Manipulating bovine granulosa cell energy metabolism limits inflammation.

Bovine granulosa cells are often exposed to energy stress, due to the energy demands of lactation, and exposed to lipopolysaccharide from postpartum bacterial infections. Granulosa cells mount innate immune responses to lipopolysaccharide, including the phosphorylation of mitogen-activated protein kinases and production of pro-inflammatory interleukins. Cellular energy depends on glycolysis, and energy stress activates intracellular AMPK (AMP-activated protein kinase), which in turn inhibits mTOR (mechanistic target of rapamycin). Here, we tested the hypothesis that manipulating glycolysis, AMPK or mTOR to mimic energy stress in bovine granulosa cells limits the inflammatory responses to lipopolysaccharide. We inhibited glycolysis, activated AMPK or inhibited mTOR in granulosa cells isolated from 4-8mm and from > 8.5 mm diameter ovarian follicles, and then challenged the cells with lipopolysaccharide and measured the production of interleukins IL-1α, IL-1ß, and IL-8. We found that inhibiting glycolysis with 2-deoxy-d-glucose reduced lipopolysaccharide-stimulated IL-1α > 80%, IL-1ß > 90%, and IL-8 > 65% in granulosa cells from 4-8 mm and from > 8.5 mm diameter ovarian follicles. Activating AMPK with AICAR also reduced lipopolysaccharide-stimulated IL-1α > 60%, IL-1ß > 75%, and IL-8 > 20%, and shortened the duration of lipopolysaccharide-stimulated phosphorylation of the mitogen-activated protein kinase ERK1/2 and JNK. However, only the mTOR inhibitor Torin 1, and not rapamycin, reduced lipopolysaccharide-stimulated IL-1α and IL-1ß. In conclusion, manipulating granulosa cell energy metabolism with a glycolysis inhibitor, an AMPK activator, or an mTOR inhibitor, limited inflammatory responses to lipopolysaccharide. Our findings imply that energy stress compromises ovarian follicle immune defences.

Anticentromere antibody induced by immunization with centromere protein and Freund's complete adjuvant may interfere with mouse early-stage embryo.

BACKGROUND: Anticentromere antibody (ACA) is a member of the antinuclear antibody spectrum (ANAs) which has been speculated to be associated with subfertility. Thus, the present study aimed to investigate the induction of ACA production and its potential interference with early-stage embryos. METHODS: Recombinant centromere protein-A (CENP-A) or centromere protein-B (CENP-B) and complete Freund's adjuvant (CFA) were used to immunize mice. Serum ACA level was then evaluated by using an indirect immunofluorescence test. Immunofluorescence assay was performed to detect IgG in follicles in ovarian tissues and early-stage embryos. RESULTS: Following treatment, serum positive ACA was observed in mice treated with CENP and CFA. Furthermore, IgG were detected in follicular fluid and early-stage embryos from mice treated with CENP and CFA. CONCLUSIONS: This study preliminarily indicated that ACA induced by CENP and CFA may penetrate into the living embryos of early-stage in mice.

Alterations in the immunoreactivity of laminin, type IV collagen and α3ß1 integrin in diabetic rat ovarian follicles.

AIM: In order to determine the possible effects of diabetes, we aimed to investigate the expression of extracellular matrix proteins in the theca and granulosa layers in different follicular stages. METHODS: Thirty-two adult Wistar albino male rats were divided into 4 groups as control and sampled groups. Four, eight and twelve weeks after inducing diabetes with an intraperitoneal injection of streptozotocin (40 mg/kg), the expressions of laminin, type IV collagen and α3ß1 integrin in ovarian tissues were evaluated by immunohistochemical method. RESULTS: In our study, in the first month of diabetes, a significant increase was observed in laminin, type IV collagen and α3ß1 integrin expressions in all follicle types compared to the control group in both the theca and granulosa layers. Laminin and type IV collagen immunoreactivity tended to increase in D2 and D3 groups also. Integrin expression did not change in the newly formed follicles in the D2 and D3 groups, however, it tended to change and increase in the developing follicles. CONCLUSIONS: The changes in the expression of laminin, type IV collagen and α3ß1 integrin, which are the extracellular matrix proteins in the follicle, along with diabetes, show that diabetes plays a role in the regulation of follicular development (Tab. 4, Fig. 36, Ref. 29).

Comparative transcriptome analysis reveals PERP upregulated during Salmonella Enteritidis challenge in laying ducks.

Salmonella Enteritidis (SE) can be transmitted to eggs through cecum or the ovary from infected layers and causes food poisoning in humans. The mechanism of cecal transmission has been extensively studied. However, the mechanism and route of transovarian transmission of SE remain unclear. In this study, the ducks were orally inoculated with SE, and the ovarian follicles and stroma were collected to detect SE infection. The immune responses were triggered and the innate and adaptive immune genes (TLR4, NOD1, AvßD7, and IL-1ß) were upregulated significantly during the SE challenge. Moreover, the ovary tissues (small follicle and stroma) of susceptible and resistant-laying ducks were performed by RNA sequencing. We obtained and identified 23 differentially expressed genes (DEGs) between susceptible and resistant-laying ducks in both small follicle and stroma tissues ( p < 0.05). The DEGs were predominately identified in the p53 signaling pathway. The expression of key genes (p53, MDM2, PERP, caspase-3, and Bcl-2) involved in the signaling pathway was significantly higher in granulosa cells (dGCs) from SE-infected ducks than those from uninfected ducks. Moreover, the overexpression of PERP resulted in further induction of p53, MDM2, caspase-3, and Bcl-2 during SE infection in dGCs. Whereas, an opposite trend was observed with the knockdown of PERP. Besides, it is further revealed that the PERP could enhance cell apoptosis, SE adhesion, and SE invasion in SE-infected dGCs overexpression. Altogether, our results demonstrate the duck PERP involved in the ovarian local immune niche through p53 signaling pathway in dGCs challenged with SE.

Immunological microenvironment alterations in follicles of women with proven severe endometriosis undergoing in vitro fertilization.

The purpose of this study was to test the hypothesis that different cytokine profiles may exist in the follicular fluid of endometriosis (EM) patients undergoing in vitro fertilization (IVF), as these differences may provide insights into the pathogenesis of the disease. This was a cross-sectional study conducted at the reproductive center of a medical university hospital. The study included 49 patients receiving IVF. 20 infertile women with proven EM and 29 women without diagnosed EM (control group) were evaluated. Follicular fluid (FF) and serum were collected at the time of follicle aspiration and the concentrations of 38 cytokines were determined by multiplexed immunoassay. The results indicated that the levels of IL-4, IL-13, IL-3 and IL-1α were significantly increased in the FF of women with EM, while levels of IFN-γ, IL-17A, MDC and MIP-1α were decreased compared with in the control subjects. In conclusions, the immune microenvironment of the FF in patients with EM is altered. This may contribute to the pathologic mechanism responsible for the poor outcome of IVF in patients with EM.

MyD88 Signaling in T Cells Is Critical for Effector CD4 T Cell Differentiation following a Transitional T Follicular Helper Cell Stage.

Activation of CD4 T cells by dendritic cells leads to their differentiation into various effector lineages. The nature of the effector lineage is determined by the innate cues provided by dendritic cells to newly primed T cells. Although the cytokines necessary for several effector lineages have been identified, the innate cues that drive T follicular helper (Tfh) lineage cell development remain unclear. Here we found that following priming, CD4 T cells undergoing clonal expansion acquire a transient Tfh-like phenotype before differentiating into other effector lineages. In addition, we found that T cell-intrinsic myeloid differentiation antigen 88 (MyD88) signaling, which occurs downstream of interleukin-1 (IL-1) and IL-18 receptors, is critical for the primed CD4 T cells to transition out of the temporary Tfh lineage. Mice with T cell-specific deletion of MyD88 have a higher proportion of Tfh cells and germinal center (GC) B cells. These exaggerated Tfh cell and GC B cell responses, however, do not lead to protective immunity against infections. We demonstrate that T cell-intrinsic MyD88 is critical for effector lineage differentiation as well as production of the cytokines that are necessary for class switching. Overall, our study establishes that following priming and clonal expansion, CD4 T cells undergo a transitional Tfh-like phase and that further differentiation into effector lineages is dictated by T cell-intrinsic MyD88-dependent cues.

IL-33 is required for disposal of unnecessary cells during ovarian atresia through regulation of autophagy and macrophage migration.

Physiological processes such as ovarian follicle atresia generate large amounts of unnecessary cells or tissue detritus, which needs to be disposed of rapidly. IL-33 is a member of the IL-1 cytokine gene family. Constitutive expression of IL-33 in a wide range of tissues has hinted at its role beyond immune defense. We have previously reported a close correlation between IL-33 expression patterns and ovarian atresia. In this study, we demonstrated that IL-33 is required for disposal of degenerative tissue during ovarian atresia using Il33(-/-) mice. Deletion of the Il33 gene impaired normal disposal of atretic follicles, resulting in massive accumulations of tissue wastes abundant with aging-related catabolic wastes such as lipofuscin. Accumulation of tissue wastes in Il33(-/-) mice, in turn, accelerated ovarian aging and functional decline. Thus, their reproductive life span was shortened to two thirds of that for Il33(+/-) littermates. IL-33 orchestrated disposal mechanism through regulation of autophagy in degenerating tissues and macrophage migration into the tissues. Our study provides direct evidence supporting an expanded role of IL-33 in tissue integrity and aging through regulating disposal of unnecessary tissues or cells.

Immunization against GnRF in adult cattle: a prospective field study.

BACKGROUND: Suppression of cyclic activity in cattle is often desired in alpine farming and for feedlot cattle, not intended for breeding. A cattle specific anti-GnRF vaccine (Bopriva™) is registered for use in heifers and bulls in different countries. In adult cows vaccinated with Bopriva™, the median period until recurrence of class III follicles was 78 days from the day of the 2nd vaccination and reversibility could be proven, as out of 11 experimental cows 10 cows became pregnant at first, and one cow at second insemination. In the present study, 76 healthy, cyclic Eringer heifers and cows were vaccinated twice with Bopriva™ 3-7 weeks apart, to prevent estrus during alpine pasturing. Blood samples were taken for progesterone and GnRF antibody titer analysis on the day of inclusion (7-9 d before the first vaccination) and at the first vaccination. At the same time, gynaecological examinations were performed. When estrus occurred in the course of the alpine pasturing season, a gynaecological examination was done including analysis of a blood sample (progesterone, anti-GnRF antibody titer). Cows were followed for fertility out to 26 months post second vaccination. RESULTS: Median duration of estrus suppression was 191 days after the second vaccination (when the 2 vaccinations were given 28-35 days apart). From n = 13 cows showing signs of estrus on the alpine pasture, n = 7 could not be confirmed in estrus (serum progesterone value >2 ng/ml, no class III follicles seen using ultrasonography). Median duration between second vaccination and next calving was 496 days (25%/75% quartiles: 478/532 days). CONCLUSION: Bopriva™ induced a reliable and reversible suppression of estrus for more than 3 months in over 90% of the cows.

Bidirectional Estrogen-Like Effects of Genistein on Murine Experimental Autoimmune Ovarian Disease.

This study was to investigate the bidirectional estrogen-like effects of genistein on murine experimental autoimmune ovarian disease (AOD). Female BALB/c mice were induced by immunization with a peptide from murine zona pellucida. The changes of estrous cycle, ovarian histomorphology were measured, and the levels of serum sex hormone were analyzed using radioimmunoassay. Proliferative responses of the ovary were also determined by immunohistochemistry. Administration of 25 or 45 mg/kg body weight genistein enhanced ovary development with changes in serum sex hormone levels and proliferative responses. Meanwhile, the proportions of growing and mature follicles increased and the incidence of autoimmune oophoritis decreased, which exhibited normal ovarian morphology in administration of 25 or 45 mg/kg body weight genistein, while a lower dose (5 mg/kg body weight genistein) produced the opposite effect. These findings suggest that genistein exerts bidirectional estrogen-like effects on murine experimental AOD, while a high dose (45 mg/kg body weight) of genistein may suppress AOD.

The impact of passive immunisation against BMPRIB and BMP4 on follicle development and ovulation in mice.

The primordial follicle reserve is the corner stone of female fertility and determines the longevity and quality of reproduction. Complete depletion of this reserve will lead to primary infertility, and the key-limiting step of follicle depletion is the transition from primordial to primary follicles. It has been reported that this process is gonadotrophin-independent, but other conflicting reports are indicated otherwise and this discrepancy needs to be unequivocally clarified. The aim of this study was to investigate the role of bone morphogenetic proteins (BMPs) in the regulation of folliculogenesis in mice passively immunised against BMP receptor 1B (BMPRIB) and BMP4. While a stereological study revealed that the numbers of primordial follicles in immunised mice were significantly higher when compared with control animals, treatment with equine chorionic gonadotrophin showed no effect. In parallel, immunofluorescence microscopy revealed the presence of BMPRIB but not FSH receptor in primordial follicles. The number of primary follicles in immunised mice were also significantly increased when compared with control animals. After puberty, the rates of depletion of primordial and primary follicles were increased with age, particularly in treated animals; however, there was no significant difference between the treatment groups of the same age. Based on these results together with our previous reports in sheep and mice, we confirm that the attenuation of BMP signalling system can be an effective approach to sustain the primordial follicle reserve while promoting the development of growing follicles, ovulation and consequently overall female fertility.

Immune-mediated destruction of ovarian follicles associated with the presence of HSP90 antibodies.

We previously established that the presence of autoantibodies to heat-shock protein 90 (HSP90) is one common causes of female infertility, and demonstrated that its presence leads to detrimental effects on ovarian and reproductive function in mice. The pathophysiological mechanism and alteration in the immune physiology, however, remain unknown. We therefore carried out detailed analysis of various immune cells in the spleen and ovary following immunization of C57BL/6 female mice to generate antibodies to HSP90 in the general circulation. We observed a significant increase in levels of CD45- cells; CD4+ T cells; Ly6G6C+ cells; and CD11b+ Ly6G+ cells in the spleen of these mice, which correlate with the increased anti-HSP90 antibody production. Ovarian- and granulosa-cell populations also showed increased infiltration of CD45+ leukocytes and neutrophil and monocyte populations, which may have led to the observed ovarian follicular degeneration that predominantly manifested as empty follicles. A decrease in the number of functional ovarian follicles was also associated with a decrease in the level of Gdf9 gene expression. Thus, changes in the immune physiology of the spleen and ovary that leads to the generation of antibodies to HSP90 can also bring about the destruction of ovarian follicles.

Feed intake alters immune cell functions and ovarian infiltration in broiler hens: implications for reproductive performance.

Leukocytes are known to participate in ovarian activities in several species, but there is a surprising lack of information for the common chicken. Broiler hens consuming feed ad libitum (AL) exhibit a number of ovarian irregularities, but leukocyte functions are unstudied. In contrast to feed-restricted (R) hens, AL feeding for 7 wk significantly reduced egg production and clutch length while increasing pause length and atretic follicle numbers (P < 0.05). Granulosa cells from F1 follicles of AL hens contained less progesterone, and follicle walls were thicker with loose fibrous morphology and had less collagenase-3-like gelatinolytic activity but more IL-1beta (P < 0.05) production, suggestive of slower maturation in ovulatory process and inflamed necrosis. Interestingly, while highly infiltrated with immune cells, particularly heterophils, IL-1beta, MMP-22-like, and gelatinase A activities were reduced in AL hen peripheral heterophils and monocytes (P < 0.05); however, AL monocytes showed an increase in phagocytosis rate (P < 0.05). Generation of reactive oxygen intermediates was also suppressed in AL heterophils but increased in AL monocytes (P < 0.05). In contrast to leukocyte-free control, both AL and R heterophils and monocytes suppressed progesterone production and increased cell death in a dose-dependent manner when coincubated with granulosa cells at different ratios (P < 0.05). AL monocytes suppressed progesterone production more, but AL heterophils were less proapoptotic when compared to their R counterparts (P < 0.05). Alterations of cellular ceramide content (P < 0.05) corresponded to the discrepancy between heterophil and monocyte functionality. In conclusion, leukocyte dysfunction contributes to impaired ovarian activities of overfed broiler hens.

Evaluation of attenuated Salmonella choleraesuis-mediated inhibin recombinant DNA vaccine in rats.

DNA vaccination has been studied intensively as a potential vaccine technology. We evaluated the effect of an attenuated Salmonella choleraesuis-mediated inhibin DNA vaccine in rats. First, 15 rats were treated with different doses of an inhibin vaccine to evaluate vaccine safety. Next, 30 rats were divided into 3 groups and injected intramuscularly with the inhibin vaccine two (T1) or three times (T2) or with control bacteria (Con) at 4-week intervals. The inhibin antibody levels increased [positive/negative well (P/N) value: T1 vs Con = 2.39 ± 0.01 vs 1.08 ± 0.1; T2 vs Con = 2.36 ± 0.1 vs 1.08 ± 0.1, P < 0.05] at week 2 and were maintained at a high level in T1 and T2 until week 8, although a small decrease in T2 was observed at week 10. Rats in the T1 group showed more corpora lutea compared with the Con group (10.50 ± 0.87 vs 7.4 ± 0.51, P < 0.05). Estradiol (0.439 ± 0.052 vs 0.719 ± 0.063 ng/mL, P < 0.05) and progesterone (1.315 ± 0.2 vs 0.737 ± 0.11 ng/mL, P < 0.05) levels differed significantly at metestrus after week 10 between rats in the T1 and Con groups. However, there were no significant differences in body, ovary, uterus weights, or pathological signs in the ovaries after immunization, indicating that this vaccine is safe. In conclusion, the attenuated S. choleraesuis-mediated inhibin vaccine may be an alternative to naked inhibin plasmids for stimulating ovarian follicular development to increase the ovulation rate in rats.

The therapeutic effect of anti-CD19 antibody on DHEA-induced PCOS mice.

Immune dysregulation has been summarized as a critical factor in the occurrence and development of Polycystic ovary syndrome (PCOS), but potential mediators and mechanisms remain unclear. Our previous study showed that CD19+ B cells were involved in the pathogenesis of dehydroepiandrosterone (DHEA)-induced PCOS mice. Here, we studied the therapeutic potential of anti-CD19 antibody (aCD19 Ab) on DHEA-induced PCOS mice. The results showed that aCD19 Ab treatment improved ovarian pathological structure and function of PCOS mice, manifested by an increased number of corpus luteum, a decreased number of cystic follicles and atretic follicles, and regular estrus cycles. The aCD19 Ab treatment reduced the proportion of splenic CD21+ CD23low marginal zone B cells as well as the level of serum IgM and decreased the percentage of peripheral blood and splenic neutrophils. In particular, aCD19 Ab treatment reduced the apoptosis of granulosa cells and macrophage infiltration in ovarian secondary follicles of PCOS mice, as well as the expression of TNF-α in ovarian tissue and serum TNF-α levels. Moreover, we confirmed that TNF-α induced the apoptosis of human ovarian granulosa tumor cell line cells in vitro. Thus, our work demonstrates that aCD19 Ab treatment improves ovarian pathological phenotype and function by reducing local and systemic inflammation in PCOS mice, which may provide a novel insight into PCOS therapy.

Ovarian leukocytes: Association with follicular persistence and cyst formation in dairy cows.

One of the initial causes of cystic ovarian disease (COD) is a failure in the normal ovulation mechanism. This study aimed to characterize the populations of immune cells (T-lymphocytes, B-lymphocytes, monocytes-macrophages and granulocytes) present in the ovary of cows with COD and induced follicular persistence, and evaluate their relation with follicular persistence and cyst formation. The follicular persistence model was developed using a progesterone (P4) slow-release intravaginal device, to obtain subluteal concentrations of P4. Results evidenced that T-lymphocytes, B-lymphocytes and monocytes-macrophages in the cortex, medulla, and theca externa and interna of dominant follicles were higher in the control group than in the COD and all persistence groups. Granulocytes in the medulla and theca externa of dominant follicles were lower in the control group than in the COD group, and those in the cortex and medulla were lower in the control group than in the persistence groups. The presence of T-lymphocytes, B-lymphocytes and granulocytes in the follicular fluid was abundant, especially that of granulocytes, without differences between control and COD cows. These results suggest that the immune system potentially plays a role in the local mechanisms of COD pathogenesis in dairy cows. In spontaneous COD and in our follicular persistence model, the distribution of the cells studied was different from that in the control group. However, to our knowledge, this is the first report describing the presence of immune cells in bovine follicular fluid samples and the expression of steroid hormone receptors in infiltrating immune cells in the bovine ovary.

Granulosa cells from emerged antral follicles of the bovine ovary initiate inflammation in response to bacterial pathogen-associated molecular patterns via Toll-like receptor pathways.

Bacterial infections of the uterus or mammary gland commonly perturb ovarian antral follicle growth and function, causing infertility in cattle. Cells of the innate immune system use Toll-like receptors (TLRs) TLR2, TLR4, and TLR5 to recognize pathogen-associated molecular patterns (PAMPs) of bacteria, leading to production of inflammatory mediators, such as IL-1beta, IL-6, and IL-8. The present study examined whether granulosa cells from emerged antral follicles have functional responses to typical bacterial PAMPs. Granulosa cells from emerged bovine antral follicles expressed mRNA for all 10 TLRs. Cellular expression of mRNA for the cytokines IL1B, IL6, IL10, and TNF, and chemokines IL8 and CCL5, increased after treatment with synthetic bacterial lipoprotein binding TLR2, lipopolysaccharide binding TLR4, or flagellin binding TLR5. Supernatants of granulosa cells accumulated IL-1beta, IL-6, and IL-8 protein in a concentration-dependent manner when treated with lipoprotein or lipopolysaccharide, but not flagellin. Accumulation of IL6 in response to lipoprotein and lipopolysaccharide was attenuated using siRNA targeting TLR2 and TLR4, respectively. Granulosa cells increased phosphorylation of mitogen-activated protein kinase (MAPK) 14 and MAPK3/1 within 30 min of treatment with lipopolysaccharide or lipoprotein, and inhibitors targeting MAPK14 reduced the accumulation of IL-6 in response to the PAMPs. Treatment with hormones follicle-stimulating hormone, luteinizing hormone, estradiol, or progesterone did not significantly affect granulosa cell responses to PAMPs. However, epidermal growth factor enhanced IL-6 accumulation in response to lipoprotein and inhibiting epidermal growth factor receptor (EGFR) abrogated the effect, whereas lipoprotein increased granulosa cell EGFR mRNA expression. In conclusion, bovine granulosa cells from emerged follicles sense bacterial PAMPs and initiate inflammatory responses via TLR2 and TLR4 pathways.

Changes in granulosa cells' gene expression associated with increased oocyte competence in bovine.

One of the challenges in mammalian reproduction is to understand the basic physiology of oocyte quality. It is believed that the follicle status is linked to developmental competence of the enclosed oocyte. To explore the link between follicles and competence in cows, previous research at our laboratory has developed an ovarian stimulation protocol that increases and then decreases oocyte quality according to the timing of oocyte recovery post-FSH withdrawal (coasting). Using this protocol, we have obtained the granulosa cells associated with oocytes of different qualities at selected times of coasting. Transcriptome analysis was done with Embryogene microarray slides and validation was performed by real-time PCR. Results show that the major changes in gene expression occurred from 20 to 44  h of coasting, when oocyte quality increases. Secondly, among upregulated genes (20-44  h), 25% were extracellular molecules, highlighting potential granulosa signaling cascades. Principal component analysis identified two patterns: one resembling the competence profile and another associated with follicle growth and atresia. Additionally, three major functional changes were identified: (i) the end of follicle growth (BMPR1B, IGF2, and RELN), involving interactions with the extracellular matrix (TFPI2); angiogenesis (NRP1), including early hypoxia, and potentially oxidative stress (GFPT2, TF, and VNN1) and (ii) apoptosis (KCNJ8) followed by iii) inflammation (ANKRD1). This unique window of analysis indicates a progressive hypoxia during coasting mixed with an increase in apoptosis and inflammation. Potential signaling pathways leading to competence have been identified and will require downstream testing. This preliminary analysis supports the potential role of the follicular differentiation in oocyte quality both during competence increase and decrease phases.

The autoimmune regulator prevents premature reproductive senescence in female mice.

Loss-of-function mutations in the autoimmune regulator (AIRE) gene are responsible for autoimmune polyglandular syndrome type 1 (APS-1), which commonly manifests as infertility in women. AIRE is a transcriptional regulator that promotes expression of tissue-restricted antigens in the thymus, including antigens specific to the ovary. Thymic expression of ovarian genes under AIRE's control may be critical for preventing ovarian autoimmune disease. Because mice lacking Aire are an important APS-1 model, we examined the reproductive properties of female Aire-deficient (Aire(-/-)) mice. Female Aire(-/-) mice on the BALB/c background were examined for reproductive parameters, including fertility, litter sizes, and ovarian follicular reserves. Although delayed puberty was observed in Aire(-/-) mice, all mice entered puberty and exhibited mating behavior. Only 50% of Aire(-/-) females gave an initial litter, and only 16% were able to produce two litters. Ovarian histopathologic examination revealed that 83% of previously bred females lost all ovarian follicular reserves. Among virgin females, follicular depletion was observed in 25% by 8 wk, and by 20 wk, 50%-60% of mice lost all follicles. This was associated with elevated serum follicle-stimulating hormone level and ovarian infiltration of proliferating CD3+ T lymphocytes. Ovulation rates of 6-wk-old Aire(-/-) mice were reduced by 22%, but this difference was not statistically significant. Finally, transplantation experiments revealed that follicular loss depended on factors extrinsic to the ovary. These results suggest that immune-mediated ovarian follicular depletion is a mechanism of infertility in Aire(-/-) mice. The results have important implications in the pathogenesis of ovarian autoimmune disease in women.

Progressive decline of residual follicle pool after clinical diagnosis of autoimmune ovarian insufficiency.

CONTEXT: In approximately 5-8% patients with primary ovarian insufficiency (POI), the disease is caused by an autoimmune process made evident by the appearance of autoantibodies against steroidogenic enzymes (SCA-POI). Anti-müllerian hormone (AMH) is the best marker of the residual follicular pool. OBJECTIVE: To evaluate the rate of loss of the residual follicle pool in women with SCA-POI after clinical diagnosis. DESIGN AND METHODS: One hundred and thirty-two women with POI were tested for 21-hydroxylase autoantibodies, 17α-hydroxylase autoantibodies and P450scc autoantibodies, and 35 patients with SCA-POI were identified. AMH was analysed at the time of the first visit in all women with POI, and in follow-up, serum samples were taken 1-3 years after in 11 women with SCA-POI and detectable AMH. RESULTS: 12/35 (35%) women with SCA-POI had AMH levels within the normal range at the time of first sampling, as compared to 6/97 (6%) with idiopathic POI (P < 0·001). 11/17 (65%) women with SCA-POI with <6 years disease duration had normal serum AMH concentration. A progressive decline in AMH concentration was observed at longitudinal follow-up in all 11 AMH-positive women with SCA-POI, at an estimated average rate of 1·6 µg/l AMH/year (corresponding to an average 57% of preserved follicle pool/previous year) (R(2)  = 0·219, P = 0·028). After 6 years of disease duration, only 1/18 (6%) women with SCA-POI had detectable levels of AMH, similar to women with idiopathic POI (5/78, 6%). CONCLUSION: Most women with SCA-POI present at clinical diagnosis with a preserved follicle pool that is progressively lost within a few years.

Immunization with ovarian autoantigens leads to reduced fertility in mice following follicular dysfunction.

Immunoproteomics using sera of women with ovarian autoimmune diseases such as primary ovarian insufficiency and IVF embryo transfer recruits led to identification of three proteins namely alpha actinin 4 (α-ACTN4), heat-shock 70 protein 5 (HSPA5), and actin beta (ACTB). This study deals with the establishment of a peptide ELISA for screening sera of antiovarian antibody (AOA)-positive patients and further delves into understanding the role of these three proteins in ovarian autoimmunity in a mouse model. Using in silico approach, antigenic peptides of these proteins were identified and used for peptide ELISA. ELISA results indicated that AOA-positive sera showed reactivity with only specific peptides. The functional significance of the dominant peptides was studied by active immunization of female mice with these peptides. All immunized mice generated high antibody titers and profound effect on ovaries with few primordial (2.4±0.1, 2.4±0.2, and 2±0.1), primary (2.4±0.5, 1.7±0.3, and 2.4±0.3), preantral (2.3±0.5, 3.4±0.3, and 2.9±0.3), antral (0.9±0.2, 1.6±0.8, and 2.3±0.6) follicles, and corpora lutea (2.8±0.8, 2.9±1.7, and 4.6±2.3), and increased number of atretic follicles (5.5±0.4, 4.9±1.8, and 7.5±1.0) in ACTN4-, HSPA5-, and ACTB-immunized mice compared with control animals (3.0±0.2, 3.5±0.6, 3±0.1, 3.6±0.2, 4.7±0.3, and 1.5±0.3) respectively. These mice when mated with fertile male mice showed an overall 25-43% reduction in fertility compared with controls. The data clearly suggest that the dominant antigenic epitopes of the three proteins play critical role in fertility and could possibly be the key autoimmune targets. These epitopes could be used to develop a more specific and sensitive diagnostic test for women with ovarian autoimmune diseases and to design therapy for disease management for reinstatement of ovarian function.