Reproductive Sciences

Endometriosis is characterized by the growth of epithelial and stromal cells outside the uterine cavity. It has a complex etiology and affects ∼10% of reproductive age women. It is accompanied by a chronic inflammatory response with substantial evidence to indicate genetic susceptibility. The causal genes and their pathways leading to endometriosis, however, are still unknown. Recently, genomewide association studies on endometriosis identified 14 genomic risk loci in women of European and Japanese ancestry. It is becoming increasingly clear that these risk regions are intergenic and thus contribute to disease susceptibility through regulatory mechanisms, most likely mediated through regulation of genes within a restricted distance from the risk variants. One endometriosis risk locus has been detected at chromosome 2q13 within an inflammatory-rich region of gene transcripts and thus may play a role in the inflammation component of the disease. We carried out detailed analysis of the genomic region 250 kb on either side of sentinel SNP rs10167914 and identified 21 transcripts which contained 6 interleukin (IL)-1 family genes, 3 previously reported coding genes that have a relationship to inflammation, 4 novel coding, or pseudogenes, and 8 noncoding RNA transcripts. Through an extensive literature search, we examined the roles these genes and their resultant proteins play in endometriosis pathogenesis. The results suggest alteration in the expression the IL-1 family transcripts either alone or as a complex milieu could have a significant influence on endometriosis and should be prioritized for future study on the implications of inflammation on endometriotic lesions.

The pipelines of pharmaceutical companies are filled with thousands of promising new compounds for a plethora of indications. Yet, a close review of the drugs that have recently been in clinical trials quickly reveals that only a handful of drugs under evaluation in women with endometriosis can be genuinely qualified as truly innovative and breakthrough drugs. Why is there such an industry-wide lukewarm interest in drug research and development for endometriosis/adenomyosis? Why are pharmaceutical companies so reluctant to initiate programs or invest in academic research in endometriosis/adenomyosis? It is evident that a substantial part of the novel druggable targets originate from research in academia. However, only the pharmaceutical industry has the resources and expertise to bring drugs to patients. In other words, we are fully dependent on the pharmaceutical industry to bring new therapeutics to the market. The aim of this editorial is to make scientists from academia aware of the enormous complexity of the drug development process, the drivers that propel pharmaceutical companies to initiate new programs and to prioritize their portfolios, the value of intellectual property rights, and also about the importance of scientific rigor, predictive translational models, and biomarkers. At the same time, the pharmaceutical industry must be made aware of the enormous opportunity at hand, as the current patient population with endometriosis/adenomyosis is just the tip of the iceberg. We hope that the insights presented here will enable the endometriosis/adenomyosis research community to find ways to valorize their knowledge and attract the interest of the industry.

In animal studies, intravenous continuous infusion or peritoneal injection of sphingosine-1-phosphate (S1P) has been shown to decrease chemotherapy- and radiotherapy-induced apoptosis on primordial follicles. Although a long-acting oral form of an S1P analogue (FTY720, fingolimod) has been recently developed and utilized in women with multiple sclerosis, there are no data exploring its ability to avoid spontaneous follicle apoptosis. Thirty 10-month-old female rats were randomly assigned to 3 groups to investigate whether fingolimod would be able to decrease the spontaneous ovarian follicle apoptosis ratio. An oral analogue form of S1P was administered for 60 days at a dose of 0.1 mg/kg (n = 10) or dose of 1 mg/kg (n = 10) per day. The control group (n = 10) received physiological serum via an orogastric feeding tube. The main outcome measures were anti-Müllerian hormone (AMH) level and nonapoptotic follicle ratio. While low-dose S1P group had comparable AMH levels to high-dose S1P group and controls, high-dose S1P group had higher mean levels of AMH, reaching marginal significance with controls (5.72 ± 0.61 vs 4.81 ± 0.85 ng/mL, P = .050). For the nonapoptotic primordial follicle ratio, both low-dose S1P group (67.0% ± 16.4% vs 29.9% ± 19.5%, P < .001) and high-dose S1P group (51.1% ± 11.5% vs 29.9% ± 19.5%, P = .023) had superior rates when compared with controls. Interestingly, low-dose S1P groups also had a statistically higher nonapoptotic primordial follicle ratio than high-dose S1P group (P = .047). Our findings suggest that a long-acting oral analogue of S1P might decrease spontaneous follicular apoptosis based on the nonapoptotic primordial follicle ratio and AMH levels when compared with placebo.

Treatments for endometriosis include pharmacological or surgical procedures that produce significant side effects. We aimed to determine how environmental enrichment (EE) could impact the progression of endometriosis using the autotransplantation rat model. Female rats were exposed to EE (endo-EE: toys and nesting materials, 4 rats per cage, larger area enclosure) or no enrichment (endo-NE: 2 rats per cage) starting on postnatal day 21. After 8 weeks, sham surgery or surgical endometriosis was induced by suturing uterine horn tissue next to the intestinal mesentery, then allowed to progress for 60 days during which EE or NE continued. At the time of killing, we measured anxiety behaviors, collected endometriotic vesicles and uterus, and processed for quantitative real-time polymerase chain reaction for corticotropin-releasing hormone (CRH), urocortin-1, CRH receptors type 1 and type 2, and glucocorticoid receptor (GR). Endometriosis did not affect anxiety-like behaviors, yet rats in enriched conditions showed lower basal anxiety behaviors than the nonenriched group. Importantly, the endo-EE group showed a 28% reduction in the number of endometriosis vesicles and the vesicles were significantly smaller compared to the endo-NE group. Endometriosis increased CRH and GR only in the vesicles of endo-NE, and this increase was dampened in the endo-EE. However, urocortin 1 was increased in the vesicles of the endo-EE group, suggesting different pathways of activation of CRH receptors in this group. Our results suggest that the use of multimodal complementary therapies that reduce stress in endometriosis could be an effective and safe treatment alternative, with minimal side effects.

To investigate the effects of trichostatin A (TSA) on nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) expression and apoptosis in human endometriotic stromal cells (HESCs), ectopic endometrial tissues were obtained from 15 patients with endometriotic cysts who underwent cystectomy. Human endometriotic stromal cells were isolated and cultured with different concentrations of TSA. Nonsteroidal anti-inflammatory drug-activated gene-1 messenger RNA (mRNA) and protein levels were evaluated by real-time polymerase chain reaction and Western blotting, respectively, and apoptosis was assessed by flow cytometry. Viability of HESCs was reduced in a dose-dependent manner by treatment with TSA. The percentage of early and late apoptotic HESCs was increased upon treatment with TSA. Nonsteroidal anti-inflammatory drug-activated gene-1 mRNA and protein expression was induced in a dose-dependent manner by TSA treatment. Gene knockdown experiments using small-interfering RNA confirmed an association between NAG-1 expression and TSA-induced apoptosis. Whether effects of TSA on NAG-1 gene expression are enhanced in the presence of 5-aza-2’-deoxycytidine (5-aza-dC) are also investigated; however, TSA-induced apoptosis was unaffected by 5-aza-dC. In conclusion, TSA induced apoptosis in HESCs via induction of NAG-1 expression. These results suggest that upregulation of NAG-1 contributes to TSA-induced apoptosis in HESCs.

The cellular function in endometriosis lesions depends on a highly estrogenic milieu. Lately, it is becoming evident that, besides the circulating levels of estrogens, the balance of synthesis versus inactivation (metabolism) of estrogens by intralesion steroid-metabolizing enzymes also determines the local net estrogen availability. In order to extend the knowledge of the role of estrogen-metabolizing enzymes in endometriosis, we investigated the gene and protein expression of a key uridine diphospho-glucuronosyltransferase (UGT) for estrogen glucuronidation, UGT1A1, in eutopic endometrial samples obtained from nonaffected and endometriosis-affected women and also from endometriotic lesions. Although UGT1A1 messenger RNA (mRNA) expression was detected at similar frequencies in endometriotic lesions and in eutopic endometrial samples, the levels of mRNA expression were greater in deep-infiltrating endometriotic lesions and in non-deep-infiltrating lesions when compared with either control endometrium or eutopic endometrium from women with endometriosis. Overall, we observed that protein expression of UGT1A1 was significantly more frequent in samples from endometriotic lesions in comparison with endometria. In addition, expression of UGT1A1 protein was greater in deep-infiltrating than in non-deep-infiltrating endometriotic lesions. We suggest that the finding of increased expression of UGT1A1 in lesions versus endometria might be related to impairment of regulatory mechanisms, in response to a highly estrogenic milieu, and that this enzyme may be a new target for therapy.

Preeclampsia (PE) is a gestational disorder with hypertension and proteinuria leading to maternal and fetal morbidity and mortality. Yes-associated protein (YAP), a transcription coactivator of Hippo pathway, was identified as an oncoprotein participated in tumorigenesis. However, the effect of YAP on trophoblast has not been investigated. In our study, YAP expression levels in first-trimester, full-term, and PE placentas were detected using quantitative real-time polymerase chain reaction (PCR), Western blot assays, and immunohistochemistry. Yes-associated protein expression was also detected in BeWo and HTR-8/SVneo. Overexpression plasmid and YAP small interfering RNA were introduced into trophoblast cells. Furthermore, we utilized a Transwell invasion assay, flow cytometry, and Cell Counting Kit-8 analysis to examine the role of YAP in the invasion, apoptosis, and proliferation of HTR-8/SVneo trophoblast cells. The result showed that both YAP messenger RNA (mRNA) and protein expression levels were less in preeclamptic placentas. Yes-associated protein mRNA and protein expression levels were more highly expressed in BeWo. Yes-associated protein enhanced cell invasion, reduced the cellular apoptotic response, and had no effect on proliferation. In addition, the overexpression of YAP activated the expression of caudal-related homeobox transcription factor 2 (CDX2), whereas reduced expression of YAP inhibited the expression of CDX2. Our results demonstrate that decreased YAP levels may contribute to the development of PE by regulating trophoblast invasion and apoptosis involving regulation of CDX2. Collectively, we proposed decreased YAP may contribute to trophoblast dysfunction, which suggests it might represent a prognostic biomarker and therapeutic target for PE.

We previously described a negative effect of xanthohumol (XN) upon placentation-related processes. We aimed to better characterize this effect by investigating the effect of XN upon the uptake of arachidonic acid (ARA), a crucial nutrient during pregnancy, by the HTR-8/SVneo human first-trimester extravillous trophoblast cell line and its relationship with the negative effect of XN upon placentation-related processes. Uptake of ¹⁴C-ARA (100 nM) was time dependent and inhibited by short-term (26 minutes) or long-term (24 hours) exposure to XN. Xanthohumol (24 hours; 5 µM) behaved as an uncompetitive inhibitor of ¹⁴C-ARA uptake; the mammalian target of rapamycin, tyrosine kinases, and c-Jun N-terminal kinases intracellular pathways were involved in this effect; and it markedly reduced long-chain acyl-CoA synthetase 1 messenger RNA levels. Moreover, the effects of XN (24 hours; 5 µM) upon cell proliferation, culture growth, migration, viability, and apoptosis index were prevented by high extracellular ARA but not by the peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist rosiglitazone. We thus conclude that ARA is an essential nutrient regulating cell viability, proliferation, culture growth, migration, and apoptosis of HTR-8/SVneo cells and that the deleterious effects of XN involve inhibition of ARA cellular uptake but appears to be independent of PPAR-γ activation.

Age-related fertility decline is hypothesized to occur mainly by the spontaneous exhaustion and deterioration of the ovarian follicle, and the accumulation of ovarian tissue damage resulting from the ovulation cycle may play roles in the process. In this study, we hypothesized that suppressing ovulation would exert protective effects against age-related fertility decline. To test this hypothesis, we established a mouse model in which oral contraceptives (OCs) were administered daily. Female C57BL/6N mice were administered OCs daily from the age of 2 months to 12 months as an ovulation suppression mouse model. Mouse fecundity was investigated by counting oocyte number after ovarian stimulation and by examining live fetuses after mating. We found that compared with control mice administered vehicle alone, 12-month-old mice administered 2-fold dose OCs used for treating humans exhibited a significantly greater average oocyte number after ovarian stimulation (8.5 ± 0.6 vs 5.9 ± 0.6, P < .01). In addition, spontaneous conception with living fetuses after mating was strikingly increased in 12-month-old mice administered OCs relative to controls (6.0 ± 1.2 vs 0.4 ± 0.3, P < .01). In the histological examination of mouse ovarian tissues, we did not detect a significant difference in ovarian follicle number, but reduced amount of brownish foamy fibrous tissues, which might reflect ovarian tissue damage, was detected in aged mice administered OCs. These results suggest the possibility that long-term OC administration might alleviate age-related fertility decline, and the improvement mechanism could be attributed to the prevention of ovarian tissue damage by suppressing ovulation.

Physiological functions of villous trophoblasts are essential for normal implantation and pregnancy, which are under fine regulations. Integrin αvβ3 has been shown to mediate cellular attachment of villous trophoblasts; however, the physiological functions of integrin αvβ3 in pregnancy have not been revealed. Here, we found that suppression of integrin αvβ3 in vivo by a small molecule inhibitor, SB-273005, resulted in abnormal pregnancy in mice. Mechanistically, suppression of integrin αvβ3 in vivo broke down the tissue homeostasis of the decidua, as revealed by disorganized histology and compromised cellular proliferation in the decidua. Compromised cellular proliferation was also observed in integrin αv knocked down human villous trophoblast cell lines, suggesting that compromised proliferation of trophoblast might contribute to the abnormal pregnancy after SB-273005 injection. Moreover, increased NK cells, as well as elevated serum levels of IFN-r in pregnant mice after SB-273005 injection, further linked aggravated pathogenic NK cell responses with the abnormal pregnancy in mice with integrin αvβ3 inhibition. Together, these data demonstrate a role of integrin αvβ3 in maintenance of decidua tissue homeostasis and of NK cell immune tolerance during pregnancy.

DJ-1 (PARK7) has been reported to be causative gene of Parkinson disease and also an oncogene. A loss in DJ-1 function can lead to cell death in neurodegenerative disease, or a gain of it can cause unregulated cell survival in cancer, respectively. DJ-1 protein is known to be expressed mainly in trophoblastic cells in the placenta with increased expression in the first trimester compared to later in term. However, its role in trophoblast regulation remains unknown. This study aimed to investigate the effect of DJ-1 regulation on a first trimester extravillous trophoblast cell line, HTR-8/SVneo. The effect of DJ-1 downregulation induced by small-interfering RNA on cell apoptosis, migration, and the pathway to regulate the cell function was assessed. Data of this study showed that DJ-1 downregulation increased apoptosis and reduced migration by regulating matrix metalloproteinase 2 and matrix metalloproteinase 9 in HTR-8/SVneo cells under both ambient and oxidative stress. Changes in cell function were demonstrated to be at least partly dependent on the AKT/S6 kinase beta-1 (S6K1) pathway. In summary, DJ-1 might play a protective role in maintaining trophoblastic cell functions through the AKT/S6K1-based pathway.

Autophagy is a survival process that maintains homeostasis in all eukaryotic cells. Recent studies show an abnormal autophagic activity in endometriosis, but the role of autophagy is controversial. Homeobox A10 (HOXA10) is a transcription factor necessary for embryonic and adult uterine development, and studies indicate that its expression decreases in endometriosis. Homeobox A10 may negatively regulate autophagy in endometriosis. To test this hypothesis, we measured the expression levels of autophagic biomarkers (beclin-1 and LC3-II) and HOXA10 proteins by Western blotting and messenger RNA (mRNA) by quantitative real-time polymerase chain reaction. Furthermore, we evaluated the serum cancer antigen 125 (CA125) levels by immunoassay. Most tested autophagic biomarker proteins and mRNAs were upregulated, whereas HOXA10 protein and mRNA were decreased in ovarian endometriomas compared with eutopic endometria of women with endometriosis and normal endometria. Compared with normal endometrium, only protein expression levels of autophagic biomarkers were increased in the eutopic endometrium of women with endometriosis. Moreover, HOXA10 was found to have a significant negative correlation with autophagy (P < .01). Serum CA125 was at a high level in endometriosis and increased with elevated revised American Fertility Society staging (I-IV). There was a significant positive correlation between serum CA125 level and LC3-II protein level and/or LC3-II/LC3-I ratio (P < .01) and a significant negative correlation between serum CA125 level and HOXA10 gene level (P < .01). In conclusion, our studies support that the deficiency of HOXA10 may induce autophagy in endometriosis, and the relationship among CA125, autophagy, and HOXA10 in endometriosis requires additional research.