Fertility and Sterility

Intrabursal injection of vascular endothelial growth factor trap in eCG-treated prepubertal rats inhibits proliferation and increases apoptosis of follicular cells involving the PI3K/AKT signaling pathway

Dalhia Abramovich, Griselda Irusta, Fernanda Parborell, Marta Tesone — 2009-03-27

Objective: To investigate the effects of local inhibition of vascular endothelial growth factor A (VEGFA) on proliferation and apoptosis of follicular cells in rat ovaries. To analyze the role of the PI3K/AKT signaling pathway on VEGFA effects.Design: Experimental study.Setting: Research laboratory.Animal(s): Female Sprague Dawley rats, 21 days old, treated with equine chorionic gonadotropin (eCG).Main Outcome Measure(s): Follicular cell proliferation, apoptosis, and activation of the PI3K/AKT signaling pathway after intrabursal injection of a VEGFA inhibitor.Result(s): Inhibition of VEGFA leads to a decrease in the expression of the proliferation marker proliferating cell nuclear antigen (PCNA) in theca and granulosa cells (GC) and an increase in the activation of caspase 3 in antral follicles. Furthermore, we observed a decrease in the phosphorylation of RAC-alpha serine/threonine-protein kinase (AKT) and its target Bcl2 antagonist of cell death (BAD). No differences were found in the levels of kinase insert domain receptor (KDR) protein or in endothelial cell density.Conclusion(s): The VEGFA prevents apoptosis and stimulates proliferation of follicular cells, regulating follicular growth and development. The PI3K/AKT signaling pathway is one of the pathways involved in this mechanism. Therefore, VEGFA has a role as an antiapoptotic and proliferative factor in follicular cells from the rat ovary.

The CB2 cannabinoid receptor regulates human sperm cell motility

2009-03-27

Objective: To analyze the expression and distribution of cannabinoid receptors in human sperm cells and evaluate the effects of activation of receptors by specific agonists and antagonists, with a special emphasis on the CB2 receptor.Design: We performed expression assays for CB1 and CB2 by reverse transcriptase PCR, Western blot, and immunofluorescence techniques in spermatozoa and performed motility analysis after incubation of semen samples with cannabinoid agonists and CB2 antagonist SR144528.Setting: Academic research laboratory.Patient(s): Semen from 50 normozoospermic, healthy human donors.Intervention(s): Spermatozoa isolated from semen by two consecutive swim-ups were used for all techniques.Main Outcome Measure(s): Reverse transcriptase PCR amplification gels, immunoblots, indirect immunofluorescence antibody assays, and percentage of motile sperm.Result(s): We have verified the presence of CB1 and CB2 receptors in human spermatozoa. The distribution of both of these receptors was distinct. Incubation with selective cannabinoid receptor agonists induced a significant reduction in the proportion of rapidly progressive motile spermatozoa, and whereas the CB1 agonist increased the proportion of immobile sperm cells, the CB2 receptor agonist increased the slow/sluggish progressive sperm cell population. The effect of the CB2 agonist was antagonized by the CB2-specific antagonist.Conclusion(s): The functional CB2 cannabinoid receptor is present in human spermatozoa and regulates the sperm motility in a more distinct manner than CB1.

Preventive role of exogenous testosterone on cisplatin-induced gonadal toxicity: an experimental placebo-controlled prospective trial

2009-04-10

Objective: To test the preventive role of exogenous T on spermatogenesis after cisplatin chemotherapy.Design: Placebo-controlled study.Setting: The animal laboratory of a medical university.Animal(s): Eighty-eight male BALB/c mice were divided into three groups; each group was subdivided into four groups.Intervention(s): Subgroups a received two or three cycles of cisplatin (2.5 mg/kg for 5 days + 16 days of recovery), subgroups b received the same chemotherapy regimen with adjuvant high-dose T enanthate (5 mg/100 g body weight) starting 1 week before chemotherapy and repeated every 21 days during chemotherapy, subgroups c received only high-dose T enanthate at the same dosage and intervals; subgroups d received a placebo.Main Outcome Measure(s): Testis spermatogenesis function was evaluated after 35 days (short term, group I) or 105 days (long term, groups II and III) of recovery, after the final dose of cisplatin, by histopathology and sperm count.Result(s): Testis tissue destruction and a significant dose-dependent decrease in spermatogenesis were identified in subgroups a. Both recovered partially during long-term recovery. Exogenous high-dose T caused damage to spermatogenesis, which was reversible (subgroups c). Adjuvant treatment with T had no additive long-term effect in animals treated with low-dose cisplatin (two cycles). However, a significant long-term preventive effect of T was seen in animals receiving high-dose cisplatin (three cycles).Conclusion(s): Hormonal intervention with exogenous T during chemotherapy had promising effects on spermatogenesis in mice receiving high-dose chemotherapy (regimens frequently used clinically). It had no additive long-term effects in animals receiving low-dose regimens.

Brain-derived neurotrophic factor is a regulator of human oocyte maturation and early embryo development

Richard A. Anderson, Rosemary A.L. Bayne, John Gardner, Paul A. De Sousa — 2009-05-21

Objective: To investigate a role for brain-derived neurotrophic factor (BDNF) in human oocyte maturation.Design: Prospective study.Setting: Research institute.Patients: Women undergoing laparoscopic sterilization.Intervention(s): Small antral follicle cumulus–oocyte complexes (COCs) were matured in vitro (IVM) to metaphase II (MII) in media with hormones (H; FSH, LH, E2), serum replacement (SR), BDNF, or blocking antibodies to BDNF (BDNF/AB and TrkB/Fc), and activated.Main Outcome Measure(s): The COCs were analyzed for expression of neurotrophin ligands/receptors and cumulus genes (HAS2, TNFAlP6, PTGS2, GREM1) by reverse transcription–polymerase chain reaction (RT-PCR), cumulus expansion, maturation to MII, and parthenogenetic embryo development.Result(s): The BDNF and truncated TrkB receptor were expressed in cumulus and mature oocytes. There was no difference in MII yields after IVM in control (H + SR) versus H + BDNF, H + SR + BDNF, or BDNF + SR media. However, both BDNF/AB and TrkB/Fc improved MII yields. After activation, normal cleavage was highest in H + SR (38%), whereas blocking antibodies yielded the highest abnormal cleavage (BDNF/AB 68%; TrkB/Fc 57%). Failure to cleave was highest in H + BDNF + SR (92%). Only H + SR yielded morulae/blastocysts (6%). Expression of GREM1 in cumulus increased after IVM in H + BDNF versus H + SR or in vivo maturation.Conclusion(s): The BDNF signaling within COCs influences oocyte maturation and early embryogenesis.

Testis atrophy and reduced sperm motility in transgenic mice overexpressing c-FLIPL

2009-03-16

Objective: To study the effect of c-FLIP overexpression in testicular germ cells.Design: A novel transgenic mouse model overexpressing the apoptotic modulator c-FLIP in the testis was generated.Setting: Animal facility and university research laboratory.Animal(s): Transgenic mice overexpressing the long isoform of c-FLIP (c-FLIPL) under the transcriptional control of a 400 bp long regulatory region of the Stra8 promoter.Intervention(s): Spermatozoa motility and testis histological, immunohistochemical, and Western blot analyses were carried out in transgenic and control derived specimens.Main Outcome Measure(s): Testis morphology, sperm motility, and germ cell apoptosis were assayed.Results: Stra8 promoter was found to activate the ectopic overexpression of c-FLIPL in round and elongated spermatids. As a consequence of such overexpression, a dramatic loss of germ cells was observed, resulting in testicular atrophy associated with reduced sperm motility.Conclusion(s): The data show that c-FLIPL forced expression in haploid male germ cells has detrimental effects on spermatogenesis and sperm quality and reveal a possible mechanism underlying the onset of testicular atrophy.

Changing P2X receptor localization on maturing sperm in the epididymides of mice, hamsters, rats, and humans: a preliminary study

Frederick C.L. Banks, Robert C. Calvert, Geoffrey Burnstock — 2009-04-01

Objective: To study using immunohistochemistry the localization of P2X receptor subtypes on the head of immature sperm in the human, mouse, hamster, and rat caput epididymidis.Design: Basic research.Setting: University-based hospital.Patient(s): Three human epididymides were obtained from patients undergoing orchidectomy for metastatic prostate cancer.Main Outcome Measure(s): P2X1, P2X2, P2X3, and P2X4 receptor immunolocalization on sperm.Result(s): In the present study, P2X1,2, and 3 receptor localization was immunohistochemically demonstrated on the head of immature sperm in the human, mouse, hamster, and rat caput epididymidis. P2X4 receptor immunostaining was also observed on the head of sperm in the caput epididymidis of mice, hamsters, and humans, but not rats. There was a subsequent loss of receptor staining on sperm in the cauda epididymidis, except in humans where staining of P2X4 receptors persisted. Comparision with peanut agglutinin (PNA) binding studies suggested the P2X receptors were located on the acrosome membrane. P2X5–7 receptors were examined but found to be absent.Conclusion(s): The change in localization of receptor subtypes is coincidental with the functionally essential morphologic and maturational changes seen in sperm as they travel through the epididymis, and is suggestive of a role for purinergic signaling in sperm maturation and possibly fertility.

Expression of the insulin-like growth factor and insulin systems in the luteinizing macaque ovarian follicle

2009-02-24

Objective: To determine intrafollicular hormone levels and characterize the mRNA expression of the insulin-like growth factor (IGF) receptors, IGF binding proteins (IGFBP), and pregnancy-associated plasma protein-A (PAPP-A) in granulosa cells before and after an ovulatory hCG stimulus.Design: Experimental animal study.Setting: Academic medical center.Animal(s): Adult rhesus macaques.Intervention(s): Animals received exogenous FSH to promote the development of multiple preovulatory follicles. Follicles were aspirated before (0 hours) or 3, 6, 12, or 24 hours after an ovulatory hCG bolus.Main Outcome Measure(s): IGF1, IGF2, and insulin levels in follicular fluid were determined by radioimmunoassay. Messenger RNA (mRNA) levels in granulosa cells were determined by real-time reverse transcriptase–polymerase chain reaction. IGFBPs and PAPP-A in follicular fluid were determined by Western blot analysis and enzyme-linked immunosorbent assay.Result(s): IGF1, IGF2, and insulin in follicular fluid did not change during luteinization. IGF1R, IGFBP1, and IGFBP2 mRNAs were unchanged by hCG. IGF2R, IGFBP3, -5, and -6 and PAPP-A mRNA levels increased after hCG administration, while insulin receptor and IGFBP4 mRNA levels decreased after hCG administration. IGFBP3 and -6 and PAPP-A protein increased after hCG administration.Conclusion(s): Dynamic changes in the expression of the IGFBPs and PAPP-A suggest tight regulation of IGF action during ovulation and corpus luteum formation.

The oocyte spindle is preserved by 1,2-propanediol during slow freezing

2009-03-26

Objective: To investigate the specific changes in oocyte spindle subjected to severe challenges of low temperature, as well as to examine the effect of cryoprotectants in preserving oocyte spindle during cryopreservation.Design: In vitro experimental study.Setting: Academic research laboratory.Animal(s): B6D2F1 (C57BL/6 X DBA/2) mice.Intervention(s): Mouse oocytes were cryopreserved using a slow freezing method in a sodium-depleted medium with 1.5 mol/l 1,2-propanediol (PROH) and 0.3 M sucrose. To examine the spindle, oocytes were fixed before, during, and after cryopreservation, and oocytes were analyzed by immunocytochemistry and confocal microscopy.Result(s): The MII spindle was preserved during the slow freezing, because the cryoprotectant PROH was found to support the organization of MII spindle in resisting the subzero temperature. In contrast, the MII spindle was disassembled gradually during the thawing process with or without PROH. Most of the oocytes were able to recover the MII spindle after thawing, but a portion of thawed oocytes could not sustain the meiotic spindle because of parthenogenetic activation.Conclusion(s): 1,2-Propanediol can support the organization of MII spindle to defy the subphysiologic temperature; however, the PROH cannot sustain oocyte spindle structure after the subsequent thawing process.

15-Epi-lipoxin A4 inhibits the progression of endometriosis in a murine model

2009-03-06

Objective: To examine the pro-resolution actions of 15-epi-lipoxin A4 (LXA4) on endometriotic lesions, on the concentrations and activities of matrix metalloproteinases (MMP-2 and MMP-9) in murine endometriosis.Design: Prospective, vehicle-controlled experimental study.Setting: Animal research facility.Animal(s): BALB/c mice.Intervention(s): Endometriosis (EM) was induced in 30 mice. Fifteen of them were administered LXA4 for 24 days (LXA4 group), whereas the other 15 served as a control group (EM group). Another 15 sham-operated mice (sham-operated group) were treated with vehicles.Main Outcome Measure(s): The weight of the endometriotic lesions was measured. The concentrations, mRNA, and activities of MMP-2 and MMP-9 were determined by enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, and gelatin zymography, respectively.Result(s): Compared with EM group, the weight of the endometriotic lesions was decreased, the concentrations of MMP-2 and MMP-9 dropped, the mRNA levels of MMP-2 and MMP-9 in the peritoneal fluid cells and the endometriotic lesions were reduced, and the activities of MMP-2 and MMP-9 were inhibited in the LXA4 group.Conclusion(s): LXA4 may inhibit the progression of endometriosis possibly by lowering the concentrations and the activities of MMP-2 and MMP-9.

Change of proinflammatory cytokines follows certain patterns after induction of endometriosis in a mouse model

2009-04-01

Objective: To examine the change in proinflammatory cytokines in the pathologic processes of endometriosis in mice.Design: A dynamic study on a murine model of endometriosis.Setting: Medical school.Animal(s): Female BALB/c mice.Intervention(s): Endometriosis was induced by injecting endometrial fragments of syngenic mice into the peritoneal cavity of model mice; in control group, phosphate-buffered saline instead of fragments was injected. The peritoneal fluid and the endometriotic lesions were harvested 1 to 21 days after the induction.Main Outcome Measure(s): The endometriotic lesions were weighed, the gene and protein levels of some proinflammatory cytokines, including interleukin 1β, tumor necrosis factor α, vascular endothelial growth factor, and monocyte chemoattractant protein 1, were determined by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively.Result(s): The levels of these cytokines reached the first peak on the first day and no endometriotic lesions were found. The lesions began to appear on the second day, presenting red color during the initial 6 days, and then they turned dark-red, brown, or bluish. The adhesion took place on the 9th day, and all the lesions evolved into white or transparent cysts on the 15th day. Corresponding to these changes, the second and the third peaks were identified during the 3rd–6th day and the 12th-15th day, respectively.Conclusion(s): The change pattern of cytokines over time might bear some relationship with the development and progression of the endometriosis.

Attenuation of ischemia–reperfusion injury with Marrubium cordatum treatment in ovarian torsion–detorsion model in rabbits

Yilmaz Cigremis, Asim Kart, Musa Karaman, Dincer Erdag — 2009-04-27

Objective: To investigate protective effects of Marrubium cordatum extract on ovary torsion–detorsion.Design: Controlled research study.Setting: Marrubium cordatum extract was obtained by methanol extraction.Animal(s): Six-month-old female New Zealand rabbits.Intervention(s): In the first phase, antioxidant activity of M. cordatum extract was evaluated. In the second phase, M. cordatum extract at doses of 0, 250, 500, and 1,000 mg/kg was studied for dose determination. In the third phase, the protective role of M. cordatum on ovarian torsion–detorsion injury was evaluated in sham control, torsion–detorsion, torsion–detorsion + M. cordatum (1,000 mg/kg).Main Outcome Measure(s): 1,1-Diphenyl-2-picrylhydrazyl, nitric oxide radical scavenging activity, reducing power capacity, and total phenolic compounds were assayed. Glutathione, malondialdehyde, catalase, and glutathione peroxidase were measured. Histopathological examination was also conducted.Result(s): Marrubium cordatum significantly inhibited 1,1-diphenyl-2-picrylhydrazyl, nitric oxide radicals, and showed a powerful reducing activity. Marrubium cordatum did not adversely affect biochemical and histopathological parameters at all doses. Malondialdehyde level and catalase activity in the torsion–detorsion group were significantly increased compared with those of the sham group, whereas the glutathione level and glutathione peroxidase activity were significantly decreased compared with those of the sham group. Marrubium cordatum treatment significantly lowered the malondialdehyde level and catalase activity but increased the glutathione level in torsion–detorsion injury. Histopathologically, severe congestion, hemorrhage, edema, and leukocyte infiltration were observed in the torsion–detorsion group. Marrubium cordatum treatment ameliorated these alterations.Conclusion(s): Marrubium cordatum attenuates ischemia–reperfusion-induced biochemical and histopathological alterations.

A differential cytokine expression profile is induced by highly purified human menopausal gonadotropin and recombinant follicle-stimulating hormone in a pre- and postovulatory mouse follicle culture model

2009-04-10

Objective: To compare the differential effects of highly purified (HP) hMG or recombinant FSH (rFSH) on cytokine expression before and after ovulation in an in vitro mouse ovarian follicle model.Design: A prospective laboratory in vitro study.Setting: A university-based reproductive biology laboratory.Material(s): Mechanically isolated mouse preantral follicles from 14-day-old prepubertal mouse ovaries (F1 hybrids: C57BL/6JxCBA/ca).Intervention(s): Randomly distributed mouse early preantral follicles were exposed to two hyperstimulation conditions with either HP-hMG or rFSH. An ovulatory stimulus was given using hCG/epidermal growth factor. Conditioned media from the two culture conditions were collected on the days before and after in vitro ovulation. Conditioned media were compared for their relative cytokine profile content as measured by a cytokine antibody array analysis.Main Outcome Measure(s): Relative concentrations of 62 cytokines in conditioned media before and after ovulation.Result(s): Statistically significant increase in the production of a number of cytokines was found after HP-hMG stimulation compared with rFSH: 14 and 24 pre- and post-rhCG, respectively. Cytokines with the largest significant difference (more than 5 times) before and after ovulation included thymus-expressed cytokine (TECK), sTNFRI, and SDF-1α. The cytokines that are most strongly related to oocyte and embryo quality and implantation and that have been related to oocyte yield and maturation were significantly higher with HP-hMG.Conclusion(s): The significant differences in follicular cytokine production induced by HP-HMG and rFSH before and after in vitro ovulation might explain the difference in treatment outcome.

Analysis of spindle characteristics and embryo quality in mice stimulated with letrozole using Polscope imaging

2009-07-30

Objective: To explore the effect of letrozole as an ovulation inducing agent on oocyte and embryo quality in mice model.Design: Prospective study.Setting: Institute of Reproductive Medicine, Kolkata, India.Animal(s): Sixty-nine sexually mature female Swiss Albino mice 6–7 weeks old.Intervention(s): Metaphase II (MII) oocytes from two groups of mice, one group injected with letrozole and the other with rFSH.Main Outcome Measure(s): Number of MII oocytes, number of oocytes with meiotic spindle (MS), different angles of MS relative to the polar body (PB), spindle characteristics, and fertilization outcome.Result(s): The MS was present in 84% and 71.2% of the oocytes in the letrozole and rFSH group, respectively. In the letrozole group, 73% of the oocytes had a 0° spindle position compared with 35.7% in the rFSH group. With letrozole, 19% of the oocytes had 0° > MS < 90° and 8% had MS >90°, compared with 46.1% oocytes with 0° > MS < 90° and 18.2% with MS >90° with rFSH. Mean spindle area retardance, spindle dimensions, and 4–8-cell embryo formation rate were significantly higher with letrozole compared with rFSH. Two-cell and blastocyst formation rates were similar in both groups.Conclusion(s): Robust birefringent spindles were obtained in mice on superovulation with letrozole. Letrozole does not appear to increase the risk of spindle assembly and preimplantation developmental arrest in mouse oocytes.

Acrosome formation-associated factor is involved in fertilization

2009-03-16

Objective: To investigate the effects of a novel acrosome formation-associated factor (Afaf) on fertilization by its regulation of acrosomal exocytosis and endosomal trafficking.Design: Controlled laboratory study.Setting: Institution-affiliated state key laboratory.Subjects: ICR mice.Intervention(s): Sperm penetration assay and in vitro fertilization experiment were performed to study the effects of the Afaf antibody on acrosome reaction and fertilization. Acrosome exocytosis (AE) with streptolysin O (SLO) permeabilization was conducted to test the Afaf's action in calcium events. Colocalization and coimmunoprecipitation was done to determine the interaction between Afaf and SNAP25 (synaptosome-associated protein of 25,000 daltons). Transferrin (Tf) uptake assay was performed to demonstrate the impact of Afaf on endosomal pathway. RNAi was used to rescue the inhibition of Afaf on Tf uptake.Main Outcome Measure(s): Number of penetrated sperms, in vitro fertilization rate. Acrosomal exocytosis index, relative Tf fluorescence.Result(s): The Afaf antibodies were capable of significantly inhibiting sperm penetration of the eggs, therefore reducing the rate of in vitro fertilization. Acrosome formation-associated factor was involved in calcium-triggered AE by acting upstream of the calcium efflux from the acrosome inside. Acrosome formation-associated factor might exert an interaction with SNAP25, which is a crucial component in both exocytosis and endosomal trafficking. Acrosome formation-associated factor was also involved in the endocytic pathway by down-regulating Tf endocytosis in the HeLa cells, and the miRNA-mediated RNAi could rescue this alternation induced by Afaf.Conclusion(s): Acrosome formation-associated factor might play an important role in membrane trafficking during acrosome formation and participate in fertilization.

Changes in circulating levels and ratios of angiopoietins during pregnancy but not during the menstrual cycle and controlled ovarian stimulation

2009-05-25

Objective: To determine whether angiopoietin (ANGPT)-1 and -2 are detectable in the circulation of nonhuman primates and women and whether these levels fluctuate in association with ovarian activity.Design: Prospective.Setting: National Primate Research Center, medical center, and infertility clinic.Patient(s): Adult female rhesus monkeys; 15 women donating oocytes for infertility treatment.Intervention(s): Controlled ovarian stimulation with gonadotropins, removal of the corpus luteum and ovaries, oocyte retrieval, and ET.Main Outcome Measure(s): Circulating levels of ANGPT-1 and ANGPT-2.Result(s): Serum ANGPT-1 and ANGPT-2 levels were detectable and invariant in maintaining an ANGPT-1 to -2 ratio >1 in [1] macaques over the course of the natural menstrual cycle, during a controlled ovulation protocol, and after removal of the corpus luteum or ovaries and [2] women undergoing controlled ovarian simulation. In contrast, the ANGPT-1 to -2 ratio was markedly decreased (<<1) at mid-to-late gestation in macaques and in the follicular fluid of women undergoing controlled ovarian simulation because of increased levels of ANGPT-2.Conclusion(s): The ovary and its dominant structures are not major contributors to circulating levels of ANGPT-1 or ANGPT-2. The physiologic importance of the rising levels of ANGPT-2 after the luteal-placental shift in pregnancy is unknown.

Myometrial cells undergo fibrotic transformation under the influence of transforming growth factor β-3

Doina S. Joseph, Minnie Malik, Sahadat Nurudeen, William H. Catherino — 2009-03-27

Objective: To examine the effect of transforming growth factor (TGF) β3 on immortalized myometrial and leiomyoma cell lines cloned from primary cell cultures of surgical specimens, and to determine whether such treatment alters myometrial cell extracellular matrix (ECM) expression.Design: Laboratory study.Setting: University hospital.Patient(s): Immortalized myometrial and leiomyoma cells from patients with symptomatic leiomyomata.Intervention(s): Tissue culture, followed by cellular, RNA, and protein analysis.Main Outcome Measure(s): Cell proliferation, alteration in ECM component expression.Result(s): Immortalized leiomyoma and myometrial cells demonstrate increased mRNA and protein production of the ECM proteins, collagen 1A1 (15.0-fold), fibronectin 1 (2.93 fold), and connective tissue growth factor (9.40-fold) with exogenous TGF-β3 stimulation. Notably, the expression of collagen 1A1, fibronectin 1, and connective tissue growth factor in myometrial cells increase to similar expression levels as those found in leiomyoma cells. In addition, TGF-β3 decreased production of genes involved in matrix resorption, including matrix metalloproteinase 2 (0.65-fold) and -11 (0.68-fold).Conclusion(s): TGF-β3 induced a molecular phenotype in myometrial cells that was similar to leiomyoma cells, with elevated production of ECM-related genes and decreased production of ECM degradation–related genes.

The effect of hyperstimulation on transforming growth factor β1 and β2 in the rat uterus: possible consequences for embryo implantation

Aleksandra Jovanović, Beverley Kramer — 2009-02-06

Objective: To investigate the effect of exogenous gonadotropins on the expression of transforming growth factor (TGF) β1 and β2 in the rat uterus and its consequences for successful embryo implantation.Design: Controlled experimental research study.Setting: School of Anatomical Sciences, University of the Witwatersrand.Patient(s): Thirty-six adult, virgin, female Sprague-Dawley rats and two fertile males.Intervention(s): Follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG) were superimposed upon the normal hormonal milieu of normal, cycling female rats before mating. Uterine tissue was collected at peri-implantation stages (i.e., at 4.5, 5.5, and 6.5 days) after mating. Enzyme-linked immunosorbent assay (ELISA) was performed to estimate the blood estrogen and progesterone levels, and immunohistochemistry was performed to localize the TGF-β1 and TGF-β2 in the uterus.Main Outcome Measure(s): Hyperstimulation affecting the expression of both TGF-β1 and TGF-β2 during the peri-implantation period.Result(s): The release of ovarian steroids was altered, causing a change in the endogenous hormonal environment. A marked increase in the expression of TGF-β2 was distinct in the glandular epithelium. Expression of both TGF-β1 and TGF-β2 was weaker in the subluminal stroma when compared with the deeper stromal region.Conclusion(s): Hyperstimulation with exogenous hormones affects the expression of both TGF-β1 and TGF-β2, which may contribute to the disruption of the endometrial environment required for successful embryo implantation.

Mouse blastocyst previtrification interventions and DNA integrity

Amr Kader, Rakesh K. Sharma, Tommaso Falcone, Ashok Agarwal — 2009-04-01

Objective: To assess the effect of implementing different previtrification interventions on the postwarming DNA integrity of vitrified blastocysts.Design: Prospective in vitro study.Setting: Center for Reproductive Medicine laboratory in a tertiary hospital setting.Animals: A total of 70 expanded and 46 nonexpanded mouse blastocysts were used for the study.Intervention(s): [1] Twenty-two expanded blastocysts were blastocele aspirated and immediately vitrified. [2] Twelve expanded blastocysts were spontaneously hatched before vitrification. [3] Twenty-two expanded blastocysts were vitrified without intervention. [4] Sixteen nonexpanded blastocysts underwent assisted hatching using acidified tyrode's solution. [5] Seventeen nonexpanded blastocysts were vitrified without intervention. [6] Thirteen nonexpanded blastocysts and 14 expanded were used as fresh controls. Vitrification was done using a cryotip loading device.Main Outcome Measure(s): DNA integrity index using terminal deoxynucleotide transferase [TdT]-mediated dUTP-digoxigenin nick-end labeling staining and confocal imaging.Result(s): [1] Intervention by blastocele aspiration for expanded blastocysts or assisted hatching of nonexpanded blastocysts significantly improved the postwarming results in each blastocyst stage. [2] Allowing spontaneous hatching before vitrification is a noninvasive technique that can improve the postwarming integrity of expanded blastocysts similar to blastocele aspiration.Conclusion(s): Blastocele aspiration or spontaneous hatching of expanded blastocysts and assisted hatching of nonexpanded blastocysts before vitrification helps minimize blastomere DNA damage.

The acrosomal protein Dickkopf-like 1 (DKKL1) is not essential for fertility

Kotaro J. Kaneko, Matthew J. Kohn, Chengyu Liu, Melvin L. DePamphilis — 2009-07-10

Objective: To determine the role of Dkkl1 on mouse development, viability, and fertility.Design: Prospective experimental study.Setting: Government research institution.Animal(s): Mice of C57BL/6 and 129X1/SvJ strains, as well as transgenic mice of mixed C57BL/6 and 129X1/SvJ strains were used for the studies.Intervention(s): Mice were constructed that lacked a functional Dkkl1 gene.Main Outcome Measure(s): Deletion of the gene was confirmed by DNA, RNA, and protein analyses; in vivo fertility was examined by continuous mating scheme.Result(s): Previous studies have shown that Dkkl1, a gene unique to mammals, is expressed predominantly, if not exclusively, in developing spermatocytes, and the DKKL1 protein accumulates in the acrosome of mature sperm. Subsequent studies (reported in the accompanying article) demonstrate that Dkkl1 also is expressed in the trophectoderm/placental lineage. Taken together, these results strongly suggested that DKKL1 protein is required for terminal differentiation either of trophoblast giant cells or of sperm, both of which are directly involved in fertility. To challenge this hypothesis, conditional targeted mutagenesis was used to ablate the Dkkl1 gene in mice. Surprisingly, Dkkl1 nullizygous embryos developed into viable, fertile adults, despite the fact that they failed to produce any portion of the DKKL1 protein.Conclusion(s): DKKL1 is a mammalian-specific acrosomal protein that is not essential either for development or fertility.

The acrosomal protein Dickkopf-like 1 (DKKL1) facilitates sperm penetration of the zona pellucida

2009-07-10

Objective: To determine the role of Dkkl1 in mouse development, viability, and fertility.Design: Prospective experimental study.Setting: Government research institution.Animal(s): Mice of C57BL/6, B6D2F1/J, and 129X1/SvJ strains, as well as transgenic mice of mixed C57BL/6 and 129X1/SvJ strains were used for the studies.Intervention(s): Expression of the Dkkl1 gene was characterized during early mouse development, and the effects of Dkkl1 ablation on reproduction and fertility were characterized in vitro and in vivo.Main Outcome Measure(s): Dkkl1 RNA expression was determined by Northern blotting hybridization as well as quantitative reverse transcriptase-polymerase chain reaction assays. In vitro fertilization assays were used to assess fertility of sperm from male mice lacking functional Dkkl1.Result(s): Dkkl1 is a gene unique to mammals that is expressed primarily in developing spermatocytes and its product localized in the acrosome of mature sperm. Here we show that Dkkl1 also is expressed in the trophectoderm/placental lineage. Surprisingly, embryos lacking DKKL1 protein developed into viable, fertile adults. Nevertheless, the ability of sperm that lacked DKKL1 protein to fertilize wild-type eggs was severely compromised in vitro. Because this defect could be overcome either by removal of the zona pellucida or by the presence of wild-type sperm, Dkkl1, either directly or indirectly, facilitates the ability of sperm to penetrate the zona pellucida. Penetration of the zona pellucida by Dkkl1− sperm was delayed in vivo as well as in vitro, but the delay in vivo was compensated by other factors during preimplantation development. Accordingly, Dkkl1−/− males offer an in vitro fertilization model for identifying factors that may contribute to infertility.Conclusion(s): DKKL1 is a mammalian-specific, acrosomal protein that strongly affects in vitro fertilization, although the effect is attenuated in vivo.

Expression and regulation of cholesterol sulfotransferase (SULT2B1b) in human endometrium

2009-02-24

Objective: To investigate the hormonal regulation of SULT2B1b in human endometrium.Design: In vitro study with human endometrial tissues and cultured human endometrial cells.Setting: University hospital.Patient(s): Thirty-seven women undergoing hysterectomy for benign disease.Intervention(s): Human endometrial tissues were collected for in situ hybridization. Culture medium of human endometrial epithelial cells (EECs) was collected for determination of secretion of cholesterol sulfate (CS). Total RNAs were extracted from human endometrial tissues and cultured cells for real-time reverse transcriptase–polymerase chain reaction (RT-PCR).Main Outcome Measure(s): The expression of SULT2B1b mRNA in human endometrial tissues and cultured cells.Result(s): In situ hybridization studies and real-time RT-PCR showed that the amount of SULT2B1b mRNA in human endometrial tissues was significantly higher during the midluteal phase than during other phases of the menstrual cycle. The secretion of CS from EECs was confirmed using [35S]-phosphoadenosine phosphosulfate. The expression of SULT2B1b mRNA was induced by cAMP or P in human endometrial stromal cells (ESCs), whereas it was induced by cAMP or relaxin in EECs. The induction of SULT2B1b mRNA by P or relaxin was abolished by the specific protein kinase A (PKA) inhibitor, Rp-adenosine 3',5' cyclic monophosphothioate (Rp-cAMPS).Conclusion(s): The expression of SULT2B1b mRNA in ESCs is induced by P and that in EECs is induced by relaxin via the cAMP pathway.

Effect of melatonin on epididymal sperm quality after testicular ischemia/reperfusion in rats

Zehra Kurcer, Askin Hekimoglu, Faruk Aral, Fusun Baba, Engin Sahna — 2009-03-27

Objective: To determine the effect of melatonin, a pineal secretory product that prevents testicular ischemia/reperfusion (IR) injury through its antioxidative properties, on epididymal sperm quality in a rat testicular IR injury model.Design: Experimental study.Setting: University pharmacology laboratory.Animal(s): Fifty-six 8-week-old male Wistar albino rats.Intervention(s): Left testicular artery and vein occluded for 1 hour; before the bilateral orchiectomy, the organ was allowed to reperfuse 30 days. Melatonin (10 mg/kg IP) or vehicle (1% ethanol in saline) was administrated for 10 minutes before reperfusion and for 1 hour after reperfusion.Main Outcome Measure(s): After 24 hours of reperfusion, the rats were decapitated, and the testicular tissue samples were obtained for histologic examination. In addition, after 30 days of reperfusion, the epididymal sperm concentration, motility, and abnormal sperm rates were determined in the sperm collected from the epididymis.Result(s): A statistically significant decrease in sperm concentration resulted from IR as well as an increase in sperm abnormalities, but the sperm motility did not change. Melatonin treatment did not prevent the IR-induced reduction in sperm concentration. However, melatonin treatment statistically significantly decreased the sperm abnormalities when compared with the IR injured samples.Conclusion(s): Melatonin may improve sperm morphology for a protective effect in IR-induced testicular injury.

Changes in the distribution of mitochondria before and after in vitro maturation of human oocytes and the effect of in vitro maturation on mitochondria distribution

Shan Liu, Yuan Li, Xuan Gao, Jun-Hao Yan, Zi-Jiang Chen — 2009-05-06

Objective: To clarify the relationship between oocyte maturation and mitochondria distribution and assess the effects of in vitro maturation (IVM) on the distribution of mitochondria in human oocytes.Design: Prospective randomized trial.Setting: Hospital-based IVF center.Patient(s): One hundred fifty-eight patients undergoing intracytoplasmic sperm injection (ICSI) treatment for male factors or combined with oviduct infertility and fifteen patients undergoing controlled ovarian hyperstimulation followed by coitus or IUI.Intervention(s): Of all the 284 immature oocytes, 140 were fixed directly. The others were prepared for IVM before they were fixed. All the 21 oocytes matured in vivo were fixed directly and stained for mitochondria. Both immature and mature oocytes were stained by Mito Tracker Green FM. The distribution of mitochondria was observed using a confocal laser scanning microscope.Main Outcome Measure(s): Mitochondrial distribution.Result(s): Three mitochondria distribution patterns were identified: peripheral, semiperipheral, and evenly diffused. A peripheral distribution of mitochondria was presented by 64.1% (50/78) of the germinal vesicle (GV) oocytes; 45.2% (28/62) of the meiosis I oocytes maintained the peripheral distribution; and 38.7% (24/62) presented a diffused status. After IVM, 75.5% (80/106) of the oocytes displayed an evenly diffused type of distribution. The mitochondria were more abundant in the inner cytoplasm than in the peripheral region in most of the oocytes matured in vivo.Conclusion(s): There are obvious changes in the distribution of mitochondria in human oocytes before and after maturation. Distribution of mitochondria in oocytes matured in vitro is slightly different from that of oocytes matured in vivo. The results may partially explain the reduced developmental potential of oocytes matured in vitro compared with those matured in vivo.

Immunization with a DNA vaccine of testis-specific sodium-hydrogen exchanger by oral feeding or nasal instillation reduces fertility in female mice

2009-04-30

Objective: To investigate the effect of immunization with a DNA vaccine of testis-specific sodium-hydrogen exchanger (tsNHE) via oral feeding or nasal instillation on fertility in female mice and to look at its potential mechanism.Design: Prospective, research study.Setting: Institution-affiliated research laboratory.Animal(s): Sexual mature BALB/c mice.Intervention(s): Female mice immunized orally or nasally with the DNA vaccine at 2-week' intervals.Main Outcome Measure(s): Number of newborns and fertility rate of the vaccinated female mice were scored.Result(s): We identified a novel testis-specific sodium-hydrogen exchanger, tsNHE, which is localized to the principal piece of sperm flagellum. Immunization of female mice with the tsNHE DNA vaccine via oral feeding or nasal instillation statistically significantly decreased fertility rate and the newborn numbers compared with the controls. The antiserum or vaginal fluid from the tsNHE cDNA vaccinated female mice could specifically recognize the principal piece of sperm tail and triggered sperm agglutination. The antibodies also showed a statistically significant inhibitory effect on in vitro sperm motility and fertilization.Conclusion(s): The sodium-hydrogen exchanger might be an excellent target molecule for developing a new contraceptive.

Damaging effect of cumulus denudation on rabbit oocytes

2009-07-08

Objective: To study the effect of cumulus denudation on in vitro maturation of rabbit oocytes.Design: Experimental animal study.Setting: Academic institution.Animal(s): Rabbits and mice.Intervention(s): Rabbit oocytes were observed compared with mouse oocytes.Main Outcome Measure(s): Developmental competence, membrane integrity, and apoptotic status of oocytes after cumulus denudation.Result(s): Although in vitro maturation of mouse cumulus-denuded oocytes was unaffected, rabbit cumulus-denuded oocytes could not mature. However, 50% of rabbit cumulus-intact oocytes matured normally when their gap junctions were sealed with 1-heptanol. Coculture with cumulus cells did not improve maturation of rabbit cumulus-denuded oocytes unless with an intact corona radiata. Staining with Hoechst 33258, Bcl-2 antibodies, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling showed membrane breaches or apoptosis of rabbit cumulus-denuded oocytes, contrary to the mouse cumulus-denuded oocytes. Ultrastructurally, rabbit oocytes showed no perivitelline space but numerous long cell junctions projecting into the egg cortex, contrary to the mouse oocytes. However, the damaging effect of cumulus denudation was much relieved after preincubation of rabbit cumulus-intact oocytes with phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, and some cumulus-denuded oocytes prepared after preincubation matured and developed into blastocysts.Conclusion(s): [1] Cumulus denudation severely damaged rabbit oocytes leading to their apoptosis or degeneration, possibly because of the deep-set junctional complexes anchoring the oocyte and corona cells; and [2] preincubation with phosphodiesterase inhibitor may provide a method to avoid the damaging effect of cumulus denudation on rabbit oocytes.

Glucose-regulated protein 78 (Grp78/BiP) is secreted by human oviduct epithelial cells and the recombinant protein modulates sperm–zona pellucida binding

2009-03-17

Objective: To determine the secretion of Grp78 by human oviduct epithelial cells, its association to spermatozoa, and its involvement in gamete interaction.Design: Prospective study.Setting: Basic research laboratory.Subject(s): Semen samples obtained from normozoospermic volunteers. Tubal tissue provided by patients undergoing hysterectomies. Oocytes collected from women undergoing IVF-ET.Intervention(s): Analysis of Grp78 expression and secretion by oviductal tissue. Gamete incubation with recombinant Grp78 (rec-Grp78).Main Outcome Measure(s): Assessment of protein expression and secretion by immunohistochemistry and Western immunoblotting, respectively. Evaluation of rec-Grp78 binding to human spermatozoa by immunocytochemistry, and analysis of its effect upon gamete interaction using the hemizona assay.Result(s): Grp78 was found in the surface of oviduct epithelial cells. Soluble Grp78 was detected in oviductal fluids from women in the periovulatory period and in oviductal tissue conditioned medium. Rec-Grp78 was able to bind to the sperm acrosomal cap, and its presence during gamete interaction led to a decrease in the number of spermatozoa bound to the zona pellucida (ZP). When calcium ions from the incubation medium were replaced by strontium, rec-Grp78 enhanced sperm–ZP interaction.Conclusion(s): Grp78 is expressed and secreted by oviduct epithelial cells. The protein would bind to the gametes and may modulate their interaction in a calcium-dependent manner.

Imprinting: RNA expression for homocysteine recycling in the human oocyte

Moncef Benkhalifa, Debbie Montjean, Paul Cohen-Bacrie, Yves Ménézo — 2009-04-09

Objective: To investigate whether homocysteine, a well known inhibitor of methylation, which is produced after imprinting and other methylation processes, can be recycled to methionine in the oocyte, at least until the stage of maternal to zygotic transition (i.e., four- to eight-cell stage); before this stage, most of the biochemical processes are carried out with the use of maternal stores of protein and mRNA.Design: A first approach using microarrays and then reverse-transcription polymerase chain reaction (RT-PCR) for methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase [MTR]), betaine-homocysteine methyltransferase (BHMT), and cystathionine-beta synthase (CBS).Setting: Two private hospitals.Patient(s): Patients involved in IVF/ICSI procedures.Intervention(s): Germinal vesicle oocytes collected at the time of oocyte retrieval, RNA extraction amplification, RT-PCR, microarrays.Main Outcome Measure(s): mRNA expression of all the enzymes involved in the chain of methylation and recycling of homocysteine to methionine.Result(s): All of the enzymes required for methylation are present in the oocyte. Homocysteine can be recycled with BHMT and MTR.Conclusion(s): The human oocyte is able to regulate its Hcy level via remethylation using MTR and BHMT but not CBS. This aspect is important, because recent studies have shown that controlled ovarian hyperstimulation affects the homocysteine concentration in follicular fluid. This may regulate, at least in part, the risk of imprinting problems during IVF procedures.

Effects of in vitro maturation and age on oocyte quality in the rhesus macaque Macaca mulatta

2009-02-26

Objective: To evaluate oocyte quality in a primate model.Design: Analysis of oocyte karyotype by chromosome spreading and oocyte spindles by confocal microscopy.Setting: Research laboratory, Caribbean Primate Research Center.Animal(s): Rhesus macaques aged 6–22 years.Intervention(s): Fourteen females underwent both Regimen A (FSH + hCG) and Regimen B (FSH only) stimulation cycles to facilitate collection of mature and immature oocytes. Immature oocytes from Regimens A and B underwent in vitro maturation (IVM) to produce metaphase II oocytes. All metaphase II oocytes underwent gradual fixation to spread chromosomes or were fixed and stained with probes specific to α-tubulin, actin, and DNA for visualization of the meiotic spindle using confocal microscopy.Main Outcome Measure(s): Karyotype and meiotic spindle architecture differences among in vivo matured (IVO) and IVM oocytes from young and old rhesus macaques.Result(s): In all, 4.7% of IVO oocytes (Regimen A) from young females were hyperhaploid versus 25.0% of IVM oocytes (Regimen B) from old females; 4.5% of IVO oocytes (Regimen A) from young females versus 51.5% of IVM oocytes (Regimen B) from old females displayed abnormal chromosome alignment on the metaphase spindle.Conclusion(s): IVM can induce meiotic anomalies in macaque oocytes, especially those obtained from older females. Results from this study provide possible explanations for the reported reduction in developmental competence of IVM primate oocytes.

Expression of angiotensin II type 1 (AT1) and angiotensin II type 2 (AT2) receptors in human granulosa-lutein (GL) cells: correlation with infertility diagnoses

2009-06-12

Objective: To correlate angiotensin II (AngII) receptor expression by granulosa-lutein (GL) cells from gonadotropin-stimulated follicles with infertility diagnosis and IVF parameters.Design: The mRNA of angiotensin receptors type 1 (AT1) and type 2 (AT2) was studied in aspirated GL cells.Setting: University laboratory and private IVF center.Patient(s): Seventy-three IVF patients.Intervention(s): Reverse-transcription polymerase chain reaction analysis for relative expression of AT1 and AT2 receptor mRNA in women with no ovarian factor (NOF), poor ovarian reserve (PR), endometriosis (ENDO), and polycystic ovary syndrome (PCOS).Main Outcome Measure(s): Expression of AT1 and AT2 receptor mRNA.Result(s): There was a constant ∼7:1 ratio between AT1 and AT2 receptors and a negative correlation between the AT1/AT2 ratio and patient age. There were statistically significant differences in AngII receptors in individual conditions: NOF showed a correlation between AT1 and AT2 receptors and a negative correlation between AT1 receptor expression, embryo fragmentation and number of metaphase II (MII) oocytes; PR showed a negative correlation between AT2 receptor expression and number of MII oocytes; PCOS AT1 receptor expression correlated negatively with the units of FSH administered and with patients' age; ENDO showed no significant correlations.Conclusion(s): Mural GL cells express AT1 receptor much more than AT2 receptor. AngII receptor expression varies with age and infertility diagnosis. Low expression of AngII receptors was associated with high-dose stimulation in women with PR. Embryo fragmentation in NOF is associated with decreased AT1 receptor expression, supporting a role for AngII in GL cell apoptosis.

Prevention of paclitaxel and cisplatin induced ovarian damage in rats by a gonadotropin-releasing hormone agonist

2009-04-01

Objective: To evaluate the protective effect of GnRH agonist for the prevention of ovarian reserve during treatment with paclitaxel and cisplatin.Design: Experimental study.Settings: University-based research laboratory.Animal(s): Seventy female Wistar-Albino rats.Intervention(s): Each group consisted of 10 rats. Group 1 served as controls. Groups without GnRH agonist (groups 2, 3, and 4) were administered paclitaxel and cisplatin, respectively; the remaining groups (groups 5, 6, and 7) were given the same regimens with GnRH agonist. The GnRH agonist (leuprolide acetate; 2.5 μg/d subcutaneously for 5 weeks) was started four weeks before chemotherapy to achieve anovulation. Paclitaxel (7.5 mg/kg) and cisplatin (5 mg/kg) were administered intraperitoneally on the 28th day as a single dose.Main Outcome Measure(s): One week after the chemotherapy, the animals were euthanized and primordial, primary, secondary, and tertiary follicle counts were evaluated.Result(s): Primordial, primary, and tertiary follicle counts in group 5 (paclitaxel plus GnRH agonist) and tertiary follicles in groups 2 and 3 had not decreased, but there was a significant decrease in other treatment groups compared with controls (P < 0.05). Binary comparison between all groups demonstrated that the primordial follicle count in group 5 was comparable to those of the controls.Conclusion(s): Paclitaxel plus GnRH agonist treatment may be an appropriate option for patients deserving further fertility in the preservation of primordial follicles.

Aberrant gene expression profile in a mouse model of endometriosis mirrors that observed in women

2009-05-26

Objective: To define the altered gene expression profile of endometriotic lesions in a mouse model of surgically induced endometriosis.Design: Autologous experimental mouse model.Setting: Medical school department.Animal(s): Adult C57Bl6 mice.Intervention(s): Endometriosis was surgically induced by autotransplantation of uterine tissue to the intestinal mesentery. Endometriotic lesions and eutopic uteri were recovered at 3 or 29 days after induction.Main Outcome Measure(s): Altered gene expression was measured in the endometriotic lesion relative to the eutopic uterus by genome-wide complementary DNA microarray analysis and was confirmed by real-time reverse transcriptase–polymerase chain reaction for six genes. Relevant categories of altered genes were identified using gene ontology analysis to determine groups of genes enriched for altered expression.Result(s): The expression of 479 and 114 genes was altered in the endometriotic lesion compared with the eutopic uterus at 3 or 29 days after induction, respectively. Gene ontology enrichment analysis revealed that genes associated with the extracellular matrix, cell adhesions, immune function, cell growth, and angiogenesis were altered in the endometriotic lesion compared with the eutopic uterus.Conclusion(s): According to gene expression analysis, the mouse model of surgically induced endometriosis is a good model for studying the pathophysiology and treatment of endometriosis.

Changes in histone methylation during human oocyte maturation and IVF- or ICSI-derived embryo development

Jie Qiao, Yuan Chen, Li-Ying Yan, Jie Yan, Ping Liu, Qing-Yuan Sun — 2009-04-27

Objective: To characterize the histone methylation pattern during human oocyte maturation and embryo development after conventional IVF and ICSI.Design: Experimental study.Setting: Reproductive center of hospital.Patient(s): Women underwent IVF or ICSI.Intervention(s): Immature and mature oocytes were collected from patients undergoing ICSI. Tripronuclear and normally fertilized embryos were obtained from patients undergoing IVF or ICSI.Main Outcome Measure(s): The distribution patterns of dimethylated histone H3 lysine 9 (H3K9) and histone H4 arginine 3 (H4R3) in oocytes and embryos were observed by indirect immunofluorescent staining and scanning confocal microscopy.Result(s): H3K9 and H4R3 were dimethylated throughout the meiotic maturation of human oocytes and the tripronuclear embryo development from two-cell to blastocyst stage. However, at the pronuclear stage, approximately half of the tripronuclear IVF zygotes displayed strong staining of MeH3K9 in one pronucleus, whereas MeH4R3 staining was always uniform in all three pronuclei. In the other half of the tripronuclear zygotes, all three pronuclei were strongly stained with MeH3K9 in some cases, and the remaining zygotes were completely unstained. Moreover, with progressively increasing fragmentation of blastomeres in ICSI-derived 2PN embryos with low morphological grade, H3K9 dimethylation decreased when compared with that of IVF embryos.Conclusion(s): Asymmetric distribution of the dimethylation form of H3K9 exists in human zygote pronuclei. The ICSI-derived embryos with low morphological grade are more likely to display H3K9 demethylation than their IVF counterparts.

Increased cell proliferation in experimentally induced endometriosis in rabbits

2009-03-23

Objective: To characterize the pattern of cell proliferation and apoptosis of eutopic and ectopic endometrium in rabbits after endometrium implantation for the experimental induction of endometriosis.Design: Animal experimental study.Setting: Sector of experimental surgery.Animal(s): Twenty-female New Zealand rabbits.Intervention(s): All animals underwent laparotomy for endometriosis induction by resection of one uterine horn, isolation of the endometrium, and fixation of tissue segment to the pelvic peritoneum. Two groups of 10 animals were sacrificed 4 and 8 weeks after endometriosis induction. The lesion was excised together with the opposite uterine horn for endometrial gland and stroma determination.Main Outcome Measure(s): Cell proliferation and apoptosis were determined in the eutopic and ectopic endometrium, and the cell proliferation index (CPI) and apoptotic index (AI) were calculated as the number of labeled cells per 1,000 cells. The tissue homeostasis index was the CPI/AI ratio. Glands and stroma were analyzed separately.Result(s): The CPI for ectopic tissue was increased compared with eutopic tissue, but there was no difference in the ectopic lesions between 4 and 8 weeks of induction. Considering only the AI, eutopic and ectopic endometrium did not differ after 4 weeks, but differed significantly in glandular tissue after 8 weeks. The tissue homeostasis index revealed cell proliferation in these tissues, with a CPI/AI more than 1.Conclusion(s): Ectopic lesions seem to have a higher CPI than eutopic endometrium, with uncontrolled tissue growth occurring in induced endometriotic lesions.

Effects of metoclopramide-induced hyperprolactinemia on the prolactin receptor of murine endometrium

2009-03-23

Objective: To evaluate the effects of metoclopramide-induced hyperprolactinemia on the prolactin receptor of murine endometrium.Design: Experimental study using the RNA extraction to detect tissue prolactin receptor isoforms by reverse-transcriptase polymerase chain reaction (RT-PCR).Setting: University-based laboratory.Animal(s): Seventy-two female swiss albino mice (Mus musculus), approximately 100 days old, were divided into six 12-animal groups: (GI) nonoophorectomized mice given vehicle; (GII) nonoophorectomized mice treated with metoclopramide; (GIII) oophorectomized mice treated with metoclopramide; (GIV) oophorectomized mice treated with metoclopramide and 17β-estradiol; (GV) oophorectomized mice treated with metoclopramide and micronized progesterone; (GVI) oophorectomized mice treated with metoclopramide and a solution of 17β-estradiol and micronized progesterone.Intervention(s): Drugs were administered for 50 days. Following euthanasia, the middle portions of the uterine horns were removed, sectioned, and immediately frozen for RT-PCR procedures. Blood was collected for the dosage of prolactin and serum estrogen and progesterone using radioimmune assay.Main Outcome Measure(s): Identification of uterine prolactin receptor isoforms.Result(s): The PRL receptor and its isoform L were identified only in GI (control group) and GII (metoclopramide), the two groups with nonoophorectomized animals. The amount of PRL receptor mRNA and that of its isoform L from GII were the largest. No other isoforms of the prolactin receptor were identified in any of the groups.Conclusion(s): Our results suggest that replacement of estrogen and progestin may not increase the mRNA of endometrial PRL receptor in metoclopromide-induced hyperprolactinemia in rats after castration.

Effect of gonadotropins on oocyte maturation in vitro: an animal model

Wei Sha, Bao-Zeng Xu, Mo Li, Di Liu, Huai L. Feng, Qing-Yuan Sun — 2009-04-09

Objective: Analysis of the effects of human-derived gonadotropin drugs, FSH and LH (Repronex) and hCG (Novarel), on oocyte maturation, using a porcine oocyte in vitro maturation system as a culture model.Design: Randomized research experimental study.Setting: Academic basic research laboratory.Animals: Prepubertal gilts that were slaughtered in the local slaughter house.Intervention(s): Oocytes will be exposed to immunofluorescent staining and confocal laser scanning microscopy: Western blot analysis on cumulus-oocyte-complexes following treatment with different concentrations of the gonadotropin drugs Repronex, Novarel, and a Repronex and Novarel combination.Main Outcome Measure(s): Analysis of porcine oocyte spindle and chromosomal configuration with α-tubulin–fluorescein isothiocyanate antibody and propidium iodide staining. Porcine oocyte mitochondrial distribution and aggregation pattern staining was assessed with Mito Tracker Red CMXRox probe. Porcine oocyte cortical granule distribution was observed via peanut agglutinin–fluorescein isothiocyannate staining; Western blot analysis detected extra-cellular signal-regulated kinase 1/2 activation in cumulus cells.Result(s): An increase of gonadotropin concentration in the culture medium resulted in an increase in the following: the percentage of oocytes reaching metaphase II, normal configuration of the spindle, normal chromosomal alignment, cortical granule migration, and mitochondrial aggregation. Levels of nuclear and cytoplasmic maturation peaked as the concentration of gonadotropins approached its threshold level.Conclusion(s): Addition of a threshold concentration of the gonadotropin drugs Repronex, Novarel, and a combination of the two can significantly improve porcine oocyte maturation in vitro.

Effects of follicular phase and oocyte–cumulus complexes quality on the protein profile and in vitro oocyte meiosis competence in Cebus apella

2009-06-29

Objective: To study the protein profile of oocytes and cumulus cells from different sized follicles throughout the follicular phase and to asses the ability of oocytes to progress from the dictyate to metaphase II (MII) stage.Design: Animal model study.Setting: Five academic basic research laboratories and the National Primate Centre.Animal(s): Eleven normal, cycling capuchin monkey (Cebus apella) females.Intervention(s): Cumulus–oocyte complexes and denuded oocytes were recovered by antral follicle aspiration.Main Outcome Measure(s): Protein profile analysis of denuded or intact oocytes.Result(s): The protein profiles of 25 denuded or intact oocytes recovered on days 5 (six denuded, five intact), 7 (four denuded, four intact), or 9 (one denuded, five intact) of the menstrual cycle were analyzed; in a second experiment, 40 intact oocytes were cultured for 24 (n = 20) or 36 hours (n = 20). The oocytes were denuded, fixed, stained, and microscopically examined to reveal the meiotic stage. The protein profile in each compartment within the cumulus–oocyte complex varied along the follicular development with a predominance of low-molecular-weight proteins in both oocyte and cumulus cells at final stages. No differences were found in the protein profile among oocytes pertaining to different sized follicles that were in the same day of the follicular phase. Oocyte MII competence was achieved only after incubation for 36 hours, and the highest maturation rate occurred in those becoming from dominant follicles.Conclusion(s): Our study shows, for the first time in a New World primate species, that the proteins contained in oocytes and cumulus cells reach an identical profile in the late follicular phase. This phenomenon could be related to the oocyte's ability to progress to the MII stage.

The mechanism responsible for the supraphysiologic gonadotropin surge in females treated with gonadotropin-releasing hormone (GnRH) agonist and primed with GnRH antagonist

2009-02-06

Objective: To elucidate the physiologic mechanism responsible for the supraphysiologic gonadotropin release from the pituitary induced by gonadotropin-releasing hormone (GnRH) agonist in female rats primed with GnRH antagonist.Design: Controlled experimental intervention.Setting: Government research facility.Animal(s): Forty 8-week-old Sprague-Dawley rats.Intervention(s): Forty oophorectomized rats were randomized into four treatment groups of 10: group A, control vehicles; group B, GnRH agonist (leuprolide acetate; 1.7 μg/kg twice a day) on day 4; group C, GnRH antagonist (Nal-Lys; 3 mg/kg each day) days 1 to 4; or group D, GnRH antagonist (Nal-Lys; 3 mg/kg each day) days 1 to 4 plus GnRH agonist (1.7 μg/kg twice a day) on day 4.Main Outcome Measure(s): Immunohistochemical methods, Northern and in situ hybridization to quantitate pituitary follicle-stimulating hormone beta (FSH-β), luteinizing hormone beta (LH-β), and GnRH receptor (GnRH-R) messenger RNA (mRNA), and receptor protein levels in all treatment groups.Result(s): Treatment with GnRH antagonist was associated with increased storage of gonadotropin in the pituitary for FSH-β and LH-β, but mRNA levels were unchanged. The GnRH-R mRNA decreased after GnRH-agonist treatment but remained stable in the GnRH-antagonist treatment groups. Levels of GnRH-R were decreased after GnRH-antagonist treatment.Conclusion(s): These data indicate that the in vivo mechanism responsible for the exaggerated release of gonadotropins in rats primed with GnRH antagonist and treated with GnRH agonist was an increase in releasable gonadotropin pools coupled with a reduction in GnRH-R, but receptor function was preserved.

Both host and graft vessels contribute to revascularization of xenografted human ovarian tissue in a murine model

2009-06-19

Objective: To characterize the human ovarian xenograft revascularization process.Design: Prospective experimental study.Setting: Gynecology research unit in a university hospital.Patient(s): Ovarian biopsies were obtained from 12 women aged 22–35 years.Intervention(s): Frozen-thawed human ovarian fragments were intraperitoneally grafted into nude mice.Main Outcome Measure(s): Graft perfusion was evaluated by Hoechst 33342 uptake. Murine and human vascularization was analyzed by CD31 and von Willebrand factor double immunostaining.Result(s): On day 3, some murine neovessels and perfused areas were located at the periphery of the fragments. Nonperfused native human vessels were present in the fragments. From day 5, perfused areas and murine endothelial areas progressively increased. Host angiogenesis initiated ovarian graft reperfusion: murine neovessels penetrated from the periphery and were colocalized with perfused areas. By day 10, the increase in perfusion and murine vascularization was significant. The center of the fragments was perfused and a significant increase was observed in human vasculature.Conclusion(s): Host and graft vessels contributed sequentially to graft revascularization: murine angiogenesis initiated reperfusion from day 5 and, by day 10, human angiogenesis was shown to participate in graft revascularization. Host and graft angiogenesis are potential targets to reduce the avascular period after grafting.

Coadministration of dextromethorphan during pregnancy and throughout lactation prevents morphine-induced hyperprolactinemia in female rats

Ling-Yi Wu, Eagle Yi-Kung Huang, Pao-Luh Tao — 2009-03-26

Objective: To investigate whether coadministration of dextromethorphan (DM) could suppress morphine-induced hyperprolactinemia in female rats during pregnancy and throughout lactation.Design: Controlled prospective study.Setting: University research laboratory.Animal(s): One hundred adult female Sprague-Dawley rats.Intervention(s): Rats were randomly divided into four groups and were subcutaneously injected with either saline, morphine, morphine + DM, or DM alone twice a day, progressively increasing by 1 mg/kg at 7-day intervals from an initial dose of 2 mg/kg for both morphine and DM. Drug administration was continued during pregnancy. After the offspring were born, the doses injected into the dams were increased by 1 mg/kg every 2 weeks.Main Outcome Measure(s): Serum prolactin (PRL) concentration and dopamine turnover rate at the hypothalamus and pituitary.Result(s): Chronic morphine administration induced higher PRL concentrations than the control animals at mating, and at early and late pregnancy. In rats receiving DM coadministration, we did not observe any increase by morphine. Our neurochemical results showed that this effect of DM may be partly through blocking the effect of morphine on inhibition of tuberoinfundibular dopaminergic (TIDA) neuronal activity.Conclusion(s): The use of DM as an adjuvant in females receiving chronic morphine treatment may prevent morphine-induced hyperprolactinemia.

Effects of fetal exposure to carbon nanoparticles on reproductive function in male offspring

2009-05-15

Objective: To investigate the effects of fetal nanoparticle exposure on reproductive function in male mice offspring.Design: Animal study.Setting: Academic research laboratory.Animal(s): Forty pregnant ICR mice and 120 male offspring.Intervention(s): Two hundred μg of 14-nm carbon nanoparticles was administered intratracheally on days 7 and 14 of gestation, and reproductive function of male offspring was determined at ages 5, 10, and 15 weeks after birth.Main Outcome Measure(s): Maternal and fetal growth, histologic changes in the testes, and daily sperm production (DSP).Result(s): Histologic examination showed partial vacuolation of seminiferous tubules. and cellular adhesion of seminiferous epithelia was reduced at all three ages. In addition, DSP was significantly decreased in fetal carbon nanoparticle–exposed mice. The DSP in the fetal carbon nanoparticle–exposed mice decreased by 47% at the age of 5 weeks, by 34% at the age of 10 weeks, and by 32% at the age of 15 weeks. On the other hand, nanoparticle administration had no marked effect on body weight, testicle weight, epididymis weight, or serum testosterone concentration.Conclusion(s): These findings suggest that fetal nanoparticle exposure affects the reproductive function of male offspring. In the future, it would be necessary to clarify the onset mechanisms of nanoparticle-induced male reproductive disorders.

Adoptive transfer of mFas ligand into dendritic cells influences the spontaneous resorption rate in the CBA/J × DBA/2 mouse model

Aimin Zhao, Miao Xiong, Yu Zhang, Shimin Bao, Jian Zhang, Lihua Qiu, Qide Lin — 2009-05-12

Objective: To investigate the effect of mFasL dendritic cells (DC) on the embryo resorption rate in the CBA/J × DBA/2 abortion mouse model.Design: Experimental study.Setting: University hospital in a major city in China.Animal(s): 101 CBA/J mice and 50 DBA/2 mice.Intervention(s): We constructed the eukaryotic expression vector pcDNA3.1-mFasL, derived the DCs from bone marrow of DBA/2 mice, and transfected the DCs with pcDNA3.1-mFasL. The abortion mouse model were established by mating female CBA/J mice with DBA/2 mice. Via the CBA/J × DBA/2 abortion mouse model, five groups were established: group 1: abortion model without treatment; group 2: abortion mouse model injected with DC culture medium (DCCM); group 3: abortion mouse model injected with DC; group 4: abortion mouse model injected with empty plasmid pcDNA3.1-DC; group 5: abortion model mouse injected with pcDNA3.1-FasL-DC.Main Outcome Measure(s): Comparison of difference in the embryo resorption rates of the CBA/J × DBA/2 abortion mouse model treated with either pcDNA3.1-mFasL-DC or different controls observed on gestation days 12 to 14.Result(s): The embryo resorption rate was statistically significantly decreased in group 3 treated with DC and group 4 with empty plasmid pcDNA3.1-DC when they compared with group 1 (no treatment for abortion mouse model) and group 2 (injected with DC culture medium, DCCM). Furthermore, abortion model group 5 (injected with pcDNA3.1-mFasL-DC) showed a statistically significantly decreased in embryo resorption rate when compared with the other four groups, including groups 1 and 2 and groups 3 and group 4.Conclusion(s): Adoptive transfer of mFasL-DC can statistically significantly reduce the embryo resorption rate in the CBA/J × DBA/2 abortion mouse model.

Evaluating the antifertility potential of an aqueous extract from Cassia fistula seeds in male rats

Alka Chauhan, Meera Agarwal — 2009-10-12

Cassia fistula suppresses fertility in male rats. Withdrawal of extract restored all the altered parameters, including organ weights, fertility, circulatory level of hormones and tissue biochemistry, to control levels after 120 days.

Superior ovarian nerve (SON) transection leads to stunted follicular maturation: a histomorphologic and morphometric analysis in the rat model

2009-11-11

Histomorphologic and morphometric effects of peripubertal superior ovarian nerve (SON) transection were evaluated during adult life in rats. Twenty Sprague-Dawley rats were randomly divided into two groups: the SON-transected (SON-t) group (n = 10) and the sham-operated control group (n = 10). Total follicle development was not influenced; however, follicular maturation was seriously stunted by the procedure, as evidenced by significantly thinner theca interna and follicular wall.

Involvement of connexin 43 in the acupuncture effect of improving rat blastocyst implantation

Guang Ying Huang, Cui Hong Zheng, Yun Xia Wu, Wei Wang — 2009-11-06

Acupuncture can improve blastocyst implantation in rats. Connexin 43 may be involved in this effect.

Increased fetal cell trafficking in murine lung following complete pregnancy loss from exposure to lipopolysaccharide

2009-10-07

To determine whether chemically induced miscarriage affects fetomaternal trafficking in a mouse model, we measured the amount of fetal DNA present in various maternal organs by polymerase chain reaction amplification following exposure to lipopolysaccharide (LPS). As the frequency of fetal cells and the number of animals with detectable microchimerism following LPS injection were significantly increased, particularly in lung tissue compared to controls, with no signs of an inflammatory response, we conclude that LPS-induced miscarriage results in increased murine fetomaternal cell trafficking, supporting a relationship between fetal loss and the establishment of fetal cell microchimerism.

Exposure to industrially polluted water resulted in regressed endometriotic lesions and enhanced adhesion formation in a rat endometriosis model: a preliminary study

2009-11-06

The effects of water collected from an industrially polluted river in a rat model with surgically induced endometriosis were investigated in this preliminary study. Exposure to industrially polluted water resulted in regressed endometriotic lesions and enhanced adhesion formation.

Editorial Board

2010-03-15

Alan H. DeCherney, M.D. Bethesda, Maryland

2010-03-15

ASRM classified announcements

2010-03-15

AMERICAN SOCIETY FOR REPRODUCTIVE MEDICINE 2010 CALENDAR For more information, contact:

Fertility and Sterility

Intrabursal injection of vascular endothelial growth factor trap in eCG-treated prepubertal rats inhibits proliferation and increases apoptosis of follicular cells involving the PI3K/AKT signaling pathway

The CB2 cannabinoid receptor regulates human sperm cell motility

Preventive role of exogenous testosterone on cisplatin-induced gonadal toxicity: an experimental placebo-controlled prospective trial

Brain-derived neurotrophic factor is a regulator of human oocyte maturation and early embryo development

Testis atrophy and reduced sperm motility in transgenic mice overexpressing c-FLIPL

Changing P2X receptor localization on maturing sperm in the epididymides of mice, hamsters, rats, and humans: a preliminary study

Expression of the insulin-like growth factor and insulin systems in the luteinizing macaque ovarian follicle

The oocyte spindle is preserved by 1,2-propanediol during slow freezing

15-Epi-lipoxin A4 inhibits the progression of endometriosis in a murine model

Change of proinflammatory cytokines follows certain patterns after induction of endometriosis in a mouse model

Attenuation of ischemia–reperfusion injury with Marrubium cordatum treatment in ovarian torsion–detorsion model in rabbits

A differential cytokine expression profile is induced by highly purified human menopausal gonadotropin and recombinant follicle-stimulating hormone in a pre- and postovulatory mouse follicle culture model

Analysis of spindle characteristics and embryo quality in mice stimulated with letrozole using Polscope imaging

Acrosome formation-associated factor is involved in fertilization

Changes in circulating levels and ratios of angiopoietins during pregnancy but not during the menstrual cycle and controlled ovarian stimulation

Myometrial cells undergo fibrotic transformation under the influence of transforming growth factor β-3

The effect of hyperstimulation on transforming growth factor β1 and β2 in the rat uterus: possible consequences for embryo implantation

Mouse blastocyst previtrification interventions and DNA integrity

The acrosomal protein Dickkopf-like 1 (DKKL1) is not essential for fertility

The acrosomal protein Dickkopf-like 1 (DKKL1) facilitates sperm penetration of the zona pellucida

Expression and regulation of cholesterol sulfotransferase (SULT2B1b) in human endometrium

Effect of melatonin on epididymal sperm quality after testicular ischemia/reperfusion in rats

Changes in the distribution of mitochondria before and after in vitro maturation of human oocytes and the effect of in vitro maturation on mitochondria distribution

Immunization with a DNA vaccine of testis-specific sodium-hydrogen exchanger by oral feeding or nasal instillation reduces fertility in female mice

Damaging effect of cumulus denudation on rabbit oocytes

Glucose-regulated protein 78 (Grp78/BiP) is secreted by human oviduct epithelial cells and the recombinant protein modulates sperm–zona pellucida binding

Imprinting: RNA expression for homocysteine recycling in the human oocyte

Effects of in vitro maturation and age on oocyte quality in the rhesus macaque Macaca mulatta

Expression of angiotensin II type 1 (AT1) and angiotensin II type 2 (AT2) receptors in human granulosa-lutein (GL) cells: correlation with infertility diagnoses

Prevention of paclitaxel and cisplatin induced ovarian damage in rats by a gonadotropin-releasing hormone agonist

Aberrant gene expression profile in a mouse model of endometriosis mirrors that observed in women

Changes in histone methylation during human oocyte maturation and IVF- or ICSI-derived embryo development

Increased cell proliferation in experimentally induced endometriosis in rabbits

Effects of metoclopramide-induced hyperprolactinemia on the prolactin receptor of murine endometrium

Effect of gonadotropins on oocyte maturation in vitro: an animal model

Effects of follicular phase and oocyte–cumulus complexes quality on the protein profile and in vitro oocyte meiosis competence in Cebus apella

The mechanism responsible for the supraphysiologic gonadotropin surge in females treated with gonadotropin-releasing hormone (GnRH) agonist and primed with GnRH antagonist

Both host and graft vessels contribute to revascularization of xenografted human ovarian tissue in a murine model

Coadministration of dextromethorphan during pregnancy and throughout lactation prevents morphine-induced hyperprolactinemia in female rats

Effects of fetal exposure to carbon nanoparticles on reproductive function in male offspring

Adoptive transfer of mFas ligand into dendritic cells influences the spontaneous resorption rate in the CBA/J × DBA/2 mouse model

Evaluating the antifertility potential of an aqueous extract from Cassia fistula seeds in male rats

Superior ovarian nerve (SON) transection leads to stunted follicular maturation: a histomorphologic and morphometric analysis in the rat model

Involvement of connexin 43 in the acupuncture effect of improving rat blastocyst implantation

Increased fetal cell trafficking in murine lung following complete pregnancy loss from exposure to lipopolysaccharide

Exposure to industrially polluted water resulted in regressed endometriotic lesions and enhanced adhesion formation in a rat endometriosis model: a preliminary study

Editorial Board

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