Ovarian follicles are resistant to monocyte perturbations-implications for ovarian health with immune disruption†.

Monocytes and macrophages are the most abundant immune cell populations in the adult ovary, with well-known roles in ovulation and corpus luteum formation and regression. They are activated and proliferate in response to immune challenge and are suppressed by anti-inflammatory treatments. It is also likely they have a functional role in the healthy ovary in supporting the maturing follicle from the primordial through to the later stages; however, this role has been unexplored until now. Here, we utilized a Cx3cr1-Dtr transgenic Wistar rat model that allows a conditional depletion of circulating monocytes, to investigate their role in ovarian follicle health. Our findings show that circulating monocyte depletion leads to a significant depletion of ovarian monocytes and monocyte-derived macrophages. Depletion of monocytes was associated with a transient reduction in circulating anti-Müllerian hormone (AMH) at 5 days postdepletion. However, the 50-60% ovarian monocyte/macrophage depletion had no effect on ovarian follicle numbers, follicle atresia, or apoptosis, within 5-21 days postdepletion. These data reveal that the healthy adult ovary is remarkably resistant to perturbations of circulating and ovarian monocytes despite acute changes in AMH. These data suggest that short-term anti-inflammatory therapies that transiently impact on circulating monocytes are unlikely to disrupt ovarian follicle health, findings that have significant implications for fertility planning relative to the experience of an immune challenge or immunosuppression.

[Monoclonal Antibody Against N Terminal 439-451 Epitopes of Anti-Mullerian Hormone and Its Properties].

OBJECTIVE: To prepare the specific monoclonal antibody against the N-terminal specific epitope peptide of anti-mullerian hormone (AMH) and to identify its specificity. METHODS: Using bioinformatics analysis software to predict the specific peptide fragment of AMH. Then synthesized four antigenic epitope peptide segments of mature N-terminal region of AMH as the screening target antigen. Synthesized AMH wholegene.Using the prokaryotic expression system to abtain recombinant AMH protein. Immunized BALB/c mice with the recombinant AMH, and prepared mouse spleen cells for fusing with SP/20 cells. Preparation of AMH monoclonal antibody by hybridoma technology. The monoclonal antibodies against AMH were screened by using four N-terminal epitope peptides (1: 439-451 RGRDPRGPGRAQ, 2: 273-285 PPRPSAELEESPP, 3: 42-54 DLDWPPGSPQEPL, 4: 494-506 WPQSDRNPRYGNH) as antigens, and indirect ELISA and Western blot were used to identify the antigen binding characteristics of the selected monoclonal antibodies. RESULTS: Two hybridoma cell lines with stable anti-AMH-1 and anti-AMH-2 antibody activities were screened. The two antibodies were named anti-AMH-1 and anti-AMH-2 respectively. The antibody titers were 1∶12 000 and 1∶1 600 after purification. Western blot confirmed that the two McAbs recognized different antigens. Anti-AMH-1 could not only recognize the N-terminal 439-451 epitope peptide of AMH, but also recognize the amino acid sequence of recombinant AMH, as well as the ovarian tissue. Anti-AMH-2 could recognize recombinant AMH and ovarian tissue. CONCLUSION: Two monoclonal antibodies against N-terminal specific epitopes of human AMH were successfully constructed.

[Preparation and application of anti-Mullerian hormone immunomagnetic beads].

Objective To prepare human anti-Mullerian hormone (AMH) immunomagnetic beads and HRP-labeled antibodies and establish a rapid double-antibody sandwich ELISA based on nanometer magnetic beads. Methods The expression vector of human AMH protein was constructed, and the recombinant AMH protein was expressed and purified. BALB/c mice were immunized with the recombinant protein to prepare the polyclonal antibody. Spleen cells were fused with myeloma Sp2/0 cells by PEG. Hybridoma cell lines which could stably secret monoclonal antibodies against AMH were screened out by ELISA. Monoclonal antibodies were produced from the ascites fluid of mice injected intraperitoneally with hybridoma cells and evaluated by Western blotting. Polyclonal antibodies purified from protein A were coupled to nano-magnetic' beads and used as capture antibodies, while HRP-labeled monoclonal antibody was prepared by sodium periodate method and used as probe antibody. A double antibody sandwich ELISA based on the nano-magnetic beads was established and optimized. Results A monoclonal antibody with good specificity for AMH was obtained,' and its subtype was IgG2b. The titers of purified polyclonal antibodies and monoclonal antibodies were up to 1:51 200. The capture antibody coupled with magnetic beads and the probe antibody labeled with HRP kept their good activity. The established method could detect AMH antigen within 1 hour and the detection limit was 50 ng/mL. Conclusion The prepared AMH immunomagnetic beads can be used for the fast and visualized detection of recombinant AMH.

Active immunization against AMH reveals its inhibitory role in the development of pre-ovulatory follicles in Zhedong White geese.

The aim of this study was to investigate the effects of active immunization against recombinant Anti-Müllerian hormone (AMH) protein on the ovarian follicular development, egg production, and molecular regulatory mechanisms in broody-prone Zhedong White geese. For this, a recombinant goose AMH protein was expressed using a prokaryotic expression system. Fifty incubating geese from the same genetic background were selected and equally divided into two groups. The immunization group was actively immunized against the recombinant goose AMH protein, whereas the control group was immunized against bovine serum albumin (BSA). Immunization against AMH accelerated ovarian follicular development and increased clutch sizes by one to two eggs in two consecutive laying-incubation cycles. Furthermore, immunization against AMH upregulated the mRNA transcription levels of the FSH-beta gene in the pituitary gland, and FSHR, 3beta-HSD, and Smad4 genes in the granulosa layer of pre-ovulatory follicles; however, immunization downregulated the expression of the OCLN gene in the granulosa layer of pre-ovulatory follicles, and Smad5 and Smad9 genes in the granulosa layer of SYFs. These results suggest that AMH might hinder ovarian follicular development by decreasing both pituitary FSH secretion as well as ovarian follicular sensitivity to FSH. The latter molecular mechanism could be fulfilled by regulating Smad5 or Smad9 signals in SYFs, as well as the FSHR and Smad4 signals that affect progesterone synthesis and yolk deposition in the pre-ovulatory follicles.

Development of anti-Müllerian hormone immunoassay based on biolayer interferometry technology.

Anti-Müllerian hormone (AMH) is a biomarker for the assessment of female fertility. The accurate measurement of the concentration of AMH is relevant for the success of assisted reproductive therapies and diagnosis of clinical cases. In this study, we show that cytokines such as fetal liver tyrosine kinase 3 ligand (Flt3L), CC subtype chemokine ligand 20 (CCL20), granulocyte-macrophage colony-stimulating factor (GM-CSF), and ß2-microglobulin (ß2M) significantly enhance the immune response against AMH. Two anti-AMH monoclonal antibodies (mAbs) with high affinity were selected by biolayer interferometry (BLI) technology for application in a fully automated magnetic chemiluminescence immunoassay (CLIA). This robust and rapid assay can efficiently detect AMH in the range of 0.125~20 ng mL-1 with a detection limit of 0.099 ng mL-1. This immunoassay showed high specificity with no cross-reaction with structurally related proteins and some of the other members of the TGF-ß super family, such as inhibin A, activin A, follicle-stimulating hormone, and luteinizing hormone. The average recovery rates of three different batches were 100.19%, 102.72%, and 103.59%, respectively, with coefficients of variation of less than 12%. The developed assay was applied in the detection of AMH in 69 serum samples from randomly selected patients. Our data showed a high correlation with those obtained using commercially available ELISA kits (correlation coefficient, 0.9831). Hence, we suggest that this immunoassay could find application in the development of POCT for the diagnosis of AMH in clinical samples. Graphical abstract.

Anti-Müllerian hormone promotes pre-antral follicle growth, but inhibits antral follicle maturation and dominant follicle selection in primates.

STUDY QUESTION: What are the direct effects and physiological role of anti-Müllerian hormone (AMH) during primate follicular development and function at specific stages of folliculogenesis? SUMMARY ANSWER: AMH actions in the primate ovary may be stage-dependent, directly promoting pre-antral follicle growth while inhibiting antral follicle maturation and dominant follicle selection. WHAT IS KNOWN ALREADY: AMH is expressed in the adult ovary, particularly in developing follicles. Studies in mice suggest that AMH suppresses pre-antral follicle growth in vitro, and inhibits primordial follicle recruitment and FSH-stimulated antral follicle steroidogenesis. STUDY DESIGN, SIZE, DURATION: For in vitro study, secondary follicles were isolated from ovaries of 12 rhesus macaques and cultured for 5 weeks. For in vivo study, intraovarian infusion was conducted on five monkeys for the entire follicular phase during two spontaneous menstrual cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: For in vitro study, individual follicles were cultured in a 5% O2 environment, in alpha minimum essential medium supplemented with recombinant human FSH. Follicles were randomly assigned to treatments of recombinant human AMH protein or neutralizing anti-human AMH antibody (AMH-Ab). Follicle survival, growth, steroid production, steroidogenic enzyme expression, and oocyte maturation were assessed. For in vivo study, ovaries were infused with control vehicle or AMH-Ab during the follicular phase of the menstrual cycle. Cycle length, serum steroid levels, and antral follicle growth were evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: AMH exposure during culture weeks 0-3 (pre-antral stage) promoted, while AMH-Ab delayed, antrum formation of growing follicles compared with controls. AMH treatment during culture weeks 3-5 (antral stage) decreased (P < 0.05) estradiol (E2) production, as well as the mRNA expression of cytochrome P450 family 19 subfamily A polypeptide 1, by antral follicles relative to controls, whereas AMH-Ab increased (P < 0.05) follicular mRNA levels of the enzyme. Intraovarian infusion of AMH-Ab during the follicular phase of the menstrual cycle increased (P < 0.05) the average levels of serum E2 compared with those of the control cycles. Three of the five AMH-Ab-treated ovaries displayed multiple (n = 2-9) medium-to-large (2-8 mm) antral follicles at the mid-cycle E2 peak, whereas only one large (4-7 mm) antral follicle was observed in all monkeys during their control cycles. The average levels of serum progesterone were higher (P < 0.05) during the luteal phase of cycles following the AMH-Ab infusion relative to the vehicle infusion. LIMITATIONS, REASONS FOR CAUTION: The in vitro study of AMH actions on cultured individual macaque follicles was limited to the interval from the secondary to small antral stage. A sequential study design was used for in vivo experiments, which may limit the power of the study. WIDER IMPLICATIONS OF THE FINDINGS: The current study provides novel information on direct actions and role of AMH during primate follicular development, and selection of a dominant follicle by the late follicular phase of the menstrual cycle. We hypothesize that AMH acts positively on follicular growth during the pre-antral stage in primates, but negatively impacts antral follicle maturation, which is different from what is reported in the mouse model. STUDY FUNDING/COMPETING INTERESTS: NIH NICHD R01HD082208, NIH ORWH/NICHD K12HD043488 (BIRCWH), NIH OD P51OD011092 (ONPRC), Collins Medical Trust. There are no conflicts of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Immunofluorescent analysis of testicular biopsies with germ cell and Sertoli cell markers shows significant MVH negative germ cell depletion with older age at orchiopexy.

PURPOSE: Undescended testis is the most common defect in male newborns. This condition is associated with increased risks of infertility and testicular malignancy due to abnormal germ cell development in the testes. Early surgery may limit such risks. We analyzed germ cell development vs age at orchiopexy using a germ cell marker and a Sertoli cell marker on testicular biopsies. MATERIALS AND METHODS: A total of 22 testicular biopsies at orchiopexy in 20 patients 5 to 24.5 months old were fixed and embedded in paraffin. Sections were processed and labeled with AMH antibody for Sertoli cells and MVH antibody for germ cells for immunofluorescent histochemical analysis. Confocal images were counted using ImageJ (National Institutes of Health, Bethesda, Maryland) for germ cells and testicular tubules. The data were analyzed using linear regression. RESULTS: Sertoli cells were clearly distinguished from MVH positive and negative germ cells located centrally or on basement membranes of tubules. Percentage of tubules with MVH negative germ cells significantly decreased with increasing age at orchiopexy (ß = -0.03, p = 0.03). Total tubular numbers and "empty" tubules without germ cells significantly increased with age at orchiopexy (ß = 1.15, p = 0.02 and ß = 0.44, p = 0.04, respectively). CONCLUSIONS: AMH antibody distinguished Sertoli cells from germ cells, and MVH antibody distinguished 2 types of germ cells at different developmental stages. Biopsy at orchiopexy in older patients showed significant germ cell depletion. These results lend support to early surgery to optimize germ cell number.

The journey from the old to the new AMH assay: how to avoid getting lost in the values.

Anti-Müllerian hormone (AMH) is set to dominate reproductive endocrinology because of its unique relationship with the ovarian reserve. To date half of the published articles have used the Diagnostic Systems Lab (DSL) assay and the other half the Immunotech (IOT) assay. Unfortunately, these assays utilize two different primary antibodies against AMH and different standards, and consequently the crude values reported can differ substantially, with the IOT assay giving values for AMH that are higher than those obtained with the DSL assay. With the recent consolidation of these two companies by Beckman Coulter, and their sole ownership of the patent to measure mammalian AMH, there is finally a single commercially available assay - the AMH Gen II assay, which will fully replace the DSL and IOT assays. The aim of this article is to briefly focus on the different assays for AMH evaluation in order to give readers hopefully helpful suggestions for a correct interpretation of the AMH measurement. A brief overview on the development and performance characteristics of the new assay, how it relates to previous values and previously developed nomograms and where the future lies for AMH is also provided.

Development of a second generation anti-Müllerian hormone (AMH) ELISA.

AMH is a glycoprotein dimer composed of two 72kDa monomers linked by disulfide bridges. It belongs to the transforming growth factor-ß family. AMH performs various physiological functions. In males, AMH is secreted by the Sertoli cells of the testis. During embryonic development, AMH is responsible for Müllerian duct regression. AMH continues to be produced by the testicles until puberty and then decreases slowly to residual post-puberty values. In females, AMH is produced in small amounts by ovarian granulosa cells after birth, until menopause, and then becomes undetectable. A two-step, sandwich-type enzymatic microplate assay has been developed to measure AMH levels in 20 µL of sample in less than 3h. AMH calibrators range from 0.2 to 28 ng/mL. The antibodies used in the assay bind to the mature region of AMH, which is more stable to proteolysis compared to prohormone region. The AMH Gen II assay (Beckman Coulter, Inc., Webster, Texas) was standardized to the Immunotech (IOT, Beckman Coulter, Inc., Marseilles, France) AMH assay. AMH Gen II, when compared to IOT using 120 serum samples in the range of 0-20.4 ng/mL yielded a correlation coefficient of 0.98 and a slope of 1.0. Total imprecision, calculated on four samples over 40 runs, four replicates per run, between two lots using CLSI EP5-A guidelines, was 5.7% at 3.8 ng/mL, 7.7% at 4.4 ng/mL, 5.8% at 14 ng/mL and 5.3% at 16.4 ng/mL. The average analytical sensitivity calculated by the interpolation of the mean plus two standard deviations of 16 replicates of the zero calibrator on two independent lots was 0.08ng/mL. Dilution and spiking studies showed an average recovery of 91-110%. Lot-to-lot comparison of two independent lots testing 38 serum samples (1.5-33 ng/mL range) yielded a slope of 1.01, intercept of -0.08 ng/mL and r of 0.99. When potential interferents (hemoglobin, triglycerides, and bilirubin) were added at two times the physiological concentrations, AMH concentrations were within ± 10% of the control. A highly specific and reproducible microplate AMH Gen II assay has been developed to standardize the measurement of AMH between methods. The performance of the AMH Gen II assay is ideal for investigation into the physiologic roles of AMH in men and women.