Reduction In Number Of Follicles Research Articles

Mechanism study of YangJing ZhongYu decoction on regulating mitochondrial dynamics of ovarian granular cells and improving diminished ovarian reserve

ObjectiveDiminished ovarian reserve (DOR) encompasses both reproductive and endocrine disorders, resulting in a decline in female fertility. This paper explored the mechanism of Yangjing Zhongyu Decoction (YJZYD) regulating mitochondrial dynamics of ovarian granulosa cells (GCs) to improve DOR.MethodsDOR patients were treated with YJZYD, with ovarian volume (OV), antral follicle count (AFC), and endometrial thickness (EMT) detected. C57BL/6 female mice were treated by cyclophosphamide (Cy) intraperitoneal injection and YJZYD solution daily gavage, with serum anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) levels determined. Ovarian GCs (KGN) were interfered with 4-Hydroperoxy-Cyclophosphamide (4-HC) and treated with the MAPK/ERK pathway inhibitor or activator.ResultsDOR patients showed increased levels of serum AMH, E2, OV, AFC and EMT, while reduced FSH and LH levels after YJZYD treatment. After Cy induction, DOR mice exhibited irregular estrous cycles, diminished serum AMH and E2 levels, elevated FSH and LH levels, reduced follicle number and atresia follicle number, disorderly arranged GCs, and severe interstitial fibrosis. After 4-HC treatment, KGN proliferation and Bcl-2, MFN1, and MFN2 were suppressed, while apoptotic rate, Bax, Cleaved-caspase-3, and p-Drp1 (Ser616) levels, and mitochondrial fission and quantity increased. YJZYD promoted 4-HC-treated KGN proliferation, boosted mitochondrial fusion, and inhibited apoptosis and mitochondrial fission via the MAPK/ERK pathway.ConclusionYJZYD promoted ovarian GC proliferation and mitochondrial fusion, suppressed cell apoptosis and mitochondrial fission, and effectively improved DOR in mice by activating the MAPK/ERK pathway, providing a theoretical basis for the clinical application value of YJZYD in DOR treatment.

Growth factor-loaded ovarian extracellular matrix hydrogels promote in vivo ovarian niche regeneration and enhance fertility in premature ovarian insufficiency preclinical models

Premature ovarian insufficiency (POI) means menopause before 40 years of age affecting about 1 % of women. Approaches based on cell therapy and the paracrine effects of stem cells or bioproducts such as platelet-rich plasma have been proposed, but concerns remain about undesired systemic effects, as well as the need to optimize delivery methods through bioengineering methods. This study explores the efficacy of decellularized bovine ovarian cortex extracellular matrix (OvaECM) hydrogels alone and as a growth factor (GF) carrier (OvaECM+GF) in a chemotherapy-induced POI murine model. In vitro assays showed a gradual release of GF from the OvaECM sustained for two weeks. Chemotherapy drastically reduced follicle numbers, but OvaECM+GF treatment restored pre-antral follicle development. Moreover, this treatment notably regenerated the ovarian microenvironment by increasing cell proliferation and microvessel density while reducing chemotherapy-induced apoptosis and fibrosis. Whole-ovary RNA sequencing and gene set enrichment analysis revealed an upregulation of regeneration-related genes and a downregulation of apoptotic pathways. The OvaECM+GF treatment also yielded significantly better outcomes following ovarian stimulation and in vitro fertilization. After two consecutive crossbreeding cycles, OvaECM+GF-treated mice showed normal reproductive function. This research showcases the biocompatibility and efficacy of OvaECM to reverse POI in mice, setting a foundation to explore innovative bioengineering-based POI therapies. Statement of significancePremature ovarian insufficiency (POI) affects about 1 % of women worldwide, causing early menopause before 40 years old. Current treatments alleviate symptoms but do not restore ovarian function. This study explores an innovative approach using ovarian cortex extracellular matrix hydrogels to deliver growth factors into the murine ovarian niche and reverse POI. In vitro release kinetic assays demonstrated a gradual and sustained release of growth factors. In a POI-induced mouse model, intraovarian injections of the hydrogel encapsulating growth factors restored pre-antral follicle development, increased cell proliferation, reduced apoptosis and fibrosis, and improved ovarian response and in vitro fertilization outcomes. Long-term benefits included larger litter sizes. This innovative technique shows promise in regenerating the ovarian environment and improving reproductive outcomes.

Melatonin Protects Against Mitochondrial Dyshomeostasis and Ovarian Damage Caused by Chronic Unpredictable Mild Stress Through the eIF2α-AFT4 Signaling Pathway in Mice.

Stress is an emotional state caused by an unexpected external environmental change or stimulus, and several experiments have demonstrated its negative impact on ovarian function, ultimately affecting reproductive ability. Melatonin (MT) has been shown to facilitate oocyte maturation and enhance ovarian function by regulating mitochondrial function. However, the specific effect and underlying molecular mechanisms of MT on stress-induced ovarian dysfunction remain largely unknown. In this study, we established a mouse model of chronic unpredictable mild stress (CUMS) to investigate its impact on ovarian function. Our findings revealed that CUMS led to premature ovarian insufficiency (POI) in mice, characterized by a reduction in follicle numbers and decreased levels of anti-Müllerian hormone (AMH) and bone morphogenetic protein 15 (BMP15). Furthermore, CUMS caused decreased expression of mitochondrial fission protein 1 (FIS1) and enhanced level of mitochondrial fusion protein optic atrophy 1(OPA1), mitofusin1(MFN1), as well as nucleus-encoded protein succinate dehydrogenase complex A (SDHA), reflecting mitochondrial dyshomeostasis. Additionally, CUMS resulted in excessive autophagy and apoptosis. However, MT reversed these effects and improved ovarian damage. Importantly, the protective effects of MT were mediated through the inhibition of the eIF2α-AFT4 pathway. Overall, this study provides valuable insights into the treatment of POI caused by CUMS.

An updated view on the role of the female reproductive tract microbiome in IVF outcomes

Introduction. In the last decade, a high-throughput 16S ribosomal RNA (rRNA) sequencing allowed to markedly extend insights into female reproductive tract microbiome. However, evidence about its role on in vitro fertilization (IVF) outcomes remains scarce and controversial.Aim: to analyze literature data for assessing an impact of the vaginal, uterine, and ovarian microbiome on IVF outcomes.Materials and Methods. The review was carried out based on publications from available in PubMed/MEDLINE, EBSCO, ResearchGate, Google Academy and еLibrary databases released over the last 20 years. For this, there was search for keywords and their combinations in Russian- and English-written publications: «vaginal microbiome», «cervical microbiome», «uterine microbiome», «ovarian microbiome», «microbiome of ovarian follicles», «IVF», «miscarriage», «early pregnancy loss», «implantation failure», «ovarian failure», «inflammasome». Only full-text original articles and reviews published in peer-reviewed journals were included in the review. Exclusion criteria were as follows: studies containing less than 10 observations per group, abstracts of conferences, studies on the male reproductive tract microbiome. Duplicate publicationswere excluded. The publications were selected independently by two co-authors, and in case of discrepancy two other co-authors were involved.Results. Vaginal, uterine, and ovarian microbiomes were characterized by providing relevant classifications and the features related to implantation failures and pregnancy loss after IVF. It was found that a decline in total Lactobacillus level and elevated proportion of L. jensenii, G. vaginalis, and Proteobacteria in the vaginal microbiome were predictors of implantation failure. In addition, IVF failure was also associated with the presence of Atopobium, Bifidobacterium, Chryseobacterium, Gardnerella, Streptococcus, Haemophilus, Staphylococcus, Brevundimonas, and Ralstonia in the uterine cavity as well as Bifidobacterium, Gardnerella, and Klebsiella in the endometrial tissue. On the other hand, Lactobacillus dominance in the uterine microbiome has a favorable effect. The colonization of the follicle by any microorganisms as well as the presence of follicular fluid anaerobic bacteria-derived metabolite trimethylamine-N-oxide (TMAO) associated with bacterial vaginosis additionally alters IVF outcomes. Moreover, the role of infectious cues in lowered ovarian reserve has also been established. Activation of the NLRP3 (NLR Family Pyrin Domain Containing 3) inflammasome by microbe-derived ligands stimulates production of pro-inflammatory cytokines and contributes to reduced follicle number. Blocking NLRP3 in mouse experiments can delay depletion of the follicle pool and result in elevated fertility.Conclusion. Favorable IVF outcomes are associated with Lactobacillus predominance in the vaginal and endometrial microbiome as well as lack of follicular fluid microorganisms. TMAO detected in the follicular fluid as well as activated NLRP3 inflammasome serve as negative predictors of IVF outcomes.

P-587 effects of erzhi-tiangui granules to NEAT1 and apoptotic factors expression in granulosa cells of women with diminished ovarian reserve and Kidney-deficiency syndrome

Abstract Study question To investigate whether erzhi-tiangui granules (ETG) can reduce granulosa cells (GCs) apoptosis levels by regulating NEAT1 expression, thereby improving the cycle outcomes of patients. Summary answer ETG significantly improved oocyte quality and decreased apoptotic factor levels in GCs, which may be related to its intervention in NEAT1 expression. What is known already The mechanism by which diminished ovarian reserve (DOR) occurs is mainly due to extensive apoptosis of GCs, which leads to follicular atresia and reduced follicle numbers. It has been found that changes in NEAT1 expression in ovarian tissues can exert influence the extent of apoptosis of GCs, which leads to diseases such as POF and PCOS. While ETG, a clinical formula used to improve female fertility, can improve the quality of oocytes and embryos by down-regulating the expression of apoptosis-related effector proteins, thus improving clinical outcomes. Whether ETG improves clinical outcomes in DOR patients via NEAT1 hasn’t been elucidated. Study design, size, duration This study was conducted in two separate parts from January 2020 to September 2021. Experiment I included 15 women each with normal ovarian reserve (NOR) and DOR, aiming to investigate the expression differences of NEAT1, key apoptosis proteins (Bcl-2 and Caspase-3). Experiment II, a randomized controlled trial, recruited 70 DOR patients, aimed to investigate if ETG could regulate the expression levels of Bcl-2 and Caspase-3 through NEAT1, thus acting to improve clinical outcomes. Participants/materials, setting, methods We detected expression levels of lncRNA NEAT1 and apoptotic proteins Bcl-2 and Caspase-3 of DOR and NOR women using RT-qPCR and Western-blotting, respectively. DOR patients were randomly assigned to ETG and control group, treated with ETG and placebo respectively. Clinical efficacy was confirmed by comparing two groups regarding changes in clinical and laboratory data. We also probed the changes in expression levels of NEAT1, Bcl-2 and Caspase-3 to investigate effects of ETG on GCs’ apoptosis. Main results and the role of chance In Experiment I, compared to the NOR group, the expression levels of NEAT1 and Bcl-2 were down-regulated while those of Caspase-3 were up-regulated in the GCs of DOR patients (P < 0.05). In Experiment II, a total of 65 of 70 patients completed the study and were included in the outcome analysis. There were no significant differences between the two groups regarding baseline characteristics. Before treatment, there was no significant difference in the Kidney-deficiency syndrome scores between the two groups. After treatment, the ETG group had significantly lower Kidney-deficiency syndrome scores compared to the control group (P < 0.01). Also, Kidney-deficiency syndrome scores were significantly lower in the ETG group post-treatment compared with that pre-treatment (P < 0.001); there was no significant change in the control group (P > 0.05). There were no significant differences between the two groups in the duration of gonadotrophin stimulation, serum estrogenand progesterone levels on the trigger day, and normal fertilization rate. Compared to the control group, the ETG group had lower gonadotropin dosage and increased number of oocytes, transferable embryos and top embryos obtained (P < 0.05). Furthermore, the expression of NEAT1 was upregulated (P < 0.05), Caspase-3 expression was decreased (P < 0.01) in GCs from the ETG group than those in the control group (P < 0.05). Limitations, reasons for caution The small number of observed cases and the lack of long-term efficacy may lead to biased study conclusions. Further studies are also expected in the future to understand the relevant pathway mechanisms. Wider implications of the findings In this study, not only to judge the therapeutic effects of ETG by using clinical symptoms and clinical laboratory indexes as observations, but also to explore the molecular mechanism of its treatment of Kidney-deficiency type DOR based on NEAT1, which may be related to affecting the apoptotic process. Trial registration number No. 82274573, No. 82174429, No. ZR2021MH255, No. tsqn202103182

Prdm9 deficiency of rat oocytes causes synapsis among non-homologous chromosomes and aneuploidy.

Aneuploidy (abnormal chromosome number) accompanies reduced ovarian function in humans and mice, but the reasons behind this concomitance remain underexplored. Some variants in the human gene encoding histone-3-lysine-4,36-trimethyltransferase PRDM9 are associated with aneuploidy, and other variants with ovarian function reduced by premature ovarian failure (POF), but no link between POF and aneuploidy has been revealed. SHR/OlaIpcv rat females lacking PRDM9 manifest POF-a reduced follicle number, litter size, and reproductive age. Here, we explored this model to test how POF relates to oocyte euploidy. The mutant rat females displayed increased oocyte aneuploidy and embryonic death of their offspring compared to controls. Because rat PRDM9 positions meiotic DNA breaks, we investigated the repair of these breaks. Fertile control rodents carry pachytene oocytes with synapsed homologous chromosomes and repaired breaks, while sterile Prdm9-deficient mice carry pachytene-like oocytes with many persisting breaks and asynapsed chromosomes. However, most PRDM9-lacking rat oocytes displayed a few persisting breaks and non-homologous synapsis (NHS). HORMAD2 protein serves as a barrier to sister-chromatid repair and a signal for the synapsis and DNA repair checkpoints. NHS but not asynapsis was associated with HORMAD2 levels similar to the levels on rat pachytene chromosomes with homologous synapsis. NHS was accompanied by crossing-over decreased below the minimum that is essential for euploidy. We argue that the increased mutant rat aneuploidy is due to NHS, which allows some oocytes to pass meiotic checkpoints without one crossing-over per chromosomal pair, leading to segregation errors, and thereby NHS links POF to aneuploidy.

HENMT1 is involved in the maintenance of normal female fertility in the mouse.

PIWI-interacting small RNAs (piRNAs) maintain genome stability in animal germ cells, with a predominant role in silencing transposable elements. Mutations in the piRNA pathway in the mouse uniformly lead to failed spermatogenesis and male sterility. By contrast, mutant females are fertile. In keeping with this paradigm, we previously reported male sterility and female fertility associated with loss of the enzyme HENMT1, which is responsible for stabilising piRNAs through the catalysation of 3'-terminal 2'-O-methylation. However, the Henmt1 mutant females were poor breeders, suggesting they could be subfertile. Therefore, we investigated oogenesis and female fertility in these mice in greater detail. Here, we show that mutant females indeed have a 3- to 4-fold reduction in follicle number and reduced litter sizes. In addition, meiosis-II mutant oocytes display various spindle abnormalities and have a dramatically altered transcriptome which includes a down-regulation of transcripts required for microtubule function. This down-regulation could explain the spindle defects observed with consequent reductions in litter size. We suggest these various effects on oogenesis could be exacerbated by asynapsis, an apparently universal feature of piRNA mutants of both sexes. Our findings reveal that loss of the piRNA pathway in females has significant functional consequences.

Efficacy and safety of herbal medicine Yijin-tang on polycystic ovary syndrome: A single-arm pilot study

IntroductionPolycystic ovary syndrome (PCOS) is one of the most common disorders in reproductive endocrinology among women of reproductive age. Recent studies suggest that complementary and alternative therapies can alleviate PCOS symptoms, but the evidence for its efficacy and safety is minimal. The objectives of this study were to examine the effect of Yijin-tang (YJT) on PCOS patients. MethodsA single-arm, prospective pilot study enrolled 15 PCOS women with oligomenorrhea at Kyung Hee University Hospital at Gangdong, Seoul. Each participant took YJT 3 times a day (1 h after meal), totaling 6 g of YJT (Pinellia ternata Breitenbach, Citrus unshiu Markovich, Poria cocos Wolf, Glycyrrhiza uralensis Fisch, and Zingiber officinale Roscoe) per day, for 12 weeks. The primary outcome was menstrual cycle length. The secondary outcomes were the levels of estradiol (E2), luteinizing hormone (LH), follicle stimulating hormone (FSH), total testosterone, LH/FSH ratio, number of ovarian follicles, phlegm pattern questionnaire scores, body weight, and body mass index. ResultsThe menstrual cycle length significantly decreased at week 12 compared to baseline (60.31 ± 15.61 to 44.40 ± 15.58; P = 0.002) without body weight reduction. Reduced menstrual cycle length was maintained at additional 12 weeks without treatment (60.31 ± 15.61 to 44.50 ± 15.70; P = 0.010). Number of ovarian follicles was reduced after 12-week treatment (12.33 ± 3.29 to 9.93 ± 2.79; P = 0.012). Levels of E2, LH, FSH, and total testosterone and LH/FSH ratio did not show significant change at week 12 compared to baseline. No serious adverse events were reported during the study. ConclusionsThis study demonstrated that YJT administration for 12 weeks improved oligomenorrhea and reduced follicle number of the ovary though there was not significant body weight reduction.

Protective impacts of gallic acid against cadmium-induced oxidative toxicity in the ovary of rats

This study evaluates the probable effects of induced reproductive toxicity by cadmium and its amelioration by gallic acid (GA). Adult female rats were daily administered with cadmium (CdCl2) (5 mg/kg) singly or combined with GA (20 mg/kg) for 14 days to evaluate the effect on antioxidant status and ovary function using biochemical and histological analysis. Cadmium-treated rats alone revealed a considerable increment in total body weight and ovaries’ weight due to muscle wasting. However, GA + cadmium reversed considerably the tread and offered protection against cadmium-induced muscle wasting. Cadmium treatment alone decreased reduced glutathione (GSH) level, activities of superoxide dismutase (SOD), and catalase (CAT) and also increased malondialdehyde (MDA) level. Besides, the administration of GA alone or in combination mitigated the cadmium-mediated decrease in GSH, SOD, and CAT, respectively. Serum concentrations of estrogen (E), testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were reduced significantly in cadmium-treated rats. Histopathology of the ovaries showed a considerable reduction in follicle number. GA treatment alone and CdCl2 + GA-treatment group decreased cell death of follicular cells in the ovaries of rats and increased serum reproductive hormones. Taken together, gallic acid prevented the deleterious impacts of CdCl2 on the biomarkers of oxidative stress, antioxidant enzymes, and ovarian histopathological structure.

Comparative gonadotoxicity of the chemotherapy drugs cisplatin and carboplatin on prepubertal mouse gonads.

The treatment of childhood cancer with chemotherapy drugs can result in infertility in adulthood. Newer generations of drugs are developed to replace parent drugs, with the potential benefits of less toxic side effects. For platinum alkylating-like drugs, in contrast to the parent compound cisplatin, the newer-generation drug carboplatin is reported to have reduced toxicity in some respects, despite being administered at 5–15 times higher than the cisplatin dose. Whether carboplatin is also less toxic than cisplatin to the reproductive system is unknown. Here we compare the gonadotoxic impact of cisplatin and carboplatin on female and male mouse prepubertal gonads. In vitro cultured CD1 mouse ovaries or testis fragments were exposed to either cisplatin or carboplatin for 24 h on Day 2 of culture and analysed by Day 6. A dose response for each drug was determined for the ovary (0.5, 1 & 5 μg/ml cisplatin and 1, 5 & 10 μg/ml carboplatin) and the testis (0.01, 0.05 & 0.1 μg/ml cisplatin and 0.1, 0.5 & 1 μg/ml carboplatin). For the ovary, unhealthy follicles were evident from 1 μg/ml cisplatin (73% unhealthy, P = 0.001) and 5 μg/ml carboplatin (84% unhealthy, P = 0.001), with a concomitant reduction in follicle number (P = 0.001). For the testis, the proliferating germ cell population was significantly reduced from 0.05 μg/ml cisplatin (73% reduction, P = 0.001) and 0.5 μg/ml carboplatin (75% reduction, P = 0.001), with no significant impact on the Sertoli cell population. Overall, results from this in vitro animal model study indicate that, at patient equivalent concentrations, carboplatin is no less gonadotoxic than cisplatin.

Insulin-like peptide 3 (INSL3) is a major regulator of female reproductive physiology.

Insulin-like peptide 3 (INSL3) is a member of the relaxin family of neohormones which has evolved to address specifically mammalian aspects of reproduction related to viviparity and internal fertilization. It was originally identified as a major product of testicular Leydig cells and has proved to be an important biomarker of Leydig cell functional capacity. However, INSL3 is also produced by theca interna cells of growing antral follicles and is secreted into the bloodstream in phases corresponding to the number and health of the follicles. Moreover, gene silencing experiments have shown that INSL3 is essentially required for androstenedione synthesis, which is the major steroid precursor for the granulosa cells of antral follicles to produce oestrogens. Knockout studies in mice confirm that loss of INSL3 or its receptor in females leads to partial infertility, with reduced follicle numbers, ovulations and litter size. Circulating INSL3 concentration corresponds to the reproductive lifespan, beginning with puberty and declining at the menopause, and thus may contribute to the physiology of other organ systems, particularly those relevant for hormone replacement strategies. A literature review was carried out by exhaustive searching of literature databases (PubMed and Google Scholar) with the search terms INSL3, RLF, Ley-IL and RXFP2. We present the first comprehensive review of INSL3 and its specific receptor RXFP2, and their roles in the context of female reproductive physiology. Moreover, we highlight the potential involvement of INSL3 in female reproductive pathology, such as PCOS, its clinical application as a valuable biomarker of reproductive processes, and its potential for therapeutic interventions. In the female mammal, INSL3 is largely produced by the theca interna cells of growing antral follicles during the follicular phase of the menstrual (oestrous) cycle. Within the follicle, INSL3 acts via its G-protein-coupled receptor, RXFP2, in an autocrine/paracrine manner to orchestrate and drive the production of the major steroid precursor androstenedione and its conversion by granulosa cells into oestrogens. These in turn create a positive feedback loop promoting the expression of more theca cell INSL3. This is countered by the follicular production of bone morphogenetic proteins and by the LH surge. Thus, the activity of the theca cell INSL3-RXFP2 system effectively determines the production of estradiol within an antral follicle through the follicular phase. INSL3 is also secreted into the circulation where it acts as a valuable biomarker to monitor the growth of antral follicles; it is consequently increased in PCOS and decreased in women with premature ovarian failure (POF). As an endocrine factor, INSL3 may also influence bone metabolism and kidney function. Additionally, INSL3 or its analogues may prove valuable as an adjunct in hormone replacement therapy or to monitor or influence IVF protocols. The INSL3-RXFP2 system represents a new regulatory pathway essential for the proper functioning of growing antral follicles. We still know very little about its involvement in pathologies such as PCOS or POF, and its role as a new biomarker of female function needs to be explored more widely to improve diagnosis and treatment of ovarian dysfunction. We need to examine how INSL3 might be used to improve IVF protocols and outcomes. Opportunities should also be investigated in regard to the systemic application of INSL3 as a rejuvenant therapy, with positive effects on bone or kidney function, and possibly also for fertility regulation. Most research to date has involved animal models; this now needs to be extended to include more human studies.

Basonuclin 1 deficiency is a cause of primary ovarian insufficiency.

Primary ovarian insufficiency (POI) leads to infertility and premature menopause in young women. The genetic etiology of this disorder remains unknown in most patients. Using whole exome sequencing of a large Chinese POI pedigree, we identified a heterozygous 5bp deletion inducing a frameshift in BNC1, which is predicted to result in a non-sense-mediated decay or a truncated BNC1 protein. Sanger sequencing identified another BNC1 missense mutation in 4 of 82 idiopathic patients with POI, and the mutation was absent in 332 healthy controls. Transfection of recombinant plasmids with the frameshift mutant and separately with the missense mutant in HEK293T cells led to abnormal nuclear localization. Knockdown of BNC1 was found to reduce BMP15 and p-AKT levels and to inhibit meiosis in oocytes. A female mouse model of the human Bnc1 frameshift mutation exhibited infertility, significantly increased serum follicle-stimulating hormone, decreased ovary size and reduced follicle numbers, consistent with POI. We report haploinsufficiency of BNC1 as an etiology of human autosomal dominant POI.

Vitamin D Receptor TaqI Polymorphism Is Associated With Reduced Follicle Number in Women Utilizing Assisted Reproductive Technologies.

Calcitriol, or 1,25-hydroxycholecalciferol, is the active form of vitamin D. It binds and activates vitamin D receptor (VDR). Infertility and defective folliculogenesis have been observed in female vdr-knockout mice; however, whether VDR polymorphisms affect human ovarian responses to controlled ovarian stimulation (COS) remains unclear. We hypothesized that VDR polymorphisms are associated with infertility and COS responses. Thus, we evaluated the association between the TaqI, BsmI, and FokI VDR polymorphisms and ovarian responses in women undergoing COS. In this study, we recruited a control group (n = 121) comprising volunteers with a history of natural conception and a second group of women undergoing COS (n = 70). TaqI, BsmI, and FokI genotyping was performed via restriction fragment length polymorphism analysis or TaqMan qPCR and Sanger sequencing. Intrafollicular 25(OH)D contents were measured in follicular fluid collected from COS patients during oocyte retrieval. Ovarian response parameters were obtained from patient medical records. There were no significant differences in the genotype frequencies of VDR polymorphisms (TaqI, BsmI and FokI) between the control and COS groups. However, the allele frequency of TaqI (C allele) was significantly lower in the COS group than in the control group (p = 0.02). Follicle number but not oocyte number was lower in patients with TaqI polymorphic (TC/CC) genotypes (p = 0.03). Importantly, the ratio between the number of follicles retrieved and intrafollicular estradiol concentrations was higher in patients with the TC/CC TaqI genotypes (p < 0.02). We identified an association between the VDR TaqI polymorphism and reduced follicle number in women undergoing COS, suggesting that VDR signaling affects the ovarian response to stimulation via unknown mechanisms.

Stromal Senp1 promotes mouse early folliculogenesis by regulating BMP4 expression

BackgroundMammalian folliculogenesis, maturation of the ovarian follicles, require both growth factors derived from oocyte and surrounding cells, including stromal cells. However, the mechanism by which stromal cells and derived factors regulate oocyte development remains unclear.ResultsWe observed that SENP1, a small ubiquitin-related modifier (SUMO)-specific isopeptidase, was expressed in sm22α-positive stromal cells of mouse ovary. The sm22α-positive stromal cells tightly associated with follicle maturation. By using the sm22α-specific Cre system, we show that mice with a stromal cell-specific deletion of SENP1 exhibit attenuated stroma-follicle association, delayed oocyte growth and follicle maturation with reduced follicle number and size at early oocyte development, leading to premature ovarian failure at late stages of ovulating life. Mechanistic studies suggest that stromal SENP1 deficiency induces down-regulation of BMP4 in stromal cells concomitant with decreased expression of BMP4 receptor BMPR1b and BMPR2 on oocytes.ConclusionsOur data support that protein SUMOylation-regulating enzyme SENP1 plays a critical role in early ovarian follicle development by regulating gene expression of BMP4 in stroma and stroma-oocyte communication.

Analgesic exposure in pregnant rats affects fetal germ cell development with inter-generational reproductive consequences.

Analgesics which affect prostaglandin (PG) pathways are used by most pregnant women. As germ cells (GC) undergo developmental and epigenetic changes in fetal life and are PG targets, we investigated if exposure of pregnant rats to analgesics (indomethacin or acetaminophen) affected GC development and reproductive function in resulting offspring (F1) or in the F2 generation. Exposure to either analgesic reduced F1 fetal GC number in both sexes and altered the tempo of fetal GC development sex-dependently, with delayed meiotic entry in oogonia but accelerated GC differentiation in males. These effects persisted in adult F1 females as reduced ovarian and litter size, whereas F1 males recovered normal GC numbers and fertility by adulthood. F2 offspring deriving from an analgesic-exposed F1 parent also exhibited sex-specific changes. F2 males exhibited normal reproductive development whereas F2 females had smaller ovaries and reduced follicle numbers during puberty/adulthood; as similar changes were found for F2 offspring of analgesic-exposed F1 fathers or mothers, we interpret this as potentially indicating an analgesic-induced change to GC in F1. Assuming our results are translatable to humans, they raise concerns that analgesic use in pregnancy could potentially affect fertility of resulting daughters and grand-daughters.

Maternal Smoke Exposure Impairs the Long-Term Fertility of Female Offspring in a Murine Model.

The theory of fetal origins of adult disease was first proposed in 1989, and in the decades since, a wide range of other diseases from obesity to asthma have been found to originate in early development. Because mammalian oocyte development begins in fetal life it has been suggested that environmental and lifestyle factors of the mother could directly impact the fertility of subsequent generations. Cigarette smoke is a known ovotoxicant in active smokers, yet disturbingly 13% of Australian and 12% of US women continue to smoke throughout pregnancy. The focus of our investigation was to characterize the adverse effects of smoking on ovary and oocyte quality in female offspring exposed in utero. Pregnant mice were nasally exposed to cigarette smoke for 12 wk throughout pregnancy/lactation, and ovary and oocyte quality of the F1 (maternal smoke exposed) generation was examined. Neonatal ovaries displayed abnormal somatic cell proliferation and increased apoptosis, leading to a reduction in follicle numbers. Further investigation found that altered somatic cell proliferation and reduced follicle number continued into adulthood; however, apoptosis did not. This reduction in follicles resulted in decreased oocyte numbers, with these oocytes found to have elevated levels of oxidative stress, altered metaphase II spindle, and reduced sperm-egg interaction. These ovarian and oocyte changes ultimately lead to subfertility, with maternal smoke-exposed animals having smaller litters and also taking longer to conceive. In conclusion, our results demonstrate that in utero and lactational exposure to cigarette smoke can have long-lasting effects on the fertility of the next generation of females.

Mate preference for dominant vs. subordinate males in young female Syrian hamsters (Mesocricetus auratus) following chemically-accelerated ovarian follicle depletion

Life history theory predicts that selectivity for mates generally declines as females age. We previously demonstrated this phenomenon in Syrian hamsters (Mesocricetus auratus), in that older females showed reduced preference for dominant over subordinate males. To test the hypothesis that decreased reproductive quality due to aging reduces mate preference, we decoupled reproductive and chronological age by treating young female hamsters with 4-vinylcyclohexene diepoxide (VCD), which destroys ovarian follicles and functionally accelerates ovarian follicle depletion without compromising the general health of rodents. In this study, VCD effectively reduced follicle numbers in young Syrian hamsters. VCD-treated and control females were allowed to choose between a dominant and a subordinate male in a Y-maze on the day of proestrus. Both VCD-treated and control females demonstrated preference for the dominant male by leaving a greater proportion of vaginal scent marks near him, which is a behavior that females display when soliciting prospective mates. However, there was no effect of treatment on the proportion of vaginal scent marks left for the dominant male. Furthermore, ovarian follicle numbers were not significantly correlated with any behaviors in either group. We conclude that accelerated ovarian follicle depletion does not reduce mate preference in young female hamsters.

Involvement of a volatile metabolite during phosphoramide mustard-induced ovotoxicity

The finite ovarian follicle reserve can be negatively impacted by exposure to chemicals including the anti-neoplastic agent, cyclophosphamide (CPA). CPA requires bioactivation to phosphoramide mustard (PM) to elicit its therapeutic effects however; in addition to being the tumor-targeting metabolite, PM is also ovotoxic. In addition, PM can break down to a cytotoxic, volatile metabolite, chloroethylaziridine (CEZ). The aim of this study was initially to characterize PM-induced ovotoxicity in growing follicles. Using PND4 Fisher 344 rats, ovaries were cultured for 4days before being exposed once to PM (10 or 30μM). Following eight additional days in culture, relative to control (1% DMSO), PM had no impact on primordial, small primary or large primary follicle number, but both PM concentrations induced secondary follicle depletion (P<0.05). Interestingly, a reduction in follicle number in the control-treated ovaries was observed. Thus, the involvement of a volatile, cytotoxic PM metabolite (VC) in PM-induced ovotoxicity was explored in cultured rat ovaries, with control ovaries physically separated from PM-treated ovaries during culture. Direct PM (60μM) exposure destroyed all stage follicles after 4days (P<0.05). VC from nearby wells depleted primordial follicles after 4days (P<0.05), temporarily reduced secondary follicle number after 2days, and did not impact other stage follicles at any other time point. VC was determined to spontaneously liberate from PM, which could contribute to degradation of PM during storage. Taken together, this study demonstrates that PM and VC are ovotoxicants, with different follicular targets, and that the VC may be a major player during PM-induced ovotoxicity observed in cancer survivors.

The effect of Vitamin C on amodiaquine‐induced oxidative stress in the ovary of Sprague‐Dawley rats

AQ‐induced oxidative stress leads to increased atresia which in turn leads to reduced follicle numbers, and reduced oocyte numbers at ovulation. This research study the effect of the co‐administration of amodiaquine hydrochloride (AQ.HCl) with Vitamin C on oestrous cycle, ovulation, oxidative stress markers and the histomorphology of the ovary of mature Sprague‐Dawley rats.Two study experimental groups were observed, Group 1: the effect of the co‐administration of AQ.HCl with Vitamin C on pattern and length of the oestrous cycle, histomorphology of the ovary and antioxidant activities of superoxide dismutase (SOD) and catalase (CAT). Group 2: the effect of the co‐administration of a single dose of AQ.HCl with Vitamin C administered in the evening of proestrus on ovulation and serum concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin (PRL).Co‐administration of AQ.HCl with Vitamin C resulted in an irregularity in cycle pattern and also increased (p &lt; 0.05) the length of the oestrous cycle by prolonging the diestrus phase compared to the control. Histological sections showed increased number of atretic follicles compared to the control. A statistical significant decrease (p &lt; 0.05) was observed in the activities of CAT and SOD. Serum levels of FSH, LH and PRL were comparable to control values however; the number of ova shed in the oviduct was significantly reduced (p &lt; 0.05). The result of this study showed that Vitamin C could not confer protection against the deleterious effect of AQ.HCl on the ovary of Sprague‐Dawley rats.

Effects of irrigation treatment and Azospirillum inoculation on yield and yield component of black cumin (Nigella sativa L.)

The aim of this study was to investigate the effects of terminating irrigation at different developmental stages and Azospirillum inoculation on quantitative yield of black cumin (Nigella sativa L.). The experiment was conducted during the growing season of 2011 at the Experimental Station of the Research Institute of Forest and Rangeland, Damavand, Iran. Treatments consisted of irrigation with three levels (W1 = normal irrigation from emergence to harvest (control), W2 = irrigation terminated at the start of budding and W3 = irrigation terminated at the start of flowering) and Azospirillum inoculation at four levels (A1 = non-inoculated, A2 = seed inoculated and A3 = spraying on the plant base at stem elongation stage, A4 = seed inoculated + spraying on the plant base at stem elongation stage). The present results have shown that irrigation terminated at the start of budding caused significant reduction in number of follicles per plant, biomass production and seed yield. Azospirillum had positive effects in all measured traits, especially when it used at two times (at seed inoculated + spraying on the plant base at stem elongation stage or A4 treatment).