Wen-Juan Wang

Adoptive transfer of pregnancy-induced CD4+CD25+ regulatory T cells reverses the increase in abortion rate caused by interleukin 17 in the CBA/J×BALB/c mouse model

STUDY QUESTION Could adoptive transfer of pregnancy-induced CD4+CD25+ regulatory T cells (Tregs) reverse the increase in abortion rate caused by interleukin 17 (IL-17) in the CBA/J × BALB/c mouse model? SUMMARY ANSWER The effects of exogenous IL-17 on increased abortion rate, as well as decreased transforming growth factor (TGF)-β and IL-10 expression, are reversed by a pre-mating transfusion of Tregs in a mouse model of pregnancy. WHAT IS KNOWN ALREADY IL-17 is a pro-inflammatory cytokine mainly expressed by T helper 17 cells, and plays a pivotal role in the pathogenesis of endometriosis, miscarriage, preterm labor and pre-eclampsia. The activity of Th17 cells is attenuated by the anti-inflammatory action of Tregs. STUDY DESIGN, SIZE, DURATION Fifty microliters of phosphate-buffered saline (PBS) (Group 1,) or recombinant IL-17 (rIL) (10 µg/mouse) supernatant (Group 2) was administered in the vaginal vaults of anesthetized pregnant CBA/J mice on Day 1 of pregnancy. Tregs (2 × 10(5) cells) purified from pregnant CBA/J × BALB/c mice were given i.v. via the tail vein 2 days before mating (Group 3) or on Day 7 of pregnancy (Group 4). PARTICIPANTS/MATERIALS, SETTING, METHODS Mice (n = 40) were randomly assigned to one of four experimental groups. The numbers of surviving and reabsorbed fetuses in each group were counted on Day 14 of pregnancy, and the expression of interferon (IFN)-γ, IL-4, TGF-β and IL-10 in the decidual tissue was assessed by real-time RT-PCR and western blotting. MAIN RESULTS AND THE ROLE OF CHANCE Normal pregnant CBA/J mice mated with BALB/c males which received transvaginal rIL-17 presented with a significantly increased abortion rate compared with the group which received PBS (27.7 versus 9.9%, respectively; P < 0.05). The transfusion of pregnancy-induced Tregs from 14-day normal pregnant mice 2 days before mating reduced the abortion rate caused by IL-17 (12.5 versus 27.7%, respectively; P < 0.05), while transfusion of Tregs on Day 7 of pregnancy had no effect. Transfusion of Tregs did not affect IFN-γ or IL-4 expression in the decidual tissue at either the mRNA or protein level. Administration of rIL-17 resulted in a decrease in production of TGF-β and IL-10 at both mRNA and protein levels (P < 0.05). Transfusion of Tregs before mating increased TGF-β and IL-10 mRNA and protein levels (P < 0.05), while Tregs transfusion at Day 7 of pregnancy had no effect on TGF-β or IL-10 expression. LIMITATIONS, REASONS FOR CAUTION These data derive from only a small number of mice. It is unclear whether the same effects would be seen in humans. WIDER IMPLICATIONS OF THE FINDINGS Abnormally elevated expression of IL-17 in the feto-maternal interface may result in miscarriage. Transfer of antigen-specific Tregs before mating takes place may have potential applications in the prevention of recurrent spontaneous abortion. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by a grant from the National Natural Science Foundation of China (81370013, 81000277 and 81300533) and Shandong Provincial Natural Science Foundation, China (ZR2013HQ002). There were no conflicts of interest.

Dysregulation of macrophage activation by decidual regulatory T cells in unexplained recurrent miscarriage patients

CD4(+)CD25(+) T cells (Treg cells) and macrophages play roles in the maintenance of maternal-fetal immunological tolerance. Treg cells suppress the function of macrophages via mechanisms mediated by cell-cell contact and production of soluble factors. The purpose of this study was to investigate regulation of macrophages by Treg cells within decidua from patients with unexplained recurrent miscarriage (RM) and normal control women during early pregnancy. Treg cells and macrophages were isolated from deciduas of unexplained RM (n=15) and control women (n=15) by magnetic cell separation and co-cultured for six days. Regulation of macrophages by Treg cells was assessed in the presence and absence of neutralizing anti-TGFβ antibodies and in transwell experiments. Expression of CD80, CD86, IL10, and IFNγ by macrophages was measured by flow cytometry or ELISA. Macrophage expression of CD80 and CD86 was higher in deciduas of unexplained RM patients compared with controls whereas the expression of IL10 was lower. There was no difference in the expression of IFNγ by macrophages between the two groups. Treg cells inhibited macrophage expression of CD80, CD86 and IFNγ and increased the expression of IL10. The regulatory effects of Treg cells were abrogated in the presence of neutralizing anti-TGFβ antibodies or by transwell culture. The phenotype of macrophages therefore differed in unexplained RM patients compared with normal early pregnant subjects. Macrophage regulation by Treg cells was shown to be mediated by cell-cell contact and TGFβ and this capacity was decreased in unexplained RM patients.

Regulation of the expression of Th17 cells and regulatory T cells by IL-27 in patients with unexplained early recurrent miscarriage

In normal pregnancy, tolerance of the maternal immune system with regard to the genetically incompatible fetus depends on the interactions of an array of cytokines secreted by maternal and fetal cells at the site of implantation. Earlier research indicating that altered immunity exists in unexplained recurrent miscarriage (RM) has been dominated by the Th1/Th2 hypothesis. Recently, the Th1/Th2 paradigm has been expanded into the Th1/Th2/Th17 and regulatory T cells paradigm. We recently demonstrated a prevalence of Th17 cells, an inverse relationship between Th17 cells and regulatory T cells and deregulation of Th17 cells by regulatory T cells in early pregnancy in unexplained RM patients. In this study, we investigated the expression of IL-27 and the role of the cytokine IL-27 in the regulation of Th17/Treg expression. Quantitative real-time RT-PCR and Western blot analyses were performed to evaluate IL-27 expression in deciduas from unexplained RM patients, spontaneous miscarriage (SM) patients and healthy women following elective abortion in the early stages of normal pregnancy (control). Regulation of IL-17, TGF-β and IL-10 expression in CD4(+) T cells in unexplained RM patients by IL-27 was assessed using enzyme-linked immunosorbent assay (ELISA). Expression of IL-27 was lower in deciduas of patients with unexplained RM compared with SM and control subjects. IL-27 inhibited IL-17 expression and enhanced IL-10 expression in a dose-dependent manner. IL-27 had no effect on TGF-β expression. IL-27 regulates the expression of IL-17 and IL-10, which are predominantly secreted by Th17 cells and regulatory T cells in unexplained RM patients.

Aged men share the sperm protein PATE1 defect with young asthenozoospermia patients

STUDY QUESTION Does a defect in the human sperm-located protein prostate and testis expressed 1 (PATE1) exist in both aged men and young asthenozoospermia patients? SUMMARY ANSWER A defect in sperm PATE1 exists in both aged men and young asthenozoospermia patients, and an antibody against PATE1 can decrease human sperm motility and zona-free hamster oocyte penetration. WHAT IS KNOWN ALREADY Both aged men and young asthenozoospermia patients have poor sperm quality. The PATE1 protein seems to mediate sperm-egg interactions; however, the mechanisms are still unknown. STUDY DESIGN, SIZE, DURATION This was a case-control study including 60 young fathers (aged 28-32 years) and 60 aged fathers (68-72 years old) who donated semen by masturbation after 7 days of sexual abstinence. Comparative sperm proteome analysis from the young fathers and aged fathers was performed to discover key proteins. The target protein PATE1 was chosen and validated by western blotting and immunohistochemistry. Quantitative assessment of sperm PATE1 protein was performed on sperm from 60 young fathers, 60 aged fathers and 110 young asthenozoospermia patients. Furthermore, an antibody against PATE1 assay was used to test whether PATE1 participated in sperm motility and penetration of zona-free hamster egg. PARTICIPANTS/MATERIALS, SETTING, METHODS Samples were pooled and separated by two-dimensional gel electrophoresis followed by identification by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Western blotting and immunohistochemistry were used to validate the confidence of proteomic data. Sperm immunofluorescence quantification experiments disclosed whether the aged men indeed shared the same PATE1 defect with 110 young asthenozoospermia patients. The sperm motility test and penetration of zona-free hamster egg assay were performed for PATE1. MAIN RESULTS AND THE ROLE OF CHANCE Twenty-two sperm proteins with significant differential expression between young adults and aged men were identified (P < 0.05, mean ratio >1.5), including 13 proteins with decreased expressions with aging. Based on bioinformatics, PATE1 was chosen for further study, and exhibited similar changes in expression level and localization on sperm from aged men and young asthenozoospermia patients. Antibody blocking revealed that PATE1 was involved in sperm-egg penetration and sperm motility. LIMITATIONS, REASONS FOR CAUTION Before any clinical application of PATE1 as a biomarker for the diagnosis of male infertility, more cases should be used to evaluate confidence in this approach. WIDER IMPLICATIONS OF THE FINDINGS This study revealed a common molecular basis underlying the decline in sperm quality in the natural aging process and in young men with asthenozoospermia. The data should greatly contribute to the development of molecular evaluation of sperm quality, and the diagnosis and treatment of asthenozoospermia. STUDY FUNDING/COMPETING INTERESTS This work was supported by grants from the National Natural Science Foundation of China (NO. 81300533, 81370013 and 81000277) and Shandong Provincial Natural Science Foundation, China (ZR2013HQ002, ZR2014HQ068). The authors declare no competing financial interests.

New insight into the castrated mouse epididymis based on comparative proteomics

The mammalian epididymis is an important male accessory gland where the spermatozoa gain the ability to fertilise the egg. To further understand the effects of testicular factors on the epididymis, the proteome of castrated adult mice and sham controls was compared using high-resolution two-dimensional gel electrophoresis following identification of proteins by matrix-assisted laser desorption ionisation time-of-flight/time-of-flight mass spectrometry. Twenty-three differentially expressed proteins (11 upregulated and 12 downregulated) were identified in epididymides from castrated. Bioinformatic analysis indicated that these castration-responsive proteins participated in energy metabolism and the antigen processing and presentation pathway. The differential expression levels were further validated by western blotting. The differentially expressed proteins may serve as potential candidates in studies of epididymal function and male infertility.

Proteome profiling of the sperm maturation milieu in the rhesus monkey (Macaca mulatta) epididymis.

The mammalian spermatozoon acquires its fertilising potential during transit through the epididymis, where it interacts with epididymal luminal fluid proteins (the sperm maturation milieu). In order to highlight the epididymal-specific function of the rhesus monkey (Macaca mulatta) in sperm maturation, two-dimensional gel electrophoresis of epididymal luminal fluid proteins was followed by identification by Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF/MS) or MALDI-TOF/TOF and revealed over five hundred spots, comprising 198 non-redundant proteins. Some mass spectrometric data were confirmed by western blotting identification. Some common epididymal fluid proteins were identified, such as clusterin, α-1-antitrypsin, malate dehydrogenase, L-lactate dehydrogenase B, α-1-acid glycoprotein 1 and α-mannosidase. More than 7% of all proteins were anti-oxidative, which might control oxidative stress within the male tract. When compared with bull and human epididymal luminal fluid proteins, those in the rhesus monkey had more overlap with the human, which provides evidence of a close evolutionary relationship between the rhesus monkey and man. This study provides new proteomic information on possible rhesus monkey epididymal functions and novel potential biomarkers for the noninvasive assessment of male fertility.

Characteristics of testis-specific phosphoglycerate kinase 2 and its association with human sperm quality

STUDY QUESTION Is there an association between the expression of phosphoglycerate kinase (PGK) 2 in spermatozoa and sperm quality in both elderly men and young asthenozoospermia patients? SUMMARY ANSWER Spermatozoa from elderly men and young asthenozoospermia patients show decreased expression of PGK2, which has a close positive relationship with sperm quality. WHAT IS KNOWN ALREADY PGK1 and PGK2 are involved in spermatogenesis and thought to be related to sperm motility. However, limited information is known about their temporal-spatial expression in human spermatogenesis and their relationship with sperm quality. STUDY DESIGN, SIZE, DURATION This was a case-control study including 30 healthy young males (aged 28-31 years), 30 elderly men (aged 68-70 years), and 30 asthenozoospermic patients (aged 25-40 years, progressive motility <32%) who donated semen samples. Furthermore, young testes samples were obtained from five fathers (27-33 years old) who had died in car accidents, while aged testes samples were obtained from five elderly fathers (78-82 years old) who were prostate cancer patients. PARTICIPANTS/MATERIALS, SETTING, METHODS Semen samples from young adults, elderly men and asthenozoospermic patients were prepared, and their parameters were assessed by Computer-Aided Sperm Analysis (CASA). Sperm proteins were extracted for western blot analysis. Immunohistochemistry was used to characterize the cellular localization of PGK1 and PGK2 in testes samples. Sperm immunofluorescence quantification experiments identified the differential expression of PGK1 and PGK2 in sperm from young adults, elderly men and asthenozoospermic patients. Antibodies against PGK1 and PGK2 were used to test their influence on sperm motility and penetration into viscous media. A modified Kremer test using methyl cellulose was adopted to assess sperm function via penetration into viscous media. MAIN RESULTS AND THE ROLE OF CHANCE Cellular localization analysis showed that PGK1 was mainly expressed in spermatogonia whereas PGK2 was mainly expressed in round spermatids. Expression levels of both PGKs were significantly decreased in the testis with ageing (P < 0.05). Western blot and immunofluorescence quantification showed markedly lower expression of PGK2 (P < 0.05) in sperm from elderly men or asthenozoospermic patients compared sperm from with healthy young men. Sperm functional analysis validated the close relationship between expression of PGK2 and sperm motility (staining percentage, r = 0.60, P < 0.05; intensity, r = 0.59, P < 0.05). Use of an anti-PGK2 antibody on sperm significantly decreased their ability to penetrate into a cervical mucus substitute (P < 0.05). LIMITATIONS, REASONS FOR CAUTION Before any clinical applications using PGK2 to assess sperm quality can be developed, more cases should be used to evaluate this approach. WIDER IMPLICATIONS OF THE FINDINGS The study provides new insights into the role of PGKs in male reproduction. The results also indicate that PGK2 is a promising molecular candidate for the assessment of sperm quality and the screening of male contraceptive targets. STUDY FUNDING/COMPETING INTERESTS This work was supported by grants from the National Natural Science Foundation of China (no. 81300533, 81370013 and 81000277) and Shandong Provincial Natural Science Foundation, China (ZR2013HQ002). The authors declare no competing financial interests.

Effect of dehydroepiandrosterone administration in Chinese women over 37 years undergoing assisted reproductive techniques

OBJECTIVE To evaluate the effect of DHEA supplementation on in vitro fertilization (IVF) parameters and pregnancy outcomes in patients older than 37 years with normal ovarian reserve. STUDY DESIGN A retrospective cohort study was conducted to evaluate the impact of DHEA supplementation on IVF outcome of infertile women over 37 years with normal ovarian reserve between January 2012 and July 2014. 243 patients (study group) received 75mg of DHEA daily (25mg three times daily) before the IVF cycle. Another 243 patients (control group) received infertility treatment, but did not receive DHEA. The IVF outcome parameters in each group were compared. RESULTS Both groups did not show statistically significant differences in terms of patient demographics characteristics, mean numbers of oocytes retrieved and mature oocytes rate. While patients in the DHEA group have the significantly higher implantation rate and live birth rate compared with controls (30.13% versus 22.70%, 43.33% versus 28.26%). We also found that the cycle cancellation rate and miscarriage rate were lower in the DHEA group (1.23% versus 5.34%, 13.33% versus 28.89%). CONCLUSION DHEA supplementation may significantly improve IVF outcomes in infertile women over the age of 37.

Periodic elevation of regulatory T cells on the day of embryo transfer is associated with better in vitro fertilization outcome.

Treg cells have been shown to be important in maintaining maternofetal tolerance, but the expression of Tregs in assisted reproductive technology (ART) in women on the day of embryo transfer (D0), 5days (D5) and 14days after ET (D14); the related factors influencing the expression levels of Tregs; the proliferation ability and the relevant cytokine epression by Tregs on D14 have not been investigated. In this study, 124 women undergoing in vitro fertilization-intracytoplasmic sperm injection (IVF/ICSI) were enrolled. Early morning fasting blood samples were obtained for the measurement of Tregs and other relevant indicators on the D0, D5and D14days after ET. we showed that the Tregs were increased on D0 and D14 in pregnant women, while there was no obvious fluctuation in non-pregnant women. IL-10 and TGF-β levels and the expansion of Tregs were significantly higher in successfully pregnant women than in non-pregnant women on D14. The levels of E2, P did not significantly differ between the groups. We suggest that periodic elevation of Tregs on the day of ET was associated with higher embryo implantation rate after ART.