during aging or chemotherapy, or synchronization of preantral follicle growth for IVF.

The Mullerian ducts are the anlagen of the female reproductive tract, which regress in the male fetus in response to MIS. This process is driven by subluminal mesenchymal cells expressing Misr2, which trigger the regression of the adjacent Mullerian ductal epithelium. In females, these Misr2+ cells are retained, yet their contribution to the development of the uterus remains unknown. Here, we report that subluminal Misr2+ cells persist postnatally in the uterus of rodents, but recede by week 37 of gestation in humans. Using single-cell RNA sequencing, we demonstrate that ectopic postnatal MIS administration inhibits these cells and prevents the formation of endometrial stroma in rodents, suggesting a progenitor function. Exposure to MIS during the first six days of life, by inhibiting specification of the stroma, dysregulates paracrine signals necessary for uterine development, eventually resulting in apoptosis of the Misr2+ cells, uterine hypoplasia, and complete infertility in the adult female.

Graft fibrosis is a common finding during protocol biopsy examinations after pediatric liver transplantation. We evaluated the clinical utility of liver stiffness measurements by acoustic radiation force impulse (ARFI) imaging, a novel ultrasound-based elastography method, for assessing graft fibrosis after pediatric living donor liver transplantation (LDLT). We performed 73 liver stiffness measurements by ARFI imaging in 65 pediatric LDLT recipients through the upper midline of the abdomen (midline value) and the right intercostal space (intercostal value) around the time of protocol biopsy examinations. Fifty-nine of these liver stiffness measurements could be compared with histopathological findings. Graft fibrosis was assessed according to the degrees of portal and pericellular fibrosis. Significant fibrosis, which was defined as F2 or worse portal fibrosis and/or moderate or worse pericellular fibrosis, was observed in 14 examinations, which had significantly higher midline (P = 0.005) and intercostal values (P < 0.001) than the others. Liver stiffness measurements by ARFI imaging significantly increased with increases in the portal and pericellular fibrosis grades. For the diagnosis of significant fibrosis, the areas under the receiver operating characteristic curve (AUROCs) were 0.760 (P = 0.005) and 0.849 (P < 0.001) for the midline and intercostal values, respectively. The optimal cutoff values were 1.30 and 1.39 m/second for midline and intercostal values, respectively. Slight but significant elevations were noted in the results of biochemical liver tests: serum levels of γ-glutamyltransferase showed the highest AUROC (0.809, P = 0.001) with an optimal cutoff value of 20 IU/L. In conclusion, liver stiffness measurements by ARFI imaging had good accuracy for diagnosing graft fibrosis after pediatric LDLT. The pericellular pattern of fibrosis was frequently observed after pediatric LDLT, and moderate pericellular fibrosis was detectable by ARFI imaging. Liver Transpl 19:1202–1213, 2013. © 2013 AASLD.

Eighty percent of the estimated 600 million domestic cats in the world are free-roaming. These cats typically experience suboptimal welfare and inflict high levels of predation on wildlife. Additionally, euthanasia of healthy animals in overpopulated shelters raises ethical considerations. While surgical sterilization is the mainstay of pet population control, there is a need for efficient, safe, and cost-effective permanent contraception alternatives. Herein, we report evidence that a single intramuscular treatment with an adeno-associated viral vector delivering an anti-Müllerian hormone transgene produces long-term contraception in the domestic cat. Treated females are followed for over two years, during which transgene expression, anti-transgene antibodies, and reproductive hormones are monitored. Mating behavior and reproductive success are measured during two mating studies. Here we show that ectopic expression of anti-Müllerian hormone does not impair sex steroids nor estrous cycling, but prevents breeding-induced ovulation, resulting in safe and durable contraception in the female domestic cat.

Müllerian-inhibiting substance (MIS), also known as anti-Müllerian hormone, is thought to be a negative regulator of primordial follicle activation. We have previously reported that treatment with exogenous MIS can induce complete ovarian suppression within 5 weeks of treatment in mice. To investigate the kinetics of the return of folliculogenesis following the reversal of suppression, we treated animals with recombinant human MIS (rhMIS) protein for 40 days in adult female Nu/Nu mice and monitored the recovery of each follicle type over time. Following cessation of MIS therapy, secondary, and antral follicles returned within 30 days, along with the normalization of reproductive hormones, including LH, FSH, MIS, and Inhibin B. Furthermore, 30 days following MIS pretreatment, the number of antral follicles were significantly higher than controls, and superovulation with timed pregnant mare serum gonadotropin and human chorionic gonadotropin stimulation at this time point resulted in an approximately threefold increased yield of eggs. Use of the combined rhMIS-gonadotropin superovulation regimen in a diminished ovarian reserve (DOR) mouse model, created by 4-vinylcyclohexene dioxide treatment, also resulted in a twofold improvement in the yield of eggs. In conclusion, treatment with rhMIS can induce a reversible ovarian suppression, following which a rapid and synchronized large initial wave of growing follicles can be harnessed to enhance the response to superovulation. Therapies modulating MIS signaling may therefore augment the response to current ovarian stimulation protocols and could be particularly useful to women with DOR or poor responders to controlled ovarian hyperstimulation during in vitro fertilization.