Seminal plasma (SP) induces a rapid transforming growth factor beta 1 (TGFß1)-independent up-regulation of epithelial-mesenchymal transdifferentiation (EMT) and myofibroblastic metaplasia-markers in endometriotic (EM) and endometrial cells.

PURPOSE: To study if short-term exposure (2 h and 6 h) of endometrial/endometriotic tissues and cells to 10% seminal plasma (SP) can induce EMT/metaplasia. METHODS: Basic research experimental study was carried out in a University hospital-based fertility center. Semen samples, peritoneal fluid (PF) from endometriosis patients, endometrial biopsy from premenopausal women, immortalized endometriotic epithelial cell line (12Z), and immortalized endometrial stromal cell line (St-T1b) were studied. Rapid stain identification test (RSID), TGFß1 immunofluorescence of washed sperms, TGFß1-ELISA of SP and PF, in vitro study (2 h and 6 h incubation) and real-time PCR of endometrial tissue and cell lines to analyze gene expression of EMT/metaplasia markers and mediators were done. RESULTS: SP is still detectable in washed semen. TGFß1 was expressed on the plasma membrane of the sperms and was significantly more concentrated in SP (88.17 ng/ml) than PF. 10% SP induced an up-regulation of alpha smooth muscle actin expression in endometrial tissue (p = 0.008) and in 12Z cells (p = 0.05), mostly TGFß1-independent. TWIST expression was persistently significantly down-regulated while Snail1 and 2 were up-regulated, though insignificant. CONCLUSION: Our results provide novel evidence to support that even in semen washed twice, SP is still detectable. The changes in EMT/metaplasia markers and mediators give a new insight into a possible effect of SP on the pathogenesis of endometriosis.

Practical recommendations for fertility preservation in women by the FertiPROTEKT network. Part I: Indications for fertility preservation.

PURPOSE: Most guidelines about fertility preservation are predominantly focused on scientific evidence, but are less practically orientated. Therefore, practically oriented recommendations are needed to support the clinician in daily practice. METHODS: A selective literature search was performed based on the clinical and scientific experience of the authors, focussing on the most relevant diseases and gynaecological cancers. This article (Part I) provides information on topics that are essential for the fertility preservation indication, such as disease prognosis, disease therapy and its associated risks to fertility, recommending disease-specific fertility preservation measures. Part II specifically focusses on fertility preservation techniques. RESULTS: In breast cancer patients, fertility preservation such as ovarian tissue and oocyte cryopreservation is especially recommended in low-stage cancer and in women < 35 years of age. In Hodgkin's lymphoma, the indication is mainly based on the chemotherapy regime as some therapies have very low, others very high gonadotoxicity. In borderline ovarian tumours, preservation of fertility usually is achieved through fertility sparing surgery, ovarian stimulation may also be considered. In cervical cancer, endometrial cancer, rheumatic diseases and other malignancies such as Ewing sarcoma, colorectal carcinoma, non-Hodgkin lymphoma, leukaemia etc., several other factors must be considered to enable an individual, stage-dependent decision. CONCLUSION: The decision for or against fertility preservation depends on the prognosis, the risks to fertility and individual factors such as prospective family planning.

Treatment strategies for severe oligoasthenoteratozoospermia (OAT) (<0.1 million/mL) patients.

Intracytoplasmic sperm injection (ICSI) using spermatozoa from patients with severe oligoasthenoteratozoospermia is still a challenge. Although spermatozoa are available, lower fertilisation rates as well as compromised pregnancy rates are observed after ICSI. We aimed at identifying respective parameters in the pre-values of ejaculate samples used for couple counselling. The clinical pre-values of 121 patients and their corresponding 228 ICSI cycles performed between 2002 and 2010 were retrospectively analysed. Patients were divided into three groups: (i) group 1 (G1, n = 51) where all patients showed at least once <0.1 million/mL and ICSI was performed using ejaculate alone; (ii) group 2 (G2, n = 14) patients had once <0.1 Mill/mL or azoospermia and a testicular biopsy before start of ICSI; (iii) group 3 (G3, n = 56) patients were azoospermic and directed immediately to testicular sperm extraction (TESE). The pre-values of G2 differed significantly from G1 in terms of volume and motility. Lutenizing hormone (LH) and follicle-stimulating hormone (FSH) values were equal in G1 and G2, but showed significant differences in comparison to G3. Testis volume was significantly higher in G3. In the corresponding ICSI cycles, the percentage of cancelled embryo transfers was highest in G3. We did not find any correlations of hormonal markers or sperm pre-values with the success rates of ICSI. In our patient cohort, spermatozoa retrieved either from ejaculate or testicular biopsies have nearly identical chances in achieving pregnancies. Patients in need of TESE before ICSI have significantly lower sperm counts. However, it is not possible to calculate threshold values as indicator for TESE.

Importance of transvaginal ultrasound applying elastography for identifying deep infiltrating endometriosis - a feasibility study.

PURPOSE: To evaluate the presence of a lesion indicative of endometriosis with transvaginal elastography. MATERIALS AND METHODS: Transvaginal ultrasound and clinical examination were carried out in 48 women with clinical symptoms indicative of endometriosis. In 31 cases strain values were measured at two regions of interest (ROIs) in the Douglas's cul-de-sac during a cycle of compression and decompression with a vaginal probe. RESULTS: A significant difference was found for the ratio of the ROI measuring points in the Douglas' cul-de-sacs of women with a palpable nodule in examination compared to women without a palpable nodule (p = 0.002). CONCLUSION: The ratio of strain values between two ROIs in the Douglas' s cul-de-sac is associated with the presence of an endometriotic lesion. In the future, these findings could allow for a more detailed pre-surgical evaluation and possibly serve as a novel diagnostic tool for predicting deep infiltrating endometriosis.

MicroRNA miR-513a-3p acts as a co-regulator of luteinizing hormone/chorionic gonadotropin receptor gene expression in human granulosa cells.

The luteinizing hormone/chorionic gonadotropin receptor (LHCGR) is essential for normal male and female reproductive processes. The spatial and temporal LHCGR gene expression is controlled by a complex system of regulatory mechanisms which are crucial for normal physiological function, especially during the female cycle. In this study, we aimed to elucidate whether microRNAs are involved in this network and play a role in regulating LHCGR expression. Computational analysis predicted that miR-513a-3p interacts with the LHCGR mRNA via three binding sites located in the 3'UTR region, enabling a synergistic action. Moreover, using a luciferase-based reporter assay we found that miR-513a-3p targets the LHCGR, resulting in a significant down-regulation of its expression. In human primary granulosa cell cultures we detected a dynamic, inversely associated expression pattern of miR-513a-3p and the LHCGR. In addition, transfection with miR-513a-3p or its specific inhibitor led to a down- or up-regulation at the LHCGR mRNA level, respectively. An increased amount of miR-513a-3p resulted in the down-regulation of the LHCGR mRNA, reflected by the attenuation of cAMP synthesis after hormonal stimulation. In conclusion, these data demonstrate that miR-513a-3p is involved in the control of the LHCGR expression by an inversely regulated mechanism at the post-transcriptional level and show for the first time that this kind of post-transcriptional process contributes to the multifaceted system of the human LHCGR regulation.

Optimizing TESE-ICSI by laser-assisted selection of immotile spermatozoa and polarization microscopy for selection of oocytes.

For most azoospermic men testicular sperm extraction (TESE) is the only treatment, however it presents challenges for the ART laboratory, as the retrieval of motile spermatozoa is difficult. In the absence of sperm movement no unequivocal distinction can be made between either dead or immotile, but vital spermatozoa. However, a single laser shot directed to the tip of the tail allows recognition of viability because the flagellum coils at the area of impact. To rank the quality and the maturity of oocytes, polarization microscopy can be used. The zona score and the visualization of the meiotic spindle correlate with implantation and pregnancy rates. We compared 65 TESE-ICSI cycles of the years 2007 and 2008 (Group 1, G1) with 58 TESE-ICSI cycles of the years 2009 and 2010 (Group 2, G2). Testicular spermatozoa were injected according to motility and morphology into selected oocytes. In G1 both, oocyte and spermatozoa were rated using light microscopy only, whereas in G2 the laser was used for sperm selection and the oocytes were rated by light and polarization microscopy. In G2 we enhanced our fertilization rate (FR) significantly in comparison to G1 (G1 42.1% vs. G2 52.7%, p < 0.001). The fertilization rate with immotile, but vital spermatozoa improved significantly when applying laser-based selection (p = 0.006). The laser selection of immotile spermatozoa and the use of polarization microscopy can enhance the FR of TESE-ICSI. No negative effect of the laser was seen on birth rates. The FR with immotile, but vital spermatozoa clearly benefits from laser selection and is a non-hazardous and safe method for the selection of viable but immotile sperm. To our knowledge this is the first report using new technology creating novel endpoints for the analysis of spermatozoa and oocytes in TESE-ICSI.

Role of the CAG repeat polymorphism of the androgen receptor gene in polycystic ovary syndrome (PCOS).

BACKGROUND: Polycystic ovary syndrome (PCOS) is a frequent heterogenic disorder with a familial background. Androgenic effects, determining the clinical features of the syndrome, are mediated by the androgen receptor (AR), whose activity is modulated by a genetic polymorphism. We investigated the role of the CAG repeat polymorphism of the androgen receptor in PCOS. METHODS: In the infertility unit of a university clinic, 72 PCOS patients were compared with 179 ovulatory controls undergoing a standardized diagnostic work-up. The number of CAG repeats was determined by PCR, labelling with IR-800 and PAGE. X-chromosome inactivation was assessed by a methylation-sensitive assay. RESULTS: Compared to controls, PCOS patients displayed a shorter mean CAG repeat length, encoding for higher AR activity (P=0.001). CAG repeat length correlated inversely with oligomenorrhea, a central androgen dependent feature of the syndrome (P=0.005). In a binomial regression analysis including BMI, LH and free testosterone, CAG repeat length was identified as an independent risk factor for PCOS (P=0.002). CONCLUSIONS: The CAG repeat polymorphism could constitute one of the genetic factors modulating the syndrome's phenotype, contributing to its clinical heterogeneity and associated metabolic consequences.

mRNA-expression of ERα, ERß, and PR in clonal stem cell cultures obtained from human endometrial biopsies.

Background. Proliferation and differentiation of the endometrium are regulated by estrogen and progesterone. The enormous regenerative capacity of the endometrium is thought to be based on the activity of adult stem cells. However, information on endocrine regulatory mechanisms in human endometrial stem cells is scarce. In the present study, we investigated the expression of ERα, ERß, and PR in clonal cultures of human endometrial stem cells derived from transcervical biopsies. Methods. Endometrial tissue of 11 patients was obtained by transcervical biopsy. Stromal cell suspensions were plated at clonal density and incubated for 15 days. Expression of ERα, ERß and PR was determined by qPCR prior to and after one cloning round, and normalized to 18 S rRNA expression. Results. Expression of ERα and ERß was downregulated by 64% and 89%, respectively (P = 0.002 and P < 0.001). In contrast, PR was not significantly downregulated, due to a more heterogenous expression pattern. Conclusions. Culture of human endometrial stroma cells results in a downregulation of ERα and ERß, while expression of PR remained unchanged in our patient collective. These results support the hypothesis that stem cells may not be subject to direct stimulation by sex steroids, but rather by paracrine mechanisms within the stem cell niche.

Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.

Adult stem cells are thought to be responsible for the high regenerative capacity of the human endometrium, and have been implicated in the pathology of endometriosis and endometrial carcinoma. The RNA-binding protein Musashi-1 is associated with maintenance and asymmetric cell division of neural and epithelial progenitor cells. We investigated expression and localization of Musashi-1 in endometrial, endometriotic and endometrial carcinoma tissue specimens of 46 patients. qPCR revealed significantly increased Musashi-1 mRNA expression in the endometrium compared to the myometrium. Musashi-1 protein expression presented as nuclear or cytoplasmic immunohistochemical staining of single cells in endometrial glands, and of single cells and cell groups in the endometrial stroma. Immunofluorescence microscopy revealed colocalization of Musashi-1 with its molecular target Notch-1 and telomerase. In proliferative endometrium, the proportion of Musashi-1-positive cells in the basalis layer was significantly increased 1.5-fold in the stroma, and three-fold in endometrial glands compared to the functionalis. The number of Musashi-1 expressing cell groups was significantly increased (four-fold) in proliferative compared to secretory endometrium. Musashi-1 expressing stromal cell and cell group numbers were significantly increased (five-fold) in both endometriotic and endometrial carcinoma tissue compared to secretory endometrium. A weak to moderate, diffuse cytoplasmic glandular staining was observed in 50% of the endometriosis cases and in 75% of the endometrioid carcinomas compared to complete absence in normal endometrial samples. Our results emphasize the role of Musashi-1-expressing endometrial progenitor cells in proliferating endometrium, endometriosis and endometrioid uterine carcinoma, and support the concept of a stem cell origin of endometriosis and endometrial carcinoma.

Relevance of anti-Mullerian hormone measurement in a routine IVF program.

BACKGROUND: Diminished ovarian reserve has become a major cause of infertility. Anti-Mullerian hormone (AMH) seems to be a promising candidate to assess ovarian reserve and predict the response to controlled ovarian hyperstimulation (COH). This prospective study was conducted to evaluate the relevance of AMH in a routine IVF program. METHODS: Three hundred and sixteen patients were prospectively enrolled to enter their first IVF/ICSI-cycle. Age, FSH-, inhibin B- and AMH-levels and their predictive values for ovarian response and clinical pregnancy rate were compared by discriminant analyses. RESULTS: A total of 132 oocyte retrievals were performed. A calculated cut-off level < or =1.26 ng/ml AMH alone detected poor responders (< or =4 oocytes) with a sensitivity of 97%, and there was a 98% correct prediction of normal response in COH if levels were above this threshold. With levels <0.5 ng/ml, a correct prediction of very poor response (< or =2 oocytes) was possible in 88% of cases. Levels of AMH > or =0.5 ng/ml were not significantly correlated with clinical pregnancy rates. CONCLUSIONS: AMH is a predictor of ovarian response and suitable for screening. Levels < or =1.26 ng/ml are highly predictive of reduced ovarian reserve and should be confirmed by a second line antral follicle count. Measurement of AMH supports clinical decisions, but alone it is not a suitable predictor of IVF success.

Enhanced oestradiol secretion briefly after embryo transfer in conception cycles from IVF.

The hypothesis was tested that conception cycles (CC) resulting from IVF can be distinguished from non-conception cycles (NC) by differences in corpora lutea function that are detectable at the earliest stage of embryo implantation. Luteal oestradiol secretion was analysed retrospectively in 409 ovarian stimulation cycles of 296 patients from the day of embryo transfer until 14 days after embryo transfer (ET+14) in IVF/intracytoplasmic sperm injection (ICSI) cycles. Human chorionic gonadotrophin (HCG) was administered in 330 of 409 cycles in addition to vaginal progesterone in all cycles. Differences in serum oestradiol concentrations between CC and NC increased from day ET+1 onward and became statistically significant on days ET+4 through ET+14, with higher oestradiol concentrations in CC compared with NC. Even though exogenous HCG administration prevented the fall in luteal oestradiol concentrations after ET+4 both in CC and NC, increasing differences in oestradiol concentrations between CC and NC after embryo transfer were observed in both groups of HCG-supplemented and non-supplemented cycles. It is concluded that luteal oestradiol secretion is affected at the earliest stage of embryo implantation. The putative early signal to the corpus luteum associated with embryo attachment and early implantation appears to be superimposed onto the effect of exogenous luteal HCG administration and is clearly distinguishable as early as 4 days after embryo transfer in conception cycles.