Cryopreservation of Ovarian and Testicular Tissue and the Influence on Epigenetic Pattern.

Ovarian tissue cryopreservation (OTC) or testicular tissue cryopreservation (TTC) are effective and often the only options for fertility preservation in female or male patients due to oncological, medical, or social aspects. While TTC and resumption of spermatogenesis, either in vivo or in vitro, has still be considered an experimental approach in humans, OTC and autotransplantation has been applied increasingly to preserve fertility, with more than 200 live births worldwide. However, the cryopreservation of reproductive cells followed by the resumption of gametogenesis, either in vivo or in vitro, may interfere with sensitive and highly regulated cellular processes. In particular, the epigenetic profile, which includes not just reversible modifications of the DNA itself but also post-translational histone modifications, small non-coding RNAs, gene expression and availability, and storage of related proteins or transcripts, have to be considered in this context. Due to complex reprogramming and maintenance mechanisms of the epigenome in germ cells, growing embryos, and offspring, OTC and TTC are carried out at very critical moments early in the life cycle. Given this background, the safety of OTC and TTC, taking into account the epigenetic profile, has to be clarified. Cryopreservation of mature germ cells (including metaphase II oocytes and mature spermatozoa collected via ejaculation or more invasively after testicular biopsy) or embryos has been used successfully for many years in medically assisted reproduction (MAR). However, tissue freezing followed by in vitro or in vivo gametogenesis has become more attractive in the past, while few human studies have analysed the epigenetic effects, with most data deriving from animal studies. In this review, we highlight the potential influence of the cryopreservation of immature germ cells and subsequent in vivo or in vitro growth and differentiation on the epigenetic profile (including DNA methylation, post-translational histone modifications, and the abundance and availability of relevant transcripts and proteins) in humans and animals.

Ribosomal DNA methylation in human and mouse oocytes increases with age.

An age-dependent increase in ribosomal DNA (rDNA) methylation has been observed across a broad spectrum of somatic tissues and the male mammalian germline. Bisulfite pyrosequencing (BPS) was used to determine the methylation levels of the rDNA core promoter and the rDNA upstream control element (UCE) along with two oppositely genomically imprinted control genes (PEG3 and GTL2) in individual human germinal vesicle (GV) oocytes from 90 consenting women undergoing fertility treatment because of male infertility. Apart from a few (4%) oocytes with single imprinting defects (in either PEG3 or GTL2), the analyzed GV oocytes displayed correct imprinting patterns. In 95 GV oocytes from 42 younger women (26-32 years), the mean methylation levels of the rDNA core promoter and UCE were 7.4±4.0% and 9.3±6.1%, respectively. In 79 GV oocytes from 48 older women (33-39 years), methylation levels increased to 9.3±5.3% (P = 0.014) and 11.6±7.4% (P = 0.039), respectively. An age-related increase in oocyte rDNA methylation was also observed in 123 mouse GV oocytes from 29 4-16-months-old animals. Similar to the continuously mitotically dividing male germline, ovarian aging is associated with a gain of rDNA methylation in meiotically arrested oocytes. Oocytes from the same woman can exhibit varying rDNA methylation levels and, by extrapolation, different epigenetic ages.

Impact of Nonylphenols and Polyhalogenated Compounds in Follicular Fluid on the Outcome of Intracytoplasmic Sperm Injection.

Endocrine-disrupting chemicals (EDCs) interfere with the mammalian hormone system and alter its endo- and paracrine regulation. The goal of the present study was to examine the presence of 14 EDCs, including the technical mixture of nonylphenols and Mirex, in human follicular fluid (FF) and to find a potential correlation between endocrine active substances and a possible impact on female fertility. Furthermore, potential sources of EDC exposition regarding patients' lifestyle and socioeconomic factors were investigated. Human FF was collected from a total of 210 women undergoing intracytoplasmic sperm injection-treatment cycles because of male subfertility. The presence of EDCs was analyzed using gas chromatography coupled with mass spectrometry. Thirteen of the 14 investigated EDCs were present in every FF sample; compounds with the highest concentrations in FF were nonylphenol and Mirex. Nearly all kinds of EDCs led to significantly reduced maturation and fertilization rate. No significant influence of EDC concentration on the clinical pregnancy rate was observed for neither of the analyzed EDCs. Patients who obtained their clothes and textiles at fashion discounters displayed a higher amount of EDCs in their FF. In contrast, patients' residential area, source of food products, and nicotine or caffeine consumed were not associated with EDC accumulation. Clinicaltrials.gov NCT01385605 (11 July 2011).

Comparison of sample preparation techniques and data analysis for the LC-MS/MS-based identification of proteins in human follicular fluid.

The proteomic analysis of complex body fluids by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis requires the selection of suitable sample preparation techniques and optimal parameter settings in data analysis software packages to obtain reliable results. Proteomic analysis of follicular fluid, as a representative of a complex body fluid similar to serum or plasma, is difficult as it contains a vast amount of high abundant proteins and a variety of proteins with different concentrations. However, the accessibility of this complex body fluid for LC-MS/MS analysis is an opportunity to gain insights into the status, the composition of fertility-relevant proteins including immunological factors or for the discovery of new diagnostic and prognostic markers for, for example, the treatment of infertility. In this study, we compared different sample preparation methods (FASP, eFASP and in-solution digestion) and three different data analysis software packages (Proteome Discoverer with SEQUEST, Mascot and MaxQuant with Andromeda) combined with semi- and full-tryptic databank search options to obtain a maximum coverage of the follicular fluid proteome. We found that the most comprehensive proteome coverage is achieved by the eFASP sample preparation method using SDS in the initial denaturing step and the SEQUEST-based semi-tryptic data analysis. In conclusion, we have developed a fractionation-free methodical workflow for in depth LC-MS/MS-based analysis for the standardized investigation of human follicle fluid as an important representative of a complex body fluid. Taken together, we were able to identify a total of 1392 proteins in follicular fluid.

Mutations in the N-terminal kinase-like domain of the repressor of photomorphogenesis SPA1 severely impair SPA1 function but not light responsiveness in Arabidopsis.

The COP1/SPA complex is an E3 ubiquitin ligase that acts as a key repressor of photomorphogenesis in dark-grown plants. While both COP1 and the four SPA proteins contain coiled-coil and WD-repeat domains, SPA proteins differ from COP1 in carrying an N-terminal kinase-like domain that is not present in COP1. Here, we have analyzed the effects of deletions and missense mutations in the N-terminus of SPA1 when expressed in a spa quadruple mutant background devoid of any other SPA proteins. Deletion of the large N-terminus of SPA1 severely impaired SPA1 activity in transgenic plants with respect to seedling etiolation, leaf expansion and flowering time. This ΔN SPA1 protein showed a strongly reduced affinity for COP1 in vitro and in vivo, indicating that the N-terminus contributes to COP1/SPA complex formation. Deletion of only the highly conserved 95 amino acids of the kinase-like domain did not severely affect SPA1 function nor interactions with COP1 or cryptochromes. In contrast, missense mutations in this part of the kinase-like domain severely abrogated SPA1 function, suggesting an overriding negative effect of these mutations on SPA1 activity. We therefore hypothesize that the sequence of the kinase-like domain has been conserved during evolution because it carries structural information important for the activity of SPA1 in darkness. The N-terminus of SPA1 was not essential for light responsiveness of seedlings, suggesting that photoreceptors can inhibit the COP1/SPA complex in the absence of the SPA1 N-terminal domain. Together, these results uncover an important, but complex role of the SPA1 N-terminus in the suppression of photomorphogenesis.

Thyroid autoantibodies per se do not impair intracytoplasmic sperm injection outcome in euthyroid healthy women.

OBJECTIVE: Autoimmune thyroid disease (AITD) has been associated with adverse pregnancy outcomes in subfertile women with spontaneous and assisted reproductive technology-induced pregnancies. The underlying pathophysiology is still elusive and an association with thyroid dysfunction or other infertility causes is discussed. However, whether thyroid autoimmunity (TAI) per se has a negative impact on female fertility has not yet been clarified. In this study, we investigated whether TAI in healthy women undergoing intracytoplasmic sperm injection (ICSI) for male infertility may affect pregnancy outcome. DESIGN: A retrospective, single-centre study. METHODS: THE ICSI OUTCOME DATA OBTAINED FROM 835 EUTHYROID WOMEN (AGE: 31.4±4.3 years, BMI: 23.7±4.2 kg/m(2)) were correlated with pre-ICSI TAI status. The known causes of female subfertility were excluded. Outcome parameters included rates of pregnancy, birth, miscarriage and preterm delivery. Blood analysis was carried out retrospectively using blood samples drawn before ICSI. TAI was defined by elevation of anti-thyroperoxidase- or anti-thyroglobulin-antibodies >100 U/l. RESULTS: TAI-POSITIVE AND -NEGATIVE GROUPS DID NOT DIFFER IN AGE, BMI OR TSH LEVELS. TAI STATUS DID NOT INFLUENCE ANY ICSI OUTCOME PARAMETERS. IN CONTRAST, INCREASING MATERNAL AGE WAS SIGNIFICANTLY CORRELATED WITH LOWER PREGNANCY RATE (ODDS RATIO (OR): 0.94 (95% CI: 0.91-0.97); P=0.0003) and birth rate (OR: 0.93 (95% CI: 0.09-0.97); P<0.0001). CONCLUSIONS: Our study suggests that TAI per se does not influence ICSI outcome. A strict definition of AITD and TAI and consideration of TAI-associated and -independent confounders are important to further elucidate the interplay between TAI and reproduction.

Acupuncture for infertility: is it an effective therapy? / 中国结合医学杂志

Acupuncture has been used to treat infertility extensively, including ovulatory dysfunction, in vitro fertilization and embryo transfer (IVF-ET), and male infertility. This review summarizes the recent studies which investigated the role of acupuncture in infertility. In conclusion, most of the existing studies suggest a positive effect of acupuncture in infertility treatment. Firstly, acupuncture may improve ovulation by modulating the central and peripheral nervous systems, the neuroendocrine and endocrine systems, the ovarian blood flow, and metabolism. Secondly, acupuncture can improve the outcome of IVF-ET, and the mechanisms may be related to the increased uterine blood flow, inhibited uterine motility, and the anesis of depression, anxiety and stress. Its effect on modulating immune function also suggests helpfulness in improving the outcome of IVF-ET. Finally, the studies suggest that acupuncture plays a positive role in male infertility, the mechanism of which is not yet clear. Even though a positive effect of acupuncture in infertility has been found, well-designed multi-center, prospective randomized controlled studies are still needed to provide more reliable and valid scientific evidence. Furthermore, it is urgent and necessary to clarify the mechanism of acupuncture for infertility.

Interaction of a plant pseudo-response regulator with a calmodulin-like protein.

Calmodulin (CaM) plays a crucial role in the regulation of diverse cellular processes by modulating the activities of numerous target proteins. Plants possess an extended CaM family including numerous CaM-like proteins (CMLs), most of which appear to be unique to plants. We previously demonstrated a role for CML9 in abiotic stress tolerance and seed germination in Arabidopsis thaliana. We report here the isolation of PRR2, a pseudo-response regulator as a CML9 interacting protein by screening an expression library prepared from Arabidopsis seedlings with CML9 as bait in a yeast two-hybrid system. PRR2 is similar to the response regulators of the two-component system, but lacks the invariant residue required for phosphorylation by which response regulators switch their output response, suggesting the existence of alternative regulatory mechanisms. PRR2 was found to bind CML9 and closely related CMLs but not a canonical CaM. Mapping analyses indicate that an almost complete form of PRR2 is required for interaction with CML9, suggesting a recognition mode different from the classical CaM-target peptide complex. PRR2 contains several features that are typical of transcription factors, including a GARP DNA recognition domain, a Pro-rich region and a Golden C-terminal box. PRR2 and CML9 as fusion proteins with fluorescent tags co-localized in the nucleus of plant cells, and their interaction in the nuclear compartment was validated in planta by using a fluorophore-tagged protein interaction assay. These findings suggest that binding of PRR2 to CML9 may be an important mechanism to modulate the physiological role of this transcription factor in plants.

Desquamated epithelial cells covered with a polymicrobial biofilm typical for bacterial vaginosis are present in randomly selected cryopreserved donor semen.

We tested whether the bacterial biofilm typical for bacterial vaginosis (BV) can be found on desquamated epithelial cells in cryopreserved donor semen. Bacteria were detected with FISH. Bacterial biofilm, covering the epithelial layer in vaginal biopsies of 20 women with BV, was evaluated on desquamated epithelial cells found in the urine of these same women and their male partners (N=20) and compared with the bacterial biofilm found on desquamated epithelial cells in randomly selected cryopreserved semen samples (N=20). Urine from 20 healthy women of laboratory and clinic personnel and urine from their partners were used as controls. Desquamated epithelial cells covered with a polymicrobial Gardnerella biofilm were identified in urine samples from all women with BV and 13 of their male partners and in none of the female controls and their partners. Gardnerella biofilm, typical for BV, was found in the semen of three of the 20 donors. Donor semen might be a vector for BV.

Genetic polymorphism in an inflammasome component, cervical mycoplasma detection and female infertility in women undergoing in vitro fertilization.

The inflammasome is an inducible cytoplasmic structure that is responsible for production and release of biologically active interleukin-1 (IL-1). A polymorphism in the inflammasome component NALP3 has been associated with decreased IL-1 levels and increased occurrence of vaginal Candida infection. We hypothesized that this polymorphism-induced variation would influence susceptibility to infertility. DNA was obtained from 243 women who were undergoing in vitro fertilization (IVF) and tested for a length polymorphism in intron 2 of the gene coding for NALP3 (gene symbol CIAS1). At the conclusion of testing the findings were analyzed in relation to clinical parameters and IVF outcome. The frequency of the 12unit repeat allele, associated with maximal inflammasome activity, was 62.3% in cases of female infertility vs. 75.6% in cases where only the male partner had a detectable fertility problem (p=0.0095). Conversely, the frequency of the 7unit repeat allele was 28.9% in those with a female fertility problem, 17.0% in women with infertile males and 18.4% in idiopathic infertility (p=0.0124). Among the women who were cervical culture-positive for mycoplasma the frequency of the 7unit repeat was 53.7% as opposed to 19.5% in those negative for this infection (p<0.0001). We conclude that the CIAS1 7unit repeat polymorphism increases the likelihood of mycoplasma infection-associated female infertility.

A prospective randomized placebo-controlled study of the effect of acupuncture in infertile patients with severe oligoasthenozoospermia.

In this first prospective, randomized, single-blind, placebo-controlled study, 28 infertile patients with severe oligoasthenozoospermia received acupuncture according to the principles of traditional Chinese medicine (TCM) and 29 infertile patients received placebo acupuncture. A significantly higher percentage of motile sperm (World Health Organization categories A-C), but no effect on sperm concentration, was found after acupuncture compared with placebo acupuncture.

Hyaluronan in follicular fluid and embryo implantation following in vitro fertilization and embryo transfer.

PURPOSE: To determine whether the concentration of hyaluronan (HA) in follicular fluid predicts implantation success following embryo transfer. METHODS: Follicular fluids from 170 IVF patients were tested by ELISA for HA concentration. RESULTS: The mean (standard error) HA concentration in follicular fluids was 158.0 (21.9) ng/ml from women whose embryos did not implant, 220.0 (21.3) ng/ml from women in which one embryo implanted and 239.3 (40.1) ng/ml from women with 2-3 implantations (implantation vs. no implantation p = .019). The HA level was unrelated to maternal age, number of oocytes harvested or fertilized or number of embryos transferred. Follicular fluids from women with an endocrine problem had a lower mean HA level (142.0 ng/ml) as compared to women undergoing IVF due to male factor infertility (257.3 ng/ml) (P = .05). CONCLUSIONS: HA in follicular fluid is decreased in women with unsuccessful implantation or with an endocrine disorder. A woman's level of HA production may influence the potential for implantation of her embryos.

Antibody to the Chlamydia trachomatis 60 kDa heat shock protein in follicular fluid and in vitro fertilization outcome.

PROBLEM: The association between 60 kDa Chlamydia trachomatis heat shock protein (CHSP60) antibodies and the etiology and outcomes of in vitro fertilization (IVF) outcomes is not well known. METHOD OF STUDY: A retrospective study with a double-blind analysis of follicular fluid from 253 IVF patients for IgG antibodies to CHSP60. RESULTS: The CHSP60 antibodies were detected in 74.1% of women without embryo implantation and in 47.9% of women with 1-3 implantations per IVF cycle (P = 0.0004). CHSP60 antibodies were detected in 69.5% of women with tubal occlusion and 49.7% of women with other causes of infertility (P = 0.01). CHSP60 antibody detection was unrelated to maternal age, number of oocytes collected, or percentage of oocytes fertilized. CONCLUSION: Detection of IgG antibody to CHSP60 may indicate persistence of C. trachomatis in the upper genital tract with low implantation rates resulting from a chronic inflammatory reaction. Alternatively, as human hsp60 is expressed in early stage embryogenesis, a cross-reacting antibody may induce destruction of the embryo.

Effect of acupuncture on the outcome of in vitro fertilization and intracytoplasmic sperm injection: a randomized, prospective, controlled clinical study.

OBJECTIVE: To determine the effect of luteal-phase acupuncture on the outcome of IVF/intracytoplasmic sperm injection (ICSI). DESIGN: Randomized, prospective, controlled clinical study. SETTING: University IVF center. PATIENT(S): Two hundred twenty-five infertile patients undergoing IVF/ICSI. INTERVENTION(S): In group I, 116 patients received luteal-phase acupuncture according to the principles of traditional Chinese medicine. In group II, 109 patients received placebo acupuncture. MAIN OUTCOME MEASURE(S): Clinical and ongoing pregnancy rates. RESULT(S): In group I, the clinical pregnancy rate and ongoing pregnancy rate (33.6% and 28.4%, respectively) were significantly higher than in group II (15.6% and 13.8%). CONCLUSION(S): Luteal-phase acupuncture has a positive effect on the outcome of IVF/ICSI.

Immunity to the 60kD heat shock proteins

A brief review on the role of the heat shock proteins (hsp), their common properties and possible consequences for early pregnancy development is described. The 60kD hsp plays an important role as immunogenic antigen of many microbial pathogens and possibly in postinfectious autoimmunity. The immune responce to hsp may cause pregnancy failure. The consequences of previous sensitization to microbial hsp and the effects of human autoantibodies to hsp, are demonstrated in a mouse embryo culture model.