Anti-Müllerian hormone induces autophagy to preserve the primordial follicle pool in mice.

The reserve pool of primordial follicles (PMFs) is finely regulated by molecules implicated in follicular growth or PMF survival. Anti-Müllerian hormone (AMH), produced by granulosa cells of growing follicles, is known for its inhibitory role in the initiation of PMF growth. We observed in a recent in vivo study that injection of AMH into mice seemed to induce an activation of autophagy. Furthermore, injection of AMH into mice activates the transcription factor FOXO3A which is also known for its implication in autophagy regulation. Many studies highlighted the key role of autophagy in the ovary at different stages of folliculogenesis, particularly in PMF survival. Through an in vitro approach with organotypic cultures of prepubertal mouse ovaries, treated or not with AMH, we aimed to understand the link among AMH, autophagy, and FOXO3A transcription factor. Autophagy and FOXO3A phosphorylation were analyzed by western blot. The expression of genes involved in autophagy was quantified by RT-qPCR. In our in vitro model, we confirmed the decrease in FOXO3A phosphorylation and the induction of autophagy in ovaries incubated with AMH. AMH also induces the expression of genes involved in autophagy. Interestingly, most of these genes are known to be FOXO3A target genes. In conclusion, we have identified a new role for AMH, namely the induction of autophagy, probably through FOXO3A activation. Thus, AMH protects the ovarian reserve not only by inhibiting the growth of PMFs but also by enabling their survival through activation of autophagy.

Anti-Müllerian Hormone promotes human osteoblast differentiation and calcification by modulating osteogenic gene expression.

OBJECTIVE: To investigate whether the Anti-Müllerian Hormone (AMH), an ovarian hormone belonging to the Transforming Growth Factor ß superfamily, may represent a possible candidate for use as a bone anabolic factor. METHODS: We performed in vitro studies on Human Osteoblasts (HOb) to evaluate the expression and the functionality of AMHRII, the AMH receptor type-2, and investigate the effects of exogenous AMH exposure on osteogenic gene expression and osteoblast functions. RESULTS: We reported the first evidence for the expression and functionality of AMHRII in HOb cells, thus suggesting that osteoblasts may represent a specific target for exogenous AMH treatment. Furthermore, the exposure to AMH exerted a stimulatory effect on HOb cells leading to the activation of osteogenic genes, including the upregulation of osteoblastic transcription factors such as RUNX and OSX, along with increased deposition of mineralized nodules. CONCLUSION: Our findings proved interesting clues on the stimulatory effects of AMH on mature osteoblasts expressing its specific receptor, AMHRII. This study may therefore have translation value in opening the perspective that AMH may be an effective candidate to counteract the bone loss in osteoporotic patients by selectively targeting osteoblast with minimal off-target effect.

The effects of intraovarian injection of autologous menstrual blood-derived mesenchymal stromal cells on pregnancy outcomes in women with poor ovarian response.

BACKGROUND: Assisted reproduction faces a significant obstacle in the form of poor ovarian response (POR) to controlled ovarian stimulation. To address this challenge, mesenchymal stem cell therapy has been proposed as a potential treatment for female infertility and/or restoration of ovarian function in POR women. Our previous research has demonstrated that menstrual blood-derived-mesenchymal stromal cells (MenSCs) injected into the ovaries of women with POR can increase pregnancy rates. The objective of this study was to examine whether MenSC therapy could enhance ovarian reserve parameters and pregnancy outcomes in a larger population of individuals with POR. METHOD: This study consisted of 180 infertile individuals with POR who declined oocyte donation. Participants were divided into two groups: those who received bilateral MenSCs intraovarian injection and those who received no intervention. Our primary aim was to compare the rates of spontaneous pregnancy between the two groups, followed by an investigation of any alterations in the ovarian reserve parameters, such as serum FSH, AMH, and AFC levels, as well as the ICSI/IVF outcomes, in both groups of participants. RESULTS: The MenSC therapy exhibited a favourable tolerability profile and did not raise any safety concerns. Following the 2-month follow-up period, women who received MenSC treatment demonstrated a significantly higher rate of spontaneous pregnancy (P < 0.005) and an improvement in anti-Müllerian hormone (AMH) levels (P = 0.0007) and antral follicle count (AFC) (P < 0.001), whereas the control group demonstrated a considerable decline in these parameters (Both P < 0.001). The MenSC therapy led to a greater number of mature oocytes and embryos among women who underwent ICSI/IVF. Our age subgroup analysis demonstrated a significant difference in the number of spontaneous pregnancies and ICSI/IVF outcomes between the treatment and control groups only among individuals below 40 years of age. CONCLUSION: The results of our study indicate that MenSCs treatment may be a viable option for treating women experiencing POR. However, in order to be widely implemented in clinical practice, the clinical effectiveness of MenSCs therapy will need to be established through rigorous prospective randomized clinical trials. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05703308. Registered 01/26/2023, retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT05703308 . IRCT, IRCT20180619040147N4. Registered 08/01/2020.

Long-term effect of chemotherapy after ovarian decortication on the ovarian function in women surviving cancer.

PURPOSE: Ovarian decortication may affect ovarian function. We investigated the status of ovarian reserve after ovarian decortication plus chemotherapy at a stage of presumed stabilized recovery in women surviving cancer. METHODS: We searched our database for cancer survivors subjected to ovarian decortication and chemotherapy at least 3 years previously. Ovarian function was explored for levels of anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), and estradiol (E2), and menstrual pattern. RESULTS: Forty women (mean age 29.6 (SD, 6.1) years) were assessed at a mean of 4.7 (1.5) years after surgery. The predecortication levels of AMH and FSH changed at post-treatment from 2.2 (1.4) to 0.5 (1.3) ng/mL for AMH (p < 0.001) and from 4.7 (2.1) to 16.7 (21. 6) IU/L for FSH (p < 0.001). Amenorrhea consistent with primary ovarian insufficiency (POI) was diagnosed in 11 women, and normal ovarian reserve (AMH ≥ 1.0 ng/mL) was found in 4 of the 21 women who recovered regular cycles. Logistic regression confirmed AMH as an independent predictor of diminished ovarian reserve (OR = 0.24, 95% CI: 0.04-0.63, p = 0.025) and POI (OR = 0.11, 95% CI: 0.01-0.52, p = 0.027), and age was predictive of POI (OR = 1.36, 95% CI: 1.08-1.96, p = 0.035) and of irregular menstrual cycle (OR = 1.20, 95% CI: 1.03-1.46, p = 0.034). CONCLUSION: Ovarian decortication plus chemotherapy had a deleterious effect when assessed at a stage of stabilized ovarian recovery, but whether ovarian decortication had a specific impact cannot be revealed from our data.

Melamine exposure during the weaning period negatively affects ovarian reserve.

In this study, it was aimed to investigate the effects of melamine exposure since the weaning period on ovarian tissue and ovarian reserve. Melamine is illegally added to milk and formula to provide high false protein positivity. Female rats (the weaning period = 21 days old, n = 18) were divided into 3 groups. 0.1 mL saline was applied to the control group by gavage for 21 days. Fifty mg/kg and seventy-five mg/kg melamine was dissolved in 0.1 mL of saline and applied by gavage for 21 days, respectively. At the end of the experiment, plasma anti-Mullerian hormone (AMH) was measured, follicle count and ovarian diameter measurement were performed in the right ovaries, and flow cytometric analysis for apoptosis was performed in the left ovaries. While a statistically significant decrease was not observed in the number of the follicle and ovarian diameter between the control and melamine-treated groups (p > 0.05), a significant decrease in the corpus luteum and a significant increase in the number of atretic follicles were observed (p < 0.05). Apoptosis (Annexin V) increased in both melamine groups and AMH plasma level decreased significantly in the 75 mg/kg group (p < 0.05). Melamine exposure from the weaning (early postnatal) period may cause a decrease in ovarian reserve in parallel with a dose increase.

The role of oxidative stress in chemotherapy-induced gonadotoxicity in a rat model, and the protective effects of Nigella Sativa oil on oxidative stress, the anti-Müllerian hormone level, and apoptosis.

OBJECTIVE: This study aimed to determine the role of oxidative stress (OS) in carboplatin-induced gonadotoxicity and whether Nigella Sativa oil (NSO), an herbal antioxidant, has a protective effect on ovarian apoptosis, OS, and the anti-Müllerian hormone (AMH) level in a rat model. MATERIALS AND METHODS: The study included 24 adult female rats that were divided into 4 treatment groups. Group A saline + saline (sham group); group B: NSO + saline; group C: saline + carboplatin; group D: NSO + carboplatin. Saline, NSO, and carboplatin were administered intraperitoneally 24 and/or 48 h before sacrification as 4 mL/kg, 4 mL/kg, and 80 mg/kg, respectively. Apoptosis, OS parameters, and AMH were measured. RESULTS: Oxidant levels and apoptosis were higher, whereas AMH and the antioxidants were lower in group C than in group A. Apoptosis, OS parameters, and AMH levels were negatively affected by chemotherapy (CTx) in group C whilst improvement in those parameters was observed in group D following NSO pretreatment. The levels of apoptosis and malondialdehyde (MDA), an OS parameter, in group D were lower than in group C as they declined from 34.3% to 8.65% (p = 0.002) and from 199.4 nmol/g tissue to 136.4 nmol/g tissue (p = 0.002), respectively. However, the slight increase in AMH level from 2.7 ng/mL to 3.5 ng/mL due to the NSO effect was not significant between groups C and D. CONCLUSIONS: The present findings show that carboplatin has adverse effects on AMH, ovarian tissue apoptosis, and OS parameters. NSO pretreatment might protect ovarian tissue and decrease CTx-induced ovarian injury by decreasing OS and apoptosis, but the protective effect of NSO on AMH is limited.

[Puberty induction in boys with congenital isolated hypogonadotropic hypogonadism].

BACKGROUND: Gonadotropin therapy in boys with congenital isolated hypogonadotropic hypogonadism helps to increase testes volume and induce spermatogenesis in comparison with testosterone therapy. However, difficulties with dose titration, partial therapy success, absence of generally accepted regimen protocols don't allow to use this therapy in order to induce puberty in adolescents with Kallmann syndrome or normosmic hypogonadotropic hypogonadism. AIM: To assess the effectiveness of combination hormonal replacement therapy via human chorionic gonadotropin and recombinant follicle stimulation hormone in adolescents with congenital isolated normosmic hypogonadotropic hypogonadism and with Kallmann syndromeMATERIALS AND METHODS: This is an open single-center prospective non-controlled study. Boys with hypogonadotropic hypogonadism were receiving hormonal replacement therapy for 12 months. Initial dose of human chorionic gonadotropin was 500 IU per week. Initial dose of recombinant follicle stimulation hormone was 37.5 IU per week. Doses were doubled in 6 months. Antropometric data, Tanner stage, testes volumes, inhibin B and anti-Mullerian hormone (AMH) levels were evaluated in all the patients before the treatment, after 6 and 12 months of the therapy. RESULTS: 8 boys with hypogonadotropic hypogonadism were included into the study. Median age before therapy initiation was 15.7 years [15.33; 16.41]. In 12 months after the therapy initiation puberty development, testosterone increase from 0.44 [0.34;0.62] to 4.39 [0.88;10.51] nmol/l (p=0.012), AMH decrease from 35.70 [18.00;59.00] to 14.41 [11.60;16.65] ng/ml were noted in all the patients (p=0.017). Testes volumes increase and inhibin B level increase were not statistically significant. CONCLUSION: Gonadotropin therapy is effective in order to puberty initiation in adolescents with congenital hypogonadotropic hypogonadism. In helps to achieve not only androgenization, but also to Sertoli cells maturation.

Protective effects of melatonin on female rat ovary treated with nonylphenol.

We investigated using histochemistry and immunohistochemistry ovarian damage caused by nonylphenol (NP) and the protective effect of melatonin treatment of NP induced ovarian damage. We used 21 female rats divided randomly into three groups: control, NP and melatonin + NP. Histopathological examination of the ovaries, and counting and classification of follicles were performed using Masson's trichrome staining. Expression of anti-Mullerian hormone (AMH), Bax, Bcl-2 and caspase-3 was detected in the ovaries using immunohistochemistry. Melatonin had an ameliorative effect on NP induced follicular atresia and absence of corpora lutea. More follicles were observed in the ovaries of animals treated with melatonin prior to treatment with NP. AMH immunoreactivity was significantly lower in the NP group than in the melatonin + NP group. NP increased immunostaining for Bax, Bcl-2 and caspase-3. Melatonin significantly reduced the increased expression of Bax, Bcl-2 and caspase-3 due to NP exposure. We found that pretreatment with melatonin is beneficial for protecting the ovaries from damage by NP.

OVARIAN TOXICITY OF FAC CHEMOTHERAPY IN RATS AND POSSIBILITY OF ITS CORRECTION WITH PLATELET-RICH PLASMA.

AIM: To compare the dynamics of changes in the hormonal status of female rats in the setting of the FAC (5-fluorouracil, doxorubicin and cyclophosphamide) chemotherapy and after local administration of platelet-rich plasma (PRP). MATERIALS AND METHODS: The study was carried out on female Wistar rats treated according to the FAC chemotherapy scheme (4 courses with a 3-week interval). The ovariotoxic effect of the FAC chemotherapy was assessed by the levels of anti-Mullerian hormone, estradiol (E2) and follicle-stimulating hormone in the proestrus phase. Three weeks after the last course of chemotherapy, 5 rats were administered with local intra- and periovarian injection of PRP (triply with a 1-week interval). RESULTS: The dynamics of all investigated hormonal markers of the ovarian reserve in experimental animals was characterized by a progressive decrease in anti-Mullerian hormone and E2 levels and an increase in follicle-stimulating hormone level. The dynamics of the studied parameters after the serial administration of PRP demonstrated an improvement in the hormonal status. CONCLUSION: FAC chemotherapy in the experiment causes premature ovarian failure, and local administration of PRP improves the hormonal parameters of the ovarian reserve.

Anti-Müllerian hormone-mediated preantral follicle atresia is a key determinant of antral follicle count in mice.

STUDY QUESTION: Does anti-Müllerian hormone (AMH) induce preantral follicle atresia in mice? SUMMARY ANSWER: The present findings suggest that AMH-mediated follicle atresia only occurs in early follicles before they become sensitive to FSH. WHAT IS KNOWN ALREADY: Most prior studies have investigated the ability of AMH to inhibit primordial follicle activation. Our previous study showed that AMH-overexpressing mice had fewer preantral follicles than expected after accounting for primordial follicle inhibition but the reason for this was not determined. STUDY DESIGN, SIZE, DURATION: Cross-sectional-control versus transgenic/knockout mouse studies were carried out. PARTICIPANTS/MATERIALS, SETTING, METHODS: Studies were conducted on female wild-type (Amh+/+), AMH-knockout (Amh-/-) and AMH overexpressing (Thy1.2-AMHTg/0) mice on a C57Bl/6J background (age: 42-120 days). The follicle counts were conducted for primordial, transitioning, primary, secondary and antral follicles in Amh-/- and Amh+/+ mice. After confirming that follicle development speeds were identical (proliferating cell nuclear antigen immunohistochemistry), the ratio of follicles surviving beyond each stage of folliculogenesis was determined in both genotypes. Evidence for increased rates of preantral follicle atresia was assessed by active caspase-3 immunohistochemistry in wild-type and Thy1.2-AMHTg/0 mice. MAIN RESULTS AND THE ROLE OF CHANCE: Amh -/- mice at 100-120 days of age had lower primordial follicle counts but higher primordial follicle activation rates compared to Amh+/+ mice. These counteracting effects led to equivalent numbers of primordial follicles transitioning to the primary stage in Amh+/+ and Amh-/- mice. Despite this, Amh+/+ mice had fewer primary, secondary, small antral and medium antral follicles than Amh-/- mice indicating differing rates of developing follicle atresia between genotypes. Cleaved caspase-3 immunohistochemistry in Thy1.2-AMHTg/0 ovaries revealed high rates of granulosa cell and oocyte apoptosis in late primary/early secondary follicles of Thy1.2-AMHTg/0 mice. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The findings were shown only in one species and additional research will be required to determine generalizability to other species. WIDER IMPLICATIONS OF THE FINDINGS: This study is consistent with prior studies showing that Amh-/- mice have increased primordial follicle activation but these new findings demonstrate that AMH-mediated preantral follicle atresia is a predominant cause of the increased small antral follicle counts in Amh-/- mice. This suggests that the role of AMH is not to conserve the ovarian reserve to prolong fertility, but instead to prevent the antral follicle pool from becoming too large. While this study may demonstrate a new function for AMH, the biological purpose of this function requires further investigation, particularly in mono-ovulatory species. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Health Research Council of New Zealand and the University of Otago. No competing interests to declare.

Systemic isotretinoin for acne treatment: Ovarian reserve is safe with the low dose.

Isotretinoin is among the most frequently used medications in the dermatologic daily practice. With a Black box warning, teratogenicity is a major concern. Female fertility may be an issue to be investigated when it comes to its use in females. The aim of this work was to assess the effect of low dose isotretinoin on the ovarian reserve in female patients with moderate to severe acne. Sixty-sex female acne patients candidate for isotretinoin therapy and 66 controls were enrolled in this prospective controlled cohort study. Low dose isotretinoin (0.25-0.4 mg/kg/day) was given to the patients group for 6 months. Serum anti-Mullarian hormone (AMH), ovarian volume (OV) and Antral follicle count (AFC) were evaluated at baseline and 6 months after the last dose in the patients' group, and 1 year after the baseline assessment for the control subjects. There was no significant difference in serum AMH between patients after isotretinoin treatment and control subjects (p = 0.898). AMH failed to show any significant change in pre- and post- treatment levels in patients' group (p = 0.747). Both OV and AFC showed no significant changes in patient group when comparing pre- post- treatment levels on both sides (p > 0.05) and in control group between baseline and 1-year-interval levels on both sides (p > 0.05). Low-dose isotretinoin in treatment of moderate to severe acne seems to be safer on ovarian reserve as indicated by non-significant change in AMH levels as a sensitive parameter of female fertility.

Somatic cell fate maintenance in mouse fetal testes via autocrine/paracrine action of AMH and activin B.

Fate determination and maintenance of fetal testes in most mammals occur cell autonomously as a result of the action of key transcription factors in Sertoli cells. However, the cases of freemartin, where an XX twin develops testis structures under the influence of an XY twin, imply that hormonal factor(s) from the XY embryo contribute to sex reversal of the XX twin. Here we show that in mouse XY embryos, Sertoli cell-derived anti-Mullerian hormone (AMH) and activin B together maintain Sertoli cell identity. Sertoli cells in the gonadal poles of XY embryos lacking both AMH and activin B transdifferentiate into their female counterpart granulosa cells, leading to ovotestis formation. The ovotestes remain to adulthood and produce both sperm and oocytes, although there are few of the former and the latter fail to mature. Finally, the ability of XY mice to masculinize ovaries is lost in the absence of these two factors. These results provide insight into fate maintenance of fetal testes through the action of putative freemartin factors.

Female sexual behavior is disrupted in a preclinical mouse model of PCOS via an attenuated hypothalamic nitric oxide pathway.

Women with polycystic ovary syndrome (PCOS) frequently experience decreased sexual arousal, desire, and sexual satisfaction. While the hypothalamus is known to regulate sexual behavior, the specific neuronal pathways affected in patients with PCOS are not known. To dissect the underlying neural circuitry, we capitalized on a robust preclinical animal model that reliably recapitulates all cardinal PCOS features. We discovered that female mice prenatally treated with anti-Müllerian hormone (PAMH) display impaired sexual behavior and sexual partner preference over the reproductive age. Blunted female sexual behavior was associated with increased sexual rejection and independent of sex steroid hormone status. Structurally, sexual dysfunction was associated with a substantial loss of neuronal nitric oxide synthase (nNOS)-expressing neurons in the ventromedial nucleus of the hypothalamus (VMH) and other areas of hypothalamic nuclei involved in social behaviors. Using in vivo chemogenetic manipulation, we show that nNOSVMH neurons are required for the display of normal sexual behavior in female mice and that pharmacological replenishment of nitric oxide restores normal sexual performance in PAMH mice. Our data provide a framework to investigate facets of hypothalamic nNOS neuron biology with implications for sexual disturbances in PCOS.

Targeting signaling pathways involved in primordial follicle growth or dormancy: potential application in prevention of follicular loss and infertility.

INTRODUCTION: Premature ovarian failure (POF) is one of the important causes of infertility in females. To date, no efficient preventive pharmacological treatment has been offered to prevent POF. Therefore, it is necessary to focus on strategies that provide a normal reproductive lifespan to females at risk of developing POF. AREAS COVERED: Recently, attention has been drawn to discovering pathways involved in primordial follicle activation, as the inhibition of this process might maintain the stock of primordial follicles and therefore, prevent POF. In vitro and animal studies have resulted in the discovery of several of these pathways that can be used to develop new treatments for POF. These studies show crosstalk of these pathways at different levels. One of the important crossing points of many of these pathways involves anti-Mullerian hormone (AMH). Herein, we discuss different aspects of this topic by reviewing related published articles indexed in PubMed and Web of Science as of December 2021. EXPERT OPINION: Although the findings seem promising, most of the studies were conducted on animals, and the interaction between these factors and the possible outcomes of their administration in the long term are still unknown. Therefore, further investigation is necessary to assess these aspects.

A Docosahexaenoic Acid Derivative (N-Benzyl Docosahexaenamide) as a Potential Therapeutic Candidate for Treatment of Ovarian Injury in the Mouse Model.

Commonly used clinical chemotherapy drugs, such as cyclophosphamide (CTX), may cause injury to the ovaries. Hormone therapies can reduce the ovarian injury risk; however, they do not achieve the desired effect and have obvious side effects. Therefore, it is necessary to find a potential therapeutic candidate for ovarian injury after chemotherapy. N-Benzyl docosahexaenamide (NB-DHA) is a docosahexaenoic acid derivative. It was recently identified as the specific macamide with a high degree of unsaturation in maca (Lepidium meyenii). In this study, the purified NB-DHA was administered intragastrically to the mice with CTX-induced ovarian injury at three dose levels. Blood and tissue samples were collected to assess the regulation of NB-DHA on ovarian function. The results indicated that NB-DHA was effective in improving the disorder of estrous cycle, and the CTX+NB-H group can be recovered to normal levels. NB-DHA also significantly increased the number of primordial follicles, especially in the CTX+NB-M and CTX+NB-H groups. Follicle-stimulating hormone and luteinizing hormone levels in all treatment groups and estradiol levels in the CTX+NB-H group returned to normal. mRNA expression of ovarian development-related genes was positive regulated. The proportion of granulosa cell apoptosis decreased significantly, especially in the CTX+NB-H group. The expression of anti-Müllerian hormone and follicle-stimulating hormone receptor significantly increased in ovarian tissues after NB-DHA treatment. NB-DHA may be a promising agent for treating ovarian injury.

Tanshinone IIA enhances the ovarian reserve and attenuates ovarian oxidative stress in aged mice.

BACKGROUND: Tanshinone IIA (TSA), a major lipophilic component extracted from the roots of Salvia miltiorrhiza Bunge, has been widely used in China for its various biological activities. However, its effect on ovarian reserve in aged mice was not studied elsewhere. OBJECTIVES: This study aimed to explore the effect of TSA on the ovarian reserve of aged mice as well as young mice. Forty weeks old mice (N = 40) were considered as aged group compared to 4 weeks old mice (N = 40), and these groups were subdivided into four subgroups (N = 10) to receive different doses of TSA (0, 10, 20, and 40 µg/g/day). METHODS: The effect of TSA was evaluated by counting follicular number by histological examination. Basal serum levels of FSH, LH, E2, and anti-Mullerian hormone (AMH) were measured by ELISA. Moreover, the expression levels of antioxidant genes (CAT, Nrf2, GPX1), gap junction (Cx37), ERK1/2, and Smad5 family gene were examined at both mRNA (qPCR) and protein levels (western blot). RESULTS: Follicular number, level of AMH and E2, and the expression of CAT, Nrf2, and GPX1 genes increased significantly (p < 0.05) in aged mice administrated with medium (20 µg/g/day) and high (40 µg/g/day) doses of TSA, whereas FSH and LH levels were significantly low compared to low dose (10 µg/g/day) and control (0 µg/g/day) aged subgroups. However, we did not observe any effect of all doses of TSA on young mice. CONCLUSIONS: Administration of TSA with medium and high doses up-regulates the expression of antioxidative genes, reduces the oxidative injury, increases levels of AMH, and E2 levels that are relatively comparable to those in young mice, and consequently results in a healthy oocyte development.

Recombinant anti-Müllerian hormone in the maturation medium improves the in vitro maturation of human immature (GV) oocytes after controlled ovarian hormonal stimulation.

BACKGROUND: In vitro maturation (IVM) of oocytes is a laboratory method that allows the maturation of immature (GV) oocytes retrieved from patients enrolled in the in vitro fertilization (IVF) programme. However, this method is still sparsely researched and used in clinical practice, leading to suboptimal clinical results. Anti-Müllerian hormone (AMH) is an important hormone with known effects on human ovaries, especially on follicles (follicular cells) during folliculogenesis. In contrast, the effect of AMH on the human oocyte itself is unknown. Therefore, we wanted to determine whether human oocytes express AMH receptor 2 (AMHR2) for this hormone. Recombinant AMH was added to the IVM medium to determine whether it affected oocyte maturation. METHODS: In total, 247 human oocytes (171 immature and 76 mature) were collected from patients enrolled in the intracytoplasmic sperm injection (ICSI) programme who were aged 20 to 43 years and underwent a short antagonist protocol of ovarian stimulation. The expression of AMHR2 protein and AMHR2 gene was analysed in immature and mature oocytes. Additionally, maturation of GV oocytes was performed in vitro in different maturation media with or without added AMH to evaluate the effect of AMH on the oocyte maturation rate. RESULTS: Immunocytochemistry and confocal microscopy revealed that AMHR2 protein is expressed in both immature and mature human oocytes. AMHR2 was expressed in a spotted pattern throughout the whole oocyte. The IVM procedure revealed that AMH in maturation medium improved GV oocyte maturation in vitro, as all oocytes were successfully matured in maturation medium containing recombinant AMH only. Furthermore, antagonism between AMH and follicle-stimulating hormone (FSH) during the maturation process was observed, with fewer oocytes maturing when both AMH and FSH were added to the maturation medium. Finally, AMHR2 gene expression was found in immature and in vitro matured oocytes but absent in mature oocytes. CONCLUSIONS: The positive AMHR2 protein and AMHR2 gene expression in human oocytes shows that AMH could directly act on human oocytes. This was further functionally confirmed by the IVM procedure. These findings suggest the potential clinical application of recombinant AMH to improve IVM of human oocytes in the future.

A novel method for purification of biologically active C-terminal fragment of human recombinant anti-mullerian hormone.

Anti-mullerian hormone (AMH) is one of the least studied members of transforming growth factor beta superfamily showing pro-apoptotic activity against cells positive for hormone type II receptor overexpressed by malignant cells in many cancer cases. Here, we propose an improved method for isolation of recombinant C-terminal AMH fragment (C-rAMH) to obtain homogeneous preparations of this protein with high biological activity. In contrast to our previously developed C-rAMH purification technology based on reversed-phase HPLC, the key stage of the new approach is hydrophobic interaction chromatography using Toyopearl Butyl-650S resin performed under more benign conditions. This modification of the previously developed method allowed highly purified C-rAMH to be obtained that is characterized by twice the specificity estimated as the ability to bind to the recombinant analog of AMH type II receptor and by significantly higher biological activity, that is, the ability to induce the death of target cells. Thus, we made the purification technology even more cost-effective and suitable for the production of drug forms based on C-rAMH.

Effects of Bushen Yiqi Huoxue Decoction in Treatment of Patients with Diminished Ovarian Reserve: A Randomized Controlled Trial.

OBJECTIVE: To explore the therapeutic effect of Bushen Yiqi Huoxue Decoction BYHD) in patients with diminished ovarian reserve (DOR). METHODS: A total of 180 patients with DOR diagnosed from December 2013 to December 2014 were equally assigned into progynova and duphaston (E+D) group, Zuogui Pill group and BYHD group with 60 cases in each by computerized randomization. Patients received E+D, Zuogui Pill or BYHD for 12 months, respectively. Follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), anti-Müllerian hormone (AMH), antral follicle count (AFC), ovarian volume, endometrial thickness, and the resistance indices (RIs) of ovarian arteries and uterine arteries were observed before and after treatment. RESULTS: Nine women (4 from the E+D group, 3 from the Zuogui Pill group, and 2 from the BYHD group) withdrew from the study. After 6 months, Zuogui Pill and BYHD significantly decreased FSH and LH and increased endometrial thickness and AMH (all P<0.01). BYHD also resulted in E2 elevation (P<0.05), ovary enlargement (P<0.05), AFC increase (P<0.01), and RI of ovarian arteries decrease (P<0.05). After 12 months, further improvements were observed in the Zuogui Pill and BYHD groups (all P<0.01), but BYHD showed better outcomes, with lower FSH, larger ovaries and a thicker endometrium compared with the Zuogui Pill group (all P<0.01). However, E+D only significantly increased endometrial thickness (P<0.01) and no significant improvements were observed in the RI of uterine arteries in the three groups. CONCLUSIONS: BYHD had a favorable therapeutic effect in patients with DOR by rebalancing hormone levels, promoting ovulation, and repairing the thin endometrium. The combination of tonifying Shen (Kidney), benefiting qi and activating blood circulation may be a promising therapeutic strategy for DOR.

Insights into in vivo follicle formation: a review of in vitro systems.

In vitro systems capable of reconstituting the process of mouse oogenesis are now being established to help develop further understanding of the mechanisms underlying oocyte/follicle development and differentiation. These systems could also help increase the production of useful livestock or genetically modified animals, and aid in identifying the causes of infertility in humans. Recently, we revealed, using an in vitro system for recapitulating oogenesis, that the activation of the estrogen signaling pathway induces abnormal follicle formation, that blocking estrogen-induced expression of anti-Müllerian hormone is crucial for normal follicle formation, and that the production of α-fetoprotein in fetal liver tissue is involved in normal in vivo follicle formation. In mouse fetuses, follicle formation is not carried out by factors within the ovaries but is instead orchestrated by distal endocrine factors. This review outlines findings from genetics, endocrinology, and in vitro studies regarding the factors that can affect the formation of primordial follicles in mammals.