Periovulatory Stage Research Articles

EGF-like growth factors upregulate pentraxin 3 expression in human granulosa-lutein cells

The epidermal growth factor (EGF)-like factors, comprising amphiregulin (AREG), betacellulin (BTC), and epiregulin (EREG), play a critical role in regulating the ovulatory process. Pentraxin 3 (PTX3), an essential ovulatory protein, is necessary for maintaining extracellular matrix (ECM) stability during cumulus expansion. The aim of this study was to investigate the impact of EGF-like factors, AREG, BTC, and EREG on the expression and production of PTX3 in human granulosa-lutein (hGL) cells and the molecular mechanisms involved. Our results demonstrated that AREG, BTC, and EREG could regulate follicular function by upregulating the expression and increasing the production of PTX3 in both primary (obtained from 20 consenting patients undergoing IVF treatment) and immortalized hGL cells. The upregulation of PTX3 expression was primarily facilitated by the activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway, induced by these EGF-like factors. In addition, we found that the upregulation of PTX3 expression triggered by the EGF-like factors was completely reversed by either pretreatment with the epidermal growth factor receptor (EGFR) inhibitor, AG1478, or knockdown of EGFR, suggesting that EGFR is crucial for activating the ERK1/2 signaling pathway in hGL cells. Overall, our findings indicate that AREG, BTC, and EREG may modulate human cumulus expansion during the periovulatory stage through the upregulation of PTX3.

181 Effect of systemic administration of β-nerve growth factor during the periovulatory stage on corpus luteum development and function in dairy heifers

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Semen Modulates Cell Proliferation and Differentiation-Related Transcripts in the Pig Peri-Ovulatory Endometrium.

Simple SummaryHomeostasis of the uterus after mating is crucial for the subsequent reproductive events. The post-mating inflammatory response is restricted to the uterus, but semen also modulates the expression of other genes involved in regulation along the female reproductive tract, including the oviduct. This study aims to determine if several ejaculate fractions of the pig may modulate cell proliferation and differentiation-related transcripts in different sections of the peri-ovulatory sow reproductive tract. Our data demonstrate that most of the mRNA expression changes of the 144 transcripts tested were induced by mating. Additionally, spermatozoa and seminal plasma also triggered differential expression of the transcripts tested. Finally, our data imply that spermatozoa, seminal plasma components, and the act of mating induce differential mechanisms in the peri-ovulatory female reproductive tract, which are essential for tissue repair.Uterine homeostasis is maintained after mating by eliminating pathogens, foreign cells, and proteins by a transient inflammation of the uterus. Such inflammation does not occur in the oviductal sperm reservoir (utero-tubal junction, UTJ), colonized by a population of potentially fertile spermatozoa before the inflammatory changes are triggered. Semen entry (spermatozoa and/or seminal plasma) modifies the expression of regulatory genes, including cell proliferation and differentiation-related transcripts. Considering pigs display a fractionated ejaculation, this study aims to determine whether different ejaculate fractions differentially modulate cell proliferation and differentiation-related transcripts in the sow reproductive tract during the peri-ovulatory stage. Using species-specific microarray analyses, the differential expression of 144 cell proliferation and differentiation-related transcripts was studied in specific segments: cervix (Cvx), distal and proximal uterus (DistUt, ProxUt), UTJ, isthmus (Isth), ampulla (Amp), and infundibulum (Inf) of the peri-ovulatory sow reproductive tract in response to semen and/or seminal plasma cervical deposition. Most mRNA expression changes were induced by mating. In addition, while mating upregulates the fibroblast growth factor 1 (FGF1, p-value DistUt = 0.0007; ProxUt = 0.0253) transcript in the endometrium, both its receptor, the fibroblast growth factor receptor 1 (FGFR1, p-value DistUt = 2.14 e−06; ProxUt = 0.0027; UTJ = 0.0458) transcript, and a potentiator of its biological effect, the fibroblast growth factor binding protein 1 (FGFBP1), were downregulated in the endometrium (p-value DistUt = 0.0068; ProxUt = 0.0011) and the UTJ (p-value UTJ = 0.0191). The FGFBP1 was downregulated in the whole oviduct after seminal depositions (p-value Isth = 0.0007; Amp = 0.0007; Inf = 6.87 e−05) and, interestingly, FGFR1 was downregulated in the endometrium in the absence of semen (p-value DistUt = 0.0097; ProxUt = 0.0456). In conclusion, the findings suggest that spermatozoa, seminal components, and the act of mating trigger, besides inflammation, differential mechanisms in the peri-ovulatory female reproductive tract, relevant for tissue repair.

Spatiotemporal profiling of the bovine oviduct fluid proteome around the time of ovulation

Understanding the composition of the oviduct fluid (OF) is crucial to better comprehend the microenvironment in which sperm capacitation, fertilization and early embryo development take place. Therefore, our aim was to determine the spatiotemporal changes in the OF proteome according to the anatomical region of the oviduct (ampulla vs. isthmus), the proximity of the ovulating ovary (ipsilateral vs. contralateral side) and the peri-ovulatory stage (pre-ovulatory or Pre-ov vs. post-ovulatory or Post-ov). Oviducts from adult cyclic cows were collected at a local slaughterhouse and pools of OF were analyzed by nanoLC-MS/MS and label-free protein quantification (n = 32 OF pools for all region × stage × side conditions). A total of 3760 proteins were identified in the OF, of which 65% were predicted to be potentially secreted. The oviduct region was the major source of variation in protein abundance, followed by the proximity of the ovulating ovary and finally the peri-ovulatory stage. Differentially abundant proteins between regions, stages and sides were involved in a broad variety of biological functions, including protein binding, response to stress, cell-to-cell adhesion, calcium homeostasis and the immune system. This work highlights the dynamic regulation of oviduct secretions and provides new protein candidates for interactions between the maternal environment, the gametes and the early embryo.

Effects of P4 Antagonist RU486 on VEGF and Its Receptors' Signaling during the In Vivo Transition from the Preovulatory to Periovulatory Phase of Ovarian Follicles.

The development of an adequate blood vessel network is crucial for the accomplishment of ovarian follicle growth and ovulation, which is necessary to support the proliferative and endocrine functions of the follicular cells. Although the Vascular Endothelial Growth Factor (VEGF) through gonadotropins guides ovarian angiogenesis, the role exerted by the switch on of Progesterone (P4) during the periovulatory phase remains to be clarified. The present research aimed to investigate in vivo VEGF-mediated mechanisms by inducing the development of periovulatory follicles using a pharmacologically validated synchronization treatment carried out in presence or absence of P4 receptor antagonist RU486. Spatio-temporal expression profiles of VEGF, FLT1, and FLK1 receptors and the two major MAPK/ERKs and PI3K/AKT downstream pathways were analyzed on granulosa and on theca compartment. For the first time, the results demonstrated that in vivo administration of P4 antagonist RU486 inhibits follicular VEGF receptors’ signaling mainly acting on the theca layer by downregulating the activation of ERKs and AKTs. Under the effect of RU486, periovulatory follicles’ microarchitecture did not move towards the periovulatory stage. The present evidence provides new insights on P4 in vivo biological effects in driving vascular and tissue remodeling during the periovulatory phase.

Unconservative_15_2570409 suppresses progesterone receptor expression in the granulosa cells of Hu sheep

Female fertility potential depends on the number of mature follicles; however, the underlying molecular mechanisms remain unclear. Based on previously generated miRNA and mRNA sequencing data of healthy ovarian follicles (>5 mm in diameter) isolated from Hu sheep with different prolificacy, we investigated the roles of a novel miRNA (unconservative_15_2570409) and the progesterone receptor (PGR) gene in follicular development. During the periovulatory phase, the expression of unconservative_15_2570409 and PGR was lower and higher, respectively, in the >5 mm follicles of high prolificacy (HP) ewes than in those of low prolificacy (LP) ewes and in the >3 mm follicles than in the smaller follicles of the HP ewes. Subsequently, the granulosa cells (GCs) of Hu sheep were used as an in vitro model. PGR overexpression significantly increased the mRNA expression of steroidogenic acute regulatory protein (StAR), 3-beta-hydroxysteroid dehydrogenase/isomerase (3β-HSD), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1), which promoted the secretion of progesterone (P4) and estradiol (E2). PGR knockdown significantly downregulated the levels of StAR and 3β-HSD mRNA and decreased the production of P4, whereas no effects on CYP19A1 mRNA expression and E2 levels were observed. We also found the negative regulatory effect of unconservative_15_2570409 on the mRNA and protein expression of PGR by targeting the 3ʹ-untranslated region. The regulation of PGR levels resulted in a corresponding change in the ADAMTS1, PPAR-γ, and CTSL gene transcripts, which are important for follicular maturation and ovulation. Additionally, PGR, ADAMTS1, and PPAR-γ were predominantly localized in the GCs. Collectively, our results suggest that by regulating PGR expression and consequently affecting the expression of target genes and steroidogenesis, unconservative_15_2570409 plays a role in follicular development during the periovulatory stage, which provides novel insights into the molecular mechanisms affecting Hu sheep prolificacy.

0831 Hot Flashes and Insomnia Throughout the Life Span of Women from the Episono Cohort

Abstract Introduction Hormonal changes may trigger sleep disturbances in women. Insomnia affects one in every three-to-four of them, most likely during pre to post menopause, and especially in association with hot flashes. Thus, the present study aimed to investigate the occurrence of hot flashes among women with and without insomnia and on different reproductive stages. Methods Sampling procedure was a three-stage clustering of the population of Sao Paulo, Brazil according to gender, age (20-80 years), and socio-economic status. A total of 574 women were interviewed, underwent polysomnographic recording (PSG), and had fasting-blood samples collected. Hormone levels and a gynecological questionnaire were used to classify reproductive stages. Premenopausal women were classified either in the follicular, luteal, or periovulatory stage or as anovulatory or under hormonal contraceptives; whereas those menopausal were classified in perimenopause or in early or late stages. Individuals reporting frequent and persistent insomnia symptoms accompanied by relevant daytime impairment were classified with insomnia syndrome. Objective insomnia was defined by increased sleep onset latency and/or awake after sleep onset, decreasing sleep duration. Results The final sample included 550 women, representing 53% of the EPISONO cohort (n=1,042). Hot flashes were reported by 9% of the premenopausal women (n=339) and by 42% of the menopausal. Complaints were more frequent among women in perimenopause (67%) and those in use of hormonal therapy (60%), and it tended to decrease in later stages (33%); whereas before menopause, hot flashes were especially reported by anovulatory women (26%), while significantly less by those using contraceptives (6%). Hot flashes were associated with a 2-fold increase in insomnia symptoms and while it predicted objective sleep alterations among premenopausal women, they did not after menopause, when alterations in sleep were better explained by an effect of aging. Conclusion Our current findings suggest that hot flashes are associated with irregular menstrual cycles among premenopausal women, and particularly with early stages of menopause, predicting both subjective and objective sleep alterations. Support This research was supported by fellowships from Associação Fundo de Incentivo à Pesquisa (AFIP) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Finance Code 001.

Therapeutic effects on infertility of ovulation failure in the patients with kidney deficiency treated with abdominal acupuncture and periodic therapy of Chinese herbal medicine

To observe the effects of abdominal acupuncture and the periodic therapy of Chinese herbal medicine on follicular development and endometrial receptivity in the patients with infertility induced by ovulation failure and differentiated as kidney deficiency in TCM. A total of 84 patients with infertility induced by ovulation failure and differentiated as kidney deficiency were randomized into a combined therapy group (27 cases), an abdominal acupuncture group (27 cases) and a western medication group (30 cases). In the combined therapy group, abdominal acupuncture and the periodic treatment of Chinese herbal medicine was provided. In the abdominal acupuncture group, the simple abdominal acupuncture therapy was used. In the western medication group, clomiphene citrate (CC) and human chorionic gonadotrophin (HCG) prescribed. The treatment for one menstrual cycle was taken as one session and 3 sessions of treatment were required except the pregnancy. The folicle development, endometrial thickness and morphology, menstrual condition and TCM symptom score were observed before and after treatment in the three groups, and the clinical efficacy was evaluated. After treatment, the ovulation was recovered to different degrees in the three groups. The ovulation rate was 59.3% (16/27) in the combined therapy group, 55.6% (15/27) in the abdominal acupuncture group and 53.3% (16/30) in the western medication group. The difference was not significant in comparison among the three groups (P>0.05). After treatment, the endometrial thickness in the periovulatory period was increased as compared with the thickness before treatment in the combined therapy group and the abdominal acupuncture group (both P<0.05). After treatment, the endometrial thickness in the combined therapy group was higher than the western medication group (P<0.05). In comparison before and after treatment, the difference in the endometrial morphology was significant in the combined therapy group and the abdominal acupuncture group (both P<0.05). In comparison between the combined therapy group and the western medication group, the difference in the endometrial morphology was significant after treatment (P<0.05). After treatment, the menstrual condition and TCM symptom score in the combined therapy group and the abdominal acupuncture group were all improved as compared with those before treatment (all P<0.05). The score of menstrual condition in the combined therapy group was higher than the western medication group (P<0.05) and TCM symptom score in the combined therapy group and abdominal acupuncture group was higher than the western medication group after treatment (all P<0.05). The total effective rate was 88.9% (24/27) in the combined therapy group and was 92.6% (25/29) in the abdominal acupuncture group, which was higher than 56.7% (17/30) in the western medication group (P<0.01). There was no adverse reaction in the combined therapy group and the abdominal acupuncture group. Abdominal acupuncture combined with the periodic therapy of Chinese herbal medicine improve the menstrual condition and relieve the clinical symptoms of infertility induced by ovulation failure of kidney deficiency in the patients and the therapeutic effects are better than the medication with CC + HCG. This combined therapy improves the ovulation rate and the endometrial receptivity at periovulatory stage to increase the pregnancy rate. There is no adverse reaction discovered in clinical practice.

Effects of co-incubation with conspecific ampulla oviductal epithelial cells and media composition on cryotolerance and developmental competence of in vitro matured sheep oocytes

Developmental potential of cryopreserved in vitro matured oocytes is very low in nearly all mammalian species studied to date. Despite relatively high cleavage rates, the vitrified/warmed metaphase II oocytes have a decreased rate of blastocyst formation, which can be attributed to the elevated cytoplasmic lipid content and lipid droplet fragmentation. Secretory products of ampulla oviductal epithelial cells (AECs) at the periovulatory stage of the ovarian cycle enhance the viability of in vitro matured oocytes. The present study was undertaken to determine if co-culture of cumulus-oophorus complexes (COCs) with conspecific AECs or reducing the lipid content of in vitro matured ovine oocytes would improve their cryotolerance and ensuing developmental competence. Ovine COCs aspirated from the slaughterhouse ovaries were matured in the following media or culture conditions: TCM199 + FBS + AECs (T1); TCM199 + FBS (T2); TCM199 + BSA (T3); TCM199 + 0.6 mg/mL of l-carnitine (T4); TCM199+ l-carnitine + FBS (T5), or TCM199 only (Ctr). Subsequently, the oocytes were vitrified and used for in vitro fertilization (IVF). The lowest degree of zona pellucida (ZP) hardening following vitrification of in vitro matured sheep oocytes was observed in T1 and T5 (P < 0.05). Cleavage, blastocyst formation and ensuing development (i.e., total cell numbers) as well as blastocyst hatching rates were all greater (P < 0.05) in T1 compared with the remaining groups; in vitro matured COCs in T4 and Ctr did not develop beyond the cleavage stage. The inner cell mass: trophectoderm cell ratio in T1 (1:3.29) was significantly greater compared with T2 (1:3.39), T3 (1:3.40) and T5 (1:3.44). The present results indicate that the ovine COCs/AECs co-culture system had the most positive influence on cryotolerance, ZP hardening, and developmental competence of in vitro matured oocytes.

SNAIL Mediates TGF-β1-Induced Downregulation of Pentraxin 3 Expression in Human Granulosa Cells.

Transforming growth factor-β (TGF-β) 1 plays a critical role in regulating follicular development, and its dysregulation has been shown to be involved in the pathogenesis of ovulation dysfunction. SNAIL is a well-known transcriptional repressor that mediates TGF-β1-induced cellular functions. Pentraxin 3 (PTX3) is a key enzyme for the assembly and stabilization of the cumulus oophorus extracellular matrix, which is essential for cumulus expansion during the periovulatory stage. The purpose of the present study was to investigate the roles of TGF-β1 and SNAIL in the regulation of PTX3 expression and to examine the underlying mechanism. An established immortalized human granulosa cell (GC) line (SVOG), a GC tumor cell line (KGN), and primary human granulosa-lutein cells were used as study models. We demonstrated that TGF-β1 treatment substantially decreased the messenger RNA and protein levels of PTX3. This suppressive effect was abolished by cotreatment with the soluble TGF-β type II receptor (TβRII) or the ALK4/5/7 inhibitor SB431542. Knockdown of ALK5, SMAD2/3, or SMAD4 reversed the effects of TGF-β1-induced SNAIL upregulation and PTX3 suppression. These results indicate that TGF-β1 upregulates SNAIL and downregulates PTX3 expression via a TβRII-ALK5-mediated SMAD-dependent signaling pathway in human GCs. Additionally, TGF-β1-induced PTX3 suppression was mediated by upregulation of the SNAIL transcription factor, as knockdown of SNAIL completely reversed the suppression of PTX3 in response to TGF-β1. These findings could inform the roles of TGF-β1 and SNAIL in the regulation of follicular function and might provide therapeutic targets for the treatment of ovulation dysfunction.

ALK2/ALK3-BMPR2/ACVR2A Mediate BMP2-Induced Downregulation of Pentraxin 3 Expression in Human Granulosa-Lutein Cells.

Bone morphogenetic protein 2 (BMP2) belongs to the transforming growth factor-β superfamily and plays a critical role in regulating ovarian follicle function. Currently, the role of BMP2 during cumulus expansion remains to be determined. The aim of this study was to investigate the effect of BMP2 on the regulation of pentraxin 3 (PTX3) expression (the major component of cumulus expansion) and the underlying mechanisms in human granulosa-lutein (hGL) cells. Both primary and immortalized hGL cells were used as research models. Our results showed that treatment with BMP2 significantly suppressed the basal and luteinizing hormone-induced upregulation of PTX3. In addition, BMP2 stimulated the phosphorylation of SMAD1/5/8, and this effect was abolished by the addition of BMP type I receptor inhibitors, dorsomorphin homolog 1, and dorsomorphin but not SB431542. Moreover, the knockdown of activin receptorlike kinase 2/3 or BMP receptor type II/activin receptor type IIB receptors completely reversed the BMP2-induced phosphorylation of SMAD1/5/8 and restored PTX3 expression. Similarly, the knockdown of SMAD4 completely reversed the suppressive effect of BMP2 on the expression of PTX3. These results improve our understanding of the molecular mechanisms of BMP2 signaling. Our findings suggest that BMP2 may be involved in the regulation of cumulus expansion during the periovulatory stage.

Improving porcine in vitro fertilization output by simulating the oviductal environment

Differences between the in vitro and in vivo environment in which fertilization occurs seem to play a key role in the low efficiency of porcine in vitro fertilization (IVF). This work proposes an IVF system based on the in vivo oviductal periovulatory environment. The combined use of an IVF medium at the pH found in the oviduct in the periovulatory stage (pHe 8.0), a mixture of oviductal components (cumulus-oocyte complex secretions, follicular fluid and oviductal periovulatory fluid, OFCM) and a device that interposes a physical barrier between gametes (an inverted screw cap of a Falcon tube, S) was compared with the classical system at pHe 7.4, in a 4-well multidish (W) lacking oviduct biological components. The results showed that the new IVF system reduced polyspermy and increased the final efficiency by more than 48%. This higher efficiency seems to be a direct consequence of a reduced sperm motility and lower capacitating status and it could be related to the action of OFCM components over gametes and to the increase in the sperm intracellular pH (pHi) caused by the higher pHe used. In conclusion, a medium at pH 8.0 supplemented with OFCM reduces polyspermy and improves porcine IVF output.

Analysis of follicular events in owl monkeys (Aotus azarai infulatus) using B-mode and Doppler ultrasound

Ultrasound (B-mode) was used to analyze follicular events in 12 trained female owl monkeys (Aotus azarai infulatus). The animals were examined every 48 hours for over 90 days to measure and map follicular growth in both ovaries and to measure (using Doppler velocimetry) local hemodynamic changes during the peri-ovulatory stage. There were 44 follicular growth events, each with two or three follicular waves, and a mean ± SEM interval between events of 17 ± 1.13 days. There were various hemodynamic changes during follicular growth; both vascular resistance index and pulsatility index decreased during the time when the follicle diameter peaked. Thus, both B-mode and Doppler ultrasound were useful for monitoring ovarian follicular events in owl monkeys.

Androgen/androgen receptor pathway regulates expression of the genes for cyclooxygenase-2 and amphiregulin in periovulatory granulosa cells

It is well known that the androgen/androgen receptor (AR) pathway is involved in both male and female fertility in mammals. AR knockout female mice are reported to exhibit various abnormalities in follicle development, and a subfertile phenotype. In exogenous gonadotropin-induced superovulation, serum androgen levels were robustly elevated in female mice at the periovulatory stage after human chorionic gonadotropin (hCG) treatment. At this stage, ovarian AR proteins were strongly expressed in cumulus cells. Because these results suggested that the androgen/AR pathway is involved in ovulation, we investigated the expression of ovulation-related genes in the mouse ovary treated with the nonaromatizable androgen, 5α-dihydrotestosterone (DHT). DHT treatment induced the expression of the genes for cyclooxyganase-2 (Cox-2 or prostaglandin endoperoxidase synthase 2) and the epidermal growth factor-like factor, amphiregulin (Areg), in the ovary, whereas their hCG-induced expression was suppressed by the AR antagonist flutamide. These genes were also induced by DHT in AR-expressing primary granulosa and granulosa tumor-derived cells. Reporter assays, electrophoretic shift mobility assays and chromatin immunoprecipitation assays demonstrated that androgen response sequence(s) existing upstream of each gene were responsible for androgen responsiveness and were occupied by the AR in periovulatory granulosa cells. Our results suggest that the androgen/AR pathway is involved in the ovulatory process via expression of the Cox-2 and Areg genes in periovulatory granulosa cells.

Oocyte Development in Cattle: Factors Affecting Competence.

The development of an oocyte which is competent to mature, be fertilized and sustain development until the embryonic genome takes control is a protracted process spanning foetal, pubertal and adult life which can be affected by factors such as nutrition, hormonal regulation, and environmental influence. By reviewing the biochemical and morphological profiles of the oocyte and its surrounding follicle as it develops from the primordial up to peri-ovulatory stage, we have concluded that the key transition stages of bovine oocyte and follicle development are the secondary follicle stage, mid antral stage and the peri-ovulatory period. In cattle, the secondary follicle stage is characterized by the activation of the oocyte transcriptome, deposition of the zona pellucida and the establishment of bidirectional communication between the granulosa cells and the oocyte, the tertiary or antral follicle stage is associated with the proliferation of oocyte organelles, intensive mRNA and rRNA transcription and the establishment of the maternal imprints and the periovulatory period is characterised by the extensive degradation or polyadenylation of mRNA transcripts, resumption of meiosis, spindle formation, chromosome alignment and segregation.We have also studied the global transcriptomic profiles of mature oocytes from several mammalian species and identified a number of common pathways that appear to be associated with the acquisition of oocyte development competence across species, these include WNT, Notch, and β-catenin signalling, polyadenylation and ubiquitinization, apoptosis, cytoskeleton rearrangement and cell cycle regulation. As a number of these pathways are regulated by progesterone (P4) responsive genes in other tissues and given the critical role of P4 in triggering oocyte maturation in frog and fish oocytes, we have investigated the role of P4 signaling during bovine oocyte maturation. Our studies indicate that P4 synthesis by the cumulus cells surrounding the oocyte during maturation is associated with oocyte competence. Initial investigations suggest that P4 protects the oocyte from the onset of apoptosis and that this is mediated via the classical progesterone receptor. However, it is likely that the affects of P4 are not confined to one pathway but are due to the regulation of some of the pathways and processes mentioned above, particularly during oocyte maturation. By understanding the essential changes that occur in the molecular and morphological profiles of the oocyte and its surrounding follicle and by identifying the key regulatory factors, we can identify critical checkpoints that may flag insult or injury to the oocyte or can be targeted to improve oocyte quality and developmental competence.

Meloxicam inhibits ovulation in non-human primates when administered during the follicular phase of the menstrual cycle to simulate emergency contraception

OBJECTIVE: To determine if a 5 day course of oral administration of the cyclooxygenase inhibitor meloxicam can prevent ovulation while maintaining normal menstrual cycles in nonhuman primates. DESIGN: Adult cynomolgus monkeys were studied in each of 4 menstrual cycles. In cycle 1, serum obtained each day was assayed for estradiol, progesterone, and luteinizing hormone (LH); first menses was also noted. In cycle 2, meloxicam was administered orally once each day for 5 days beginning at either mid follicular, late follicular, or periovulatory stage of the menstrual cycle; serum and menses were assessed as in cycle 1. In cycle 3, the ovary bearing the ovulatory follicle was removed 2 days after the expected day of ovulation. In cycle 4, monkeys received 5 days of oral meloxicam beginning at either mid follicular, late follicular, or periovulatory stage; the remaining ovary was removed 2 days after the expected day of ovulation. Ovarian sections were examined for the presence of an oocyte within the follicle. MATERIALS AND METHODS: Estradiol and progesterone were assessed by immunoassay; LH was assayed by RIA. Hormones levels were compared by ANOVA. Menstrual cycle length was compared by paired t-test. Ovaries were fixed, serial sectioned, and stained. RESULTS: All monkeys showed the expected pattern of changing estradiol, progesterone, and LH levels during cycle 1; meloxicam treatment in cycle 2 did not alter hormone levels or luteal phase length. Ovaries removed in cycle 3 did not contain oocytes within follicles, indicating successful ovulation. In contrast, large follicles did contain oocytes, indicating ovulation failure, after treatment with meloxicam during the mid follicular (67%), late follicular (100%), or periovulatory (50%) stage of cycle 4. CONCLUSIONS: A 5 day course of oral meloxicam can reduce the rate of oocyte release without alteration of reproductive hormones or menstrual cycle length. Meloxicam may be an effective emergency contraceptive.

Effects of growth differentiation factor 9 on cell cycle regulators and ERK42/44 in human granulosa cell proliferation

GDF-9 stimulates granulosa cell proliferation and plays important roles during folliclogenesis. However, its molecular mechanisms are still far from clear, particularly its roles in human granulosa cells around the periovulatory stage. Therefore, we investigated the effects of GDF-9 on cell cycle distribution, regulatory molecules, and signaling pathways involved in human luteinized granulosa (hLG) cells in vitro. Primary cultures of hLG cells obtained from women undergoing IVF and treated with and without recombinant GDF-9 were evaluated with and without a specific inhibitor to activin receptor-like kinase 5 (ALK5; SB-431542), ERK42/44 (PD-098059), or Smad3 (SIS3). Cell proliferation, cell cycle distribution, mRNA expression, and protein expression of relevant cell cycle molecules were determined by [(3)H]thymidine incorporation, flow cytometry, quantitative PCR, and immunoblotting, respectively. GDF-9 stimulated [(3)H]thymidine incorporation, enhanced cell transition from G(0)/G(1) to S and G(2)/M phases (whereas both SB-431542 and PD-098059 attenuated these changes), increased mRNA and protein expression of cyclin D(1) and E, and decreased those of the cyclin-dependent kinase (CDK) inhibitors p15(INK4B) and p16(INK4A). GDF-9 also activated Rb protein (a critical G(1) to S-phase regulator), ERK42/44, and Smad3. PD-098059 blocked Rb protein phorsphorylation and the increase in cyclin D(1) and E but not the decrease in p15(INK4B) and p16(INK4A) induced by GDF-9. In contrast, SIS3 reversed the decrease in p15(INK4B) and p16(INK4A) but not the increase in cyclin D(1) and E induced by GDF-9. GDF-9 stimulates hLG cell proliferation by stimulating cyclin D(1) and E and suppressing p15(INK4B) and p16(INK4A) via both Smad-dependent and Smad-independent pathways.

Comparative concentrations of steroid hormones and proteins in human peri-ovulatory peritoneal and follicular fluids

Despite the fact that both peritoneal (PF) and follicular (FF) fluids have a common ovarian origin, FF is a natural inducer of sperm acrosome reaction (AR) while PF is not. To better understand these effects, concentrations of oestradiol, progesterone and proteins in peri-ovulatory PF and FF were determined and compared. PF was aspirated by laparoscopy at the peri-ovulatory stage from women with unexplained infertility. FF was collected from patients undergoing IVF and pooled. PF and FF were tested for the presence of antisperm antibodies. Oestradiol and progesterone were measured by enzyme immunoassay, and total protein concentration was determined and analysed. The AR was determined in spermatozoa that were exposed to PF alone, progesterone-supplemented PF, progesterone, control medium, or ethanol. No antisperm antibodies were found in any fluid tested. Oestradiol and progesterone and concentrations in PF were significantly lower than in FF. Protein concentration was also significantly lower in PF than in FF, but no differences were observed between the electrophoretic patterns. When capacitated spermatozoa were exposed to progesterone-supplemented PF there was a significant increase in the percentage of AR with respect to those in PF, control medium or ethanol. These results suggest that the lack of AR-stimulating activity of PF was related to its lower progesterone concentration compared with FF.

The effect of exogenous estradiol treatment on the mRNA expression of vascular endothelial growth factor and its receptors in cultured human oviduct mucosal cells

To evaluate the responses of cultured oviduct mucosal cells to exogenous estradiol treatment in regulating the mRNA expression of vascular endothelial growth factor (VEGF) and its receptors. The mucosal layer of the ampullary regions of the human oviduct was isolated and cultured with (study groups) or without (control group) the addition of exogenous estradiol in five different concentrations. Semiquantitative reverse-transcriptase-polymerase chain reaction was performed on the oviduct mucosal cells before and after the 6-day culture. There were no significant differences in the mRNA expression of VEGF and its receptors, both KDR and flt-1, between the five study groups and the control group. The mRNA expression of VEGF and its receptors is not altered by exogenous estradiol treatment in cultured oviduct. This helps to explain the mechanism of temporal regulation of VEGF in human oviduct, which reaches the peak level in the peri-ovulatory stage when both the serum estradiol and gonadotropins concentrations are high.

Detection of implantation-related cytokines in cervicovaginal secretions and peripheral blood of fertile women during ovulatory menstrual cycles

To determine whether cytokines implicated in uterine receptivity are detectable in cervicovaginal secretions and/or serum of fertile women, and whether their concentrations undergo hormonal regulation during the menstrual cycle. Prospective, observational study. Academic medical center. Six fertile volunteers studied over two menstrual cycles. Cervicovaginal lavages (CVLs) and peripheral blood specimens were obtained during the menstrual, proliferative, periovulatory, and midsecretory phases of the cycle. Endometrial biopsies were obtained during the midsecretory phase. Concentrations of leukemia inhibitory factor (LIF), macrophage-colony stimulating factor (M-CSF), interleukin-1beta (IL-1beta), transforming growth factor-beta1 (TGF-beta1), TGF-beta2, and epidermal growth factor (EGF) in CVL and peripheral blood of fertile women; endometrial cytokine messenger (m)RNA levels in midsecretory phase; and serum E(2) and P levels throughout the menstrual cycle. Macrophage-colony stimulating factor and EGF were detectable in all CVL samples. Macrophage-colony stimulating factor concentrations were positively correlated with serum E2 levels and the E2/P ratio. Whereas EGF concentrations in serum and CVL samples remained constant throughout the menstrual cycle, individual concentrations during the secretory phase of the menstrual cycle were positively correlated with endometrial EGF mRNA levels. Interleukin-1beta, TGF-beta1, and TGF-beta2 were detected in most CVL samples. Interleukin-1beta concentrations in CVL were significantly higher at menses than at the periovulatory stage of the menstrual cycle; TGF-beta2 levels were higher at menses than at the periovulatory and secretory stages. Leukemia inhibitory factor was undetectable in CVL except at menses. In serum, TGF-beta1, TGF-beta2, M-CSF, and EGF were detectable, but there was no menstrual cycle effect. There were no correlations between cytokine levels in CVL and serum. This study demonstrates that several endometrial cytokines that have been implicated in uterine receptivity are detectable in cervicovaginal secretions and peripheral blood of reproductive-aged women, and it provides normal concentration ranges of these cytokines in CVL and serum of fertile women at four stages of the menstrual cycle. Macrophage-colony stimulating factor and EGF warrant further study in CVLs of fertile and infertile women to determine their predictive value as minimally invasive markers of uterine receptivity.