Reproductive Sciences

Endometriosis, defined by the presence of ectopic endometrial lesions, is a common disease in reproductive-age women that profoundly affects patients’ quality of life. Various pathogenic models have been proposed, but the origin of endometriosis remains elusive. In this article, we propose that the mesothelial barrier, which protects the underlying stroma from endometrial transplants present in retrograde menstrual fluid, can be compromised by activation of the epithelial to mesenchymal transition (EMT) repair mechanism that lead to temporary loss of barrier integrity. Absent of the mesothelial barrier, endometrial cells can more readily adhere to the underlying peritoneal stroma and establish endometrial lesions. The hypothesis is based on the clinical and experimental observations that correlate the location of endometrial lesions with areas of mesothelial damage, together with genetic evidence that 4 genes associated with endometriosis are direct regulators of the actin-cytoskeleton, which coordinates mesothelial barrier integrity. It supports past observations that implicate the peritoneum in the pathogenesis of endometriosis and unifies previously disparate theories that endometriosis may be triggered by infection, mechanical damage, and inflammation since each of these mechanisms can induce EMT in the mesothelium. If the hypothesis is correct, inhibition of EMT in the mesothelial barrier provides a novel paradigm for the prevention and treatment of endometriosis.

Peroxiredoxin (PRX) defines a family that provides antioxidant defense in different cell types by removing reactive oxygen species (ROS) through conserved active cysteines, with the support of other types of antioxidants such as thioredoxin, glutaredoxin, and glutathione peroxidase. By regulation of intracellular ROS levels, the mammalian PRXs influence a variety of reproductive processes including gamete maturation, fertilization, and embryo development. Experimental mice lacking PRXs developed normally, but some showed accelerated decrease in fertility with aging, suggesting that deficiency of PRXs did not have lethal consequences for reproduction. The aim of this review is to summarize the role of mammalian PRXs in the reproductive performance.

Second-trimester amniotic fluid supernatant (AFS) contains cell-free fetal RNA (cffRNA) transcripts that can provide information about fetal gene expression. In a retrospective case–control study, we measured second-trimester fetal gene expression using cffRNA extracted from AFS in women who had spontaneous preterm birth (sPTB) <34 weeks and in women who delivered >37 weeks. We extracted cffRNA from AFS of women with singletons who had second-trimester genetic amniocenteses. Twenty-one gravidas who had sPTB and 21 term controls were matched 1:1 for maternal age, fetal sex, race, gestational age (GA) at the time of amniocentesis, and medication exposure. Cell-free fetal RNA was extracted and hybridized to a customized 65-gene NanoString panel containing genes related to oxidative stress, inflammation, and hypothalamic–pituitary–adrenal (HPA) axis and included 15 housekeeping genes. Two models were run, 1 examining sPTB in relation to case/control status and 1 examining sPTB in relation to GA as a continuous variable. Among cases, the gene expression of nitric oxide synthase 1 (NOS1), d-aspartate oxidase (DDO), and Beta-2-microglobulin (B2M) was higher than controls (P value < .05; false discovery rate–corrected Q value of ≤0.10). Nitric oxide synthase 1 and DDO are genes associated with oxidative stress; B2M is a marker of the fetal inflammatory response. Fetal HPA gene expression is not associated with GA at delivery or sPTB in second-trimester AFS. Alterations of fetal gene expression related to inflammation and oxidative stress antedate clinical symptoms and may be useful for early identification of patients at risk of having an sPTB.

Hyperinnervation in endometriosis is now well documented, but so far only a few neurotrophins have been identified. Since endometriotic stromal cells secrete thromboxane A2 (TXA₂), we sought to determine whether TXA₂, derived from endometriotic stromal cells, induces neurite outgrowth. Using primary sensory neurons derived from rat dorsal root ganglia (DRG) and ectopic endometrial stromal cells (EESCs) derived from human ovarian endometrioma tissues, we treated the primary neurons with different concentrations of U-46619, a stable TXA₂ mimetic, and performed a neuronal growth assay. The primary neurons were also cocultured with a vehicle, nerve growth factor (NGF, serving as a positive control), the supernatant of EESC culture medium, or the supernatant of EESCs pretreated with ozagrel, a thromboxane synthase inhibitor, and a neuronal growth assay was performed. The total neurite length was evaluated through immunofluorescence microscopy. We found that U-46619 significantly increased the neurite outgrowth in DRG neurons in a concentration-dependent fashion (P < .001). It also increased the number of neurite ends in a concentration-dependent fashion. Ozagrel treatment alone had no effect on the neurite growth (P > .05), and the treatment with the supernatant of EESCs induced neurite outgrowth just as potently as that treated with NGF (positive control; P > .05). Remarkably, treatment with the EESC supernatant increased the neurite outgrowth by nearly 3-fold as compared with the control (P < .01), but the pretreatment with ozagrel abolished the stimulatory effect of the EESC by 31.3% (P < .05). These findings indicate that EESCs potently induce neurite outgrowth, and endometriosis-derived TXA₂ is responsible, at least in part, for this neurotrophic effect.

Estradiol and its nuclear receptors, estrogen receptor (ER) α and ER-β, have important functions in endometriosis, and the transcriptional activity of these receptors is modulated by coactivators and corepressors. The steroid receptor RNA activator 1 (SRA1) produces SRA long noncoding RNA (lncRNA) and SRA protein (SRAP), which regulate ER expression at the RNA and protein levels in some hormone-dependent tumors via an alternative splicing event. However, only a few are reported on their expressions in endometriosis. Here, we observed that low expression levels of SRA lncRNA and ER-α but relatively high expression levels of SRAP and ER-β were detected in ovarian endometriotic tissues versus normal endometrial tissues. Steroid receptor RNA activator 1–small interfering RNA treatment significantly increased ER-α levels but reduced ER-β levels in endometriotic stromal cells (ESCs). Furthermore, the treatment can also attenuate the proliferation and promote early apoptosis in these cells. Our results indicate that the regulation of ER via SRA in ovarian endometriosis may play a significant role in the growth of ESCs.

The aims of the present study were to analyze if the superior ovarian nerve (SON) plays a role in the neural signals from suprachiasmatic nucleus (SCN) that lead to ovulation and ovarian steroids secretion on proestrus day. Rats on proestrus day were treated at 11.00 to 11.30 or 17.00 to 17.30 hours with 1 of the 3 experimental procedures (1) unilateral or bilateral SON sectioning, (2) unilateral or bilateral injury to the SCN, or (3) unilateral injury to the SCN followed by unilateral sectioning of the SON ipsilateral to the treated SCN. Treatments were evaluated 24 hours after surgical procedures. Compared to laparotomized animals, right or bilateral SON sectioning treatment at 17.00 hours resulted in lower ovulation rates and number of ova shed by the right ovary. The ovaries of nonovulating animals showed early follicular luteinization signs and trapped ova. Bilateral SCN injury treatment at 11.00 hours resulted in anovulation; whereas right SCN injury treatment, with or without right SON sectioning, resulted in a lower number of ova shed. Injecting luteinizing hormone-releasing hormone to animals with bilateral SCN injury restored ovulation. In rats with unilateral or bilateral SON sectioning, or with injury to the SCN with or without unilateral sectioning of the SON, the effects on hormone levels depended of the hormone studied and the time of day treatment was performed. The present results suggest that on proestrus day, the role of the right or both SON in ovulation and steroid hormone secretion regulation takes place through different neuroendocrine mechanisms from SCN.

Preeclampsia (PE) (gestational proteinuric hypertension) is the leading cause of maternal and perinatal mortality worldwide. Although placental endothelial dysfunction and oxidative stress are known to contribute to PE, the exact pathological basis for this disorder remains unclear. Previously, we demonstrated that DNA damage at the maternal–fetal interface is more common in the placentas of women with PE than normotensive controls. In this study, we utilized an in vivo comparative study, including 20 preeclamptic women and 8 healthy control subjects, and an in vitro hypoxia/reperfusion model to mimic the effects of oxidative stress at the maternal–fetal interface. We tracked the spatial pattern of expression of 2 base excision repair proteins, 8-oxoguanine glycosylase (OGG1) and apurinic/apyrimidinic endonuclease-1 (APE1), at the maternal–fetal interface in response to oxidative stress. In vivo, we found a significant increase in OGG1 and APE1 concentrations in PE placental tissues as compared to normotensive controls (P < .0001). Further, our in vitro study revealed that OGG1 and APE1 expression is much greater in maternal cells (decidua) than in fetal cells (cytotrophoblasts) of placental tissue subjected to oxidative stress (P < .0001). Our results suggest that OGG1 and APE1 likely protect decidual cells from oxidative base damage.

Twins are often born small and early and have increased risk of obesity and diabetes later in life. Twin conception in sheep, regardless of whether the pregnancy continues as twins or is reduced to singleton in early gestation, alters offspring growth trajectory and body composition in young adulthood. We hypothesized that twin conception would result in insulin resistance in adulthood, with insulin-resistant adipose tissue and skeletal muscle phenotypes. At 3 years of age, body weight was not different among singletons, twins, and reductions; females weighed less than males. Singletons were leaner than reductions, with twins intermediate. Twins and reductions had decreased insulin sensitivity compared with singletons (singletons: mean [standard error of the mean]: 4.75 [0.4], twins: 3.34 [0.3], reductions: 3.67 [0.2] mg·I μU⁻¹·kg⁻¹·min⁻¹, P < .01). There were no group differences in adipocyte size, adipose tissue, or circulating tumor necrosis factor α, monocyte chemoattractant protein 1, or interleukin 6 concentrations. In males, omental and subcutaneous adipose SLC2A4 was 1.5- to 2.0-fold greater in twins and reductions than in singletons (P < .01) and SLC2A1 was greater in reductions than in singletons. Skeletal muscle IRS-1 was decreased in male twins but increased in female twins, compared to singletons (P ≤ .01), with no effect on reductions in either sex. Skeletal muscle SLC2A4 was decreased in female twins and reductions but elevated in male twins and reductions compared to singletons (P ≤ .01). We conclude that adult twin insulin resistance is not due to adipose tissue phenotype, but potentially phenotypic effects in skeletal muscle, and obesity is a result of twin conception per se with its origins in early gestation.

Regulatory T cells (Treg) and T helper 17 cells (Th17) are 2 distinct subsets of CD4⁺ T cells, which are mutually antagonistic in the immune response. Recently, dysregulation of these 2 cell subsets have been described in the pathogenesis of unexplained recurrent spontaneous abortion (URSA). The purpose of this study was to investigate whether the Treg/Th17 balance was perturbed in URSA patients and to explore contributing factors. We found that the proportion of Treg cells and expression of forkhead box P3 (Foxp3) messenger RNA (mRNA) were significantly lower in URSA patients than in healthy controls. However, the proportion of Th17 cells and expression of retinoid-related orphan nuclear receptor-γt (ROR-γt) mRNA were higher in URSA patients than in controls, revealing inverse correlation with Treg. The ratio of Treg/Th17 and Foxp3/ROR-γt decreased in patients with URSA compared to healthy controls. The serum levels of interleukin (IL)-6 and IL-17A were significantly higher, whereas IL-10 was lower in URSA patients compared with controls, and the level of IL-6 showed a positive correlation with Th17, ROR-γt and inverse correlation with Treg, Foxp3. The present study indicated that an imbalance between Treg and Th17 cells might be implicated in the pathogenesis of URSA and this seems to relate to elevation in serum IL-6 level.

Children born preterm are reported to be at increased risk of developing noncommunicable diseases in later life. Altered placental DNA methylation patterns are implicated in fetal programming of adult diseases. Our earlier animal studies focus on micronutrients (folic acid, vitamin B₁₂) and long-chain polyunsaturated fatty acids (LCPUFAs) that interact in the 1 carbon cycle, thereby influencing methylation reactions. Our previous studies in women delivering preterm show altered plasma levels of micronutrients and lower plasma LCPUFA levels. We postulate that alterations in the micronutrient metabolism may affect the regulation of enzymes, methionine adenosyltransferase (MAT2A), and SAH-hydrolase (AHCY), involved in the production of methyl donor S-adenosylmethionine (SAM), thereby influencing the methylation potential (MP) in the placenta of women delivering preterm. The present study, therefore, examines the mRNA, protein levels of enzymes (MAT2A and AHCY), SAM, S-adenosylhomocysteine (SAH) levels, and global DNA methylation levels from preterm (n = 73) and term (n = 73) placentae. The enzyme messenger RNA (mRNA) levels were analyzed by real-time quantitative polymerase chain reaction, protein levels by enzyme-linked immunosorbent assay, and SAM–SAH levels by high-performance liquid chromatography. The mRNA levels for MAT2A and AHCY are higher (P < .05 for both) in the preterm group as compared to the term group. S-Adenosylmethionine and SAH levels were similar in both groups, although SAM:SAH ratio was lower (P < .05) in the preterm group as compared to the term group. The global DNA methylation levels were higher (P < .05) in women delivering small for gestation age infants as compared to women delivering appropriate for gestation age infants at term. Our data showing lower MP in the preterm placenta may have implications for the epigenetic programming of the developing fetus.

Polycystic ovary syndrome (PCOS) is a complex, heterogeneous endocrine and metabolic disorder affecting 5% to 10% of reproductive-age women. A high rate of granulosa cell (GC) proliferation contributes to the abnormal folliculogenesis in patients with PCOS. Evidence has proved that dysregulation of microRNAs is involved in the pathogenesis of PCOS. In this study, we investigated the effect of miR-145 on cell proliferation and the underlying mechanism of miR-145 in isolated human GCs from the aspirated follicular fluid in women with PCOS. Our findings showed that miR-145 is downregulated in human GCs from PCOS. The miR-145 mimics suppress cell proliferation and promoted cell apoptosis in human GCs from PCOS. However, miR-145 inhibitor promotes cell proliferation and inhibited cell apoptosis. Moreover, using a dual-luciferase reporter assay, we confirmed that the insulin receptor substrate 1 (IRS1) gene is a direct target of miR-145. The miR-145 mimics inhibited messenger RNA and protein IRS1 expression levels, and silencing of IRS1 by small interfering RNA inhibits human GC proliferation, but IRS1 overexpression abrogates the suppressive effect of miR-145 mimics. Furthermore, miR-145 mimics can inhibit the activation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase (ERK). The IRS1 overexpression abrogates the suppressive effect of miR-145 mimics on MAPK/ERK signaling pathways. Together, miR-145 mimics suppress cell proliferation by targeting and inhibiting IRS1 expression to inhibit MAPK/ERK signaling pathways. Our study further found that high concentrations of insulin decreases the miR-145 expression, upregulates IRS1, and promotes cell proliferation. These observations showed that miR-145 is a novel and promising molecular target for improving the dysfunction of GCs in PCOS.

Approximately 20% of pregnant women smoke despite intentions to quit. Smoking cessation drugs, such as nicotine replacement therapy (NRT) and bupropion, are recommended treatments. Adverse cardiovascular outcomes in offspring have raised concerns about NRT’s safety during pregnancy. However, the effect of bupropion is unknown. Using a rat model, we determined whether NRT and bupropion interventions during pregnancy are safer than continued smoking on offspring’s cardiovascular function. Male offspring of controls and dams exposed to cigarette smoke (1.6 packs/day, inhalation), nicotine (2 mg/kg/d subcutaneously), and bupropion (13 mg/kg twice daily orally) were assessed for fetoplacental weight, cardiac function, blood pressure, and vascular reactivity. Fetoplacental weights were decreased and spontaneous beating and intracellular calcium in neonatal cardiomyocytes were increased in smoking, nicotine, and bupropion offspring; however, these effects were more accentuated in smoking followed by nicotine and bupropion offspring. Increased heart rate and decreased cardiac output, stroke volume, and left ventricular percent posterior wall thickening were observed in smoking, nicotine, and bupropion offspring. The left ventricular mass was reduced in smoking and nicotine but not in bupropion offspring. Blood pressure was higher with decreased endothelium-dependent relaxation and exaggerated vascular contraction to angiotensin II in smoking and nicotine offspring, with more pronounced dysfunctions in smoking than nicotine offspring. Maternal bupropion did not impact offspring’s blood pressure, endothelium-dependent relaxation, and vascular contraction. In conclusion, maternal nicotine intervention adversely affects offspring’s cardiovascular outcomes, albeit less severely than continued smoking. However, bupropion causes cardiac derangement in offspring but does not adversely affect blood pressure and vascular function.

Inflammasomes are cytosolic signaling platforms that regulate the activation of caspase (CASP)-1, which induces the maturation of interleukin (IL)-1β and IL-18. Herein, we determined whether the chorioamniotic membranes from women in spontaneous labor at term with acute histologic chorioamnionitis express major inflammasome components and whether these changes are associated with the activation of CASP-1 and CASP-4 and the release of mature IL-1β and IL-18. When comparing the chorioamniotic membranes from women in spontaneous labor at term with acute histologic chorioamnionitis to those without this placental lesion, we found that (1) the messenger RNA (mRNA) abundance of NLR family pyrin domain containing 3 (NLRP3), NLR family CARD domain containing 4 (NLRC4), absent in melanoma 2 (AIM2), and nucleotide binding oligomerization domain 2 (NOD2) was higher; (2) the NLRP3 and NLRC4 protein quantities were increased; (3) the mRNA and protein expressions of CASP-1 and its active forms were greater; (4) CASP-4 was increased at the mRNA level only; (5) the mRNA and protein expressions of IL-1β and its mature form were higher; and (6) a modest increase in the total protein concentration and abundance of the mature form of IL-18 was observed. In vitro incubation of the chorioamniotic membranes with the CASP-1 inhibitor, VX765, decreased the release of endotoxin-induced IL-1β and IL-18 (2-fold) but not IL-6 or tumor necrosis factor α. In conclusion, spontaneous labor at term with acute histologic chorioamnionitis is characterized by an upregulation of inflammasome components which, in turn, may participate in the activation of CASP-1 and lead to the release of mature IL-1β by the chorioamniotic membranes. These results support a role for the inflammasome in the mechanisms responsible for spontaneous labor at term with acute histologic chorioamnionitis.