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The cryopreservation of ovarian tissue with subsequent transplantation of the tissue represents an established method of fertility protection for female patients who have to undergo gonadotoxic therapy. The procedure can be performed at any point in the cycle and thus generally does not lead to any delay in oncological therapy. With the aid of this procedure, more than 130 births to date worldwide have been able to be recorded. The birth rate is currently approximately 30% and it can be assumed that this will increase through the further optimisation of the cryopreservation and surgical technique. The concept paper presented here is intended to provide guidance for managing cryopreservation and transplantation of ovarian tissue to German-speaking reproductive medicine centres.

Aim: The aim of this official guideline published by the German Society of Gynecology and Obstetrics (DGGG) and coordinated with the German Society of Urology (DGU) and the German Society of Reproductive Medicine (DGRM) is to provide consensus-based recommendations, obtained by evaluating the relevant literature, on counseling and fertility preservation for prepubertal girls and boys as well as patients of reproductive age. Statements and recommendations for girls and women are presented below. Statements or recommendations for boys and men are not the focus of this guideline., Methods: This S2k guideline was developed at the suggestion of the guideline commission of the DGGG, DGU and DGRM and represents the structured consensus of representative members from various professional associations (n = 40)., Recommendations: The guideline provides recommendations on counseling and fertility preservation for women and girls which take account of the patient's personal circumstances, the planned oncologic therapy and the individual risk profile as well as the preferred approach for selected tumor entities.

Objective: To identify and estimate the importance of risk factors on pregnancy loss until the end of the second trimester after clinical pregnancy was achieved by either in vitro fertilisation (IVF) or intra-cytoplasmic sperm injection (ICSI)., Study Design: Retrospective cohort study including 588 cycles with fresh embryo transfers and 150 cycles with frozen-thawed embryo transfers using logistic regression., Results: The rate of miscarriages subsequent to a fresh embryo transfer was significantly increased by a diagnosis of endometriosis (p=0.02), as well as significantly influenced by the age of the female patient at the time of oocyte retrieval (p<0.01) and the serum level of testosterone (p=0.02). The time between freezing and thawing of the pronuclear stages for a frozen-thawed embryo transfer revealed a trend to a higher rate of miscarriages (p=0.06)., Conclusion: Endometriosis significantly decreases the chance of having a baby even with IVF or ICSI., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The aim of this paper was to determine the influence of different in vitro culture media on mRNA expression of Hedgehog genes, il-6, and important genes regarding reactive oxygen species in single mouse embryos., Methods: Reverse transcription of single embryos either cultured in vitro from day 0.5 until 3.5 (COOK's Cleavage medium or Vitrolife's G-1 PLUS medium) or in vivo until day 3.5 post coitum. PCR was carried out for β-actin followed by nested-PCR for shh, ihh, il-6, nox, gpx4, gpx1, and prdx2., Results: The number of murine blastocysts cultured in COOK medium which expressed il-6, gpx4, gpx1, and prdx2 mRNA differed significantly compared to the in vivo group. Except for nox, the mRNA profile of the Vitrolife media group embryos varied significantly from the in vivo ones regarding the number of blastocysts expressing the mRNA of shh, ihh, il-6, gpx4, gpx1 and prdx2., Conclusions: The present study shows that different in vitro culture media lead to different mRNA expression profiles during early development. Even the newly developed in vitro culture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies.

Purpose: Based on the reported tocolytic action of the hormone relaxin (RLX) in rodents, locally produced in reproductive tissues and the corpus luteum in mammals, the present study aimed to evaluate the influence of RLX on contraction-mediating cyclooxygenases-1 and -2 (COX) and the contractile prostaglandin PGE(2) in human myometrial and decidual cells. Primary cultured cells were obtained from uteri and placentas of term and preterm women undergoing elective caesarean section., Methods: In vitro culture of primary myometrial and decidual cells, immunocytochemistry, reverse transcription and real-time PCR, Western blot, ELISA., Results: We demonstrate for the first time an activating effect of RLX for human COX-1 and COX-2 in primary myometrial and decidual cells in vitro., Conclusions: These effects might potentially contribute to birth-associated induction of contractions in vivo.

Objective: To evaluate the effect of N-acetyl cysteine (NAC) on gestational age at delivery in women with previous preterm labor and bacterial vaginosis., Methods: A randomized, double-blind, placebo-controlled trial with 280 women between 16 and 18 weeks of pregnancy who had 1 previous preterm birth and had just been successfully treated for bacterial vaginosis with metronidazole for 1 week. The women were randomized to receive 0.6 g of NAC per day plus 17-hydroxyprogesterone caproate (17-OHPC) or placebo plus 17-OHPC until 36 completed weeks of pregnancy or active labor. A vaginal swab was taken during labor., Results: Reaching 36 weeks of pregnancy was more frequent (P<0.05) and gestational age at delivery was significantly higher in the NAC than in the placebo group (37.4 weeks+/-0.4 weeks vs 34.1 weeks+/-1.2 weeks, P<0.05). The discontinuation rate was 11.4% in the NAC group., Conclusions: Oral NAC was found to reduce the recurrence of preterm birth in patients with bacterial vaginosis.

Objective: To report an intracytoplasmatic sperm injection (ICSI) pregnancy achieved in a couple with male primary cilia dyskinesia (PCD) with viable sperm that were detected using a 1.48 microm wavelength diode laser., Design: Case report., Setting: University hospital., Patient(s): A 37-year-old man with infertility due to primary cilia dyskinesia; semen analysis revealed a severe oligoasthenoteratozoospermia with absence of motile spermatozoa. A 34-year-old healthy woman with a 10-year history of primary infertility., Intervention(s): Selection of viable spermatozoa using the hypo-osmotic swelling (HOS) test or a 1.48 microm wavelength diode laser and subsequent ICSI., Main Outcome Measure(s): Sperm analysis. Fertilization and cleavage rates and pregnancy., Result(s): Semen samples showed no motile spermatozoa and high percentages of spermatozoa with curled flagella resembling HOS-reactive spermatozoa. To identify viable spermatozoa we used the HOS test or a 1.48 microm diode laser. The ICSI using HOS-selected spermatozoa resulted in two fertilized out of four oocytes (50%), and injection of laser-selected spermatozoa resulted in four fertilized out of seven oocytes (57%). The transfer of two frozen/thawed oocytes of the laser group led to a singleton pregnancy., Conclusion(s): Use of a noncontact diode laser for sperm viability assessment may be a useful alternative, especially in cases where the HOS test is not informative.

Experimental Objectives: Activation of the oxytocin receptor (OTR) induces phospholipase C induced PIP(2) turnover in the human uterus. Relaxin (RLX), a polypeptide hormone produced in the corpus luteum of pregnancy as well as in the placenta and decidua inhibits PIP(2) turnover and subsequent signaling in human myometrium. The purpose of this study was to evaluate a possible effect of RLX on OTR regulation in human uterine smooth muscle cells. Primary cultures of myometrium from term pregnant women undergoing elective caesarean section were incubated for different time periods (0-96 h) and with different concentrations of RLX [10 pg/ml-20 microg/ml]. The effects on OTR binding, mRNA and protein expression were evaluated by means of (125)I-OVT binding assay, RT-PCR and flow cytometry., Results: Prolonged RLX incubation was able to inhibit 30-40% of OTR binding while binding affinity remained unchanged. Oxytocin receptor mRNA and protein expression were down regulated by RLX about 50% and 35% respectively., Conclusion: We report for the first time an effect of RLX on OTR regulation in human uterine myometrial cells. The above results indicate that high local uterine RLX concentrations may be involved in uterine quiescence during human pregnancy by down regulating the OTR.

The aim of our study was to detect and characterize mRNA expression of VEGF isoforms VEGF(121), VEGF(145), VEGF(165), VEGF(189), and VEGF(206) in human blastocysts. We recently demonstrated VEGF mRNA expression during human preimplantation embryo development, and further information regarding the alternatively spliced mRNAs resulting in freely secreted proteins or proteins bound to cell surface heparan-sulphate proteoglycans is needed to better understand the process of angiogenesis during implantation. Human blastocysts unsuitable for transfer obtained from the IVF programme at Stanford University were examined by reverse transcription/hemi-nested polymerase chain reaction for their expression of VEGF mRNA splice variants. VEGF mRNA was expressed in 17 out of 19 (89%) blastocysts. Of the 17 blastocysts, VEGF(121) mRNA was detected in 88%, VEGF(145) mRNA in 100%, VEGF(165) mRNA in 71%, and VEGF(189) mRNA in 24% of blastocysts. There was co-expression of mRNA for VEGF(121) and VEGF(145) only in 29% blastocysts, of mRNA for VEGF(165) and VEGF(145) only in 12%, and of mRNA for VEGF(121), VEGF(145) and VEGF(165) in 59% blastocysts. VEGF(206) mRNA could not be detected. In conclusion, we demonstrated that blastocysts express the mRNAs encoding for the free VEGF proteins, enabling the implanting embryo to immediately induce angiogenesis at the implantation site.

The advent of assisted reproductive techniques such as intracytoplasmic sperm injection has markedly reduced the problem of unsuccessful fertilization in modern IVF. However, pregnancy rates and 'take-home-baby' rates remain unsatisfactorily low. Attempts to overcome low pregnancy rates by transferring a larger number of embryos to the mother often result in multiple pregnancies. The preimplantation embryo synthesizes several proteins that may signal its presence to the maternal system, and the interaction between the embryo and the endometrium is controlled, at least in part, by cytokines and growth factors. However, little is known about the interactions between the embryonic and maternal proteins. A better understanding of normal preimplantation embryo development may lead to improved in vitro culture conditions and higher pregnancy rates. This review gives an overview of the current knowledge of the embryonic factors produced during the preimplantation period. The development of the interleukin 1 system for screening human preimplantation embryos is also discussed. Current biochemical embryonic screening procedures are highly experimental, but increasing knowledge of the physiology of embryonic development might enable these screening procedures to be used to identify embryos that are capable of successful implantation.

Myoglobin may serve a variety of functions in muscular oxygen supply, such as O(2) storage, facilitated O(2) diffusion, and myoglobin-mediated oxidative phosphorylation. We studied the functional consequences of a myoglobin deficiency on cardiac function by producing myoglobin-knockout (myo(-/-)) mice. To genetically inactivate the myoglobin gene, exon 2 encoding the heme binding site was deleted in embryonic stem cells via homologous recombination. Myo(-/-) mice are viable, fertile, and without any obvious signs of functional limitations. Hemoglobin concentrations were significantly elevated in myo(-/-) mice. Cardiac function and energetics were analyzed in isolated perfused hearts under resting conditions and during beta-adrenergic stimulation with dobutamine. Myo(-/-) hearts showed no alteration in contractile parameters either under basal conditions or after maximal beta-adrenergic stimulation (200 nM dobutamine). Tissue levels of ATP, phosphocreatine ((31)P-NMR), and myocardial O(2) consumption were not altered. However, coronary flow [6.4 +/- 1.3 ml.min(-1).g(-1) [wild-type (WT)] vs. 8.5 +/- 2.4 ml.min(-1).g(-1) [myo(-/-)] [and coronary reserve [17.1 +/- 2.1 (WT) vs. 20.8 +/- 1.1 (myo(-/-) ml. min(-1).g(-1) were significantly elevated in myo(-/-) hearts. Histological examination revealed that capillary density also was increased in myo(-/-) hearts [3,111 +/- 400 mm(-2) (WT) vs. 4,140 +/- 140 mm(-2) (Myo(-/-)]. These data demonstrate that disruption of myoglobin results in the activation of multiple compensatory mechanisms that steepen the pO(2) gradient and reduce the diffusion path length for O(2) between capillary and the mitochondria; this suggests that myoglobin normally is important for the delivery of oxygen.

The aim of this study was to quantify and localize the mRNA expression of the vascular endothelial growth factor (VEGF) receptors Flt1, KDR and sflt, in human endometrium throughout the menstrual cycle. Since neoangiogenesis is crucial during embryonic implantation, we postulate that endometrial receptivity to VEGF may be altered during the luteal phase in order to support implantation. Human endometrium was collected and specified as early proliferative (n = 3), mid-proliferative (n = 4), late proliferative (n = 3), early secretory (n = 2), mid-secretory (n = 4), and late secretory (n = 4). Competitive reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate the mRNA values throughout the menstrual cycle. Additionally, four samples were separated into epithelial and stromal-enriched cell fractions and competitive RT-PCR was carried out to specify the distribution of the mRNA expression. While mRNA for the transmembraneous receptors Flt1 and KDR was shown to be present at almost constant values throughout the menstrual cycle, the soluble receptor, sflt, had a three-fold higher level of transcription during mid-proliferative and late proliferative when compared with early proliferative and the entire secretory phase. The expression of Flt1, KDR and sflt mRNA was detected in both isolated endometrial epithelial and stromal cell fractions. In conclusion, the down-regulation of sflt, which functions as a soluble antagonist, during the luteal phase may act to sensitize the maternal endothelial receptors to angiogenetic stimuli secreted by the implanting embryo.

Objective: To determine the levels of epidermal growth factor (EGF) and leukemia inhibitory factor (LIF) in follicular fluid, if any, and to assess the association of these cytokines with the outcome of in vitro fertilization (IVF)., Study Design: EGF and LIF levels determined by enzyme-linked immunosorbent assay in 60 preovulatory follicular fluids were compared with 25 IVF outcomes., Results: Immunoreactive EGF and LIF could be detected in human follicular fluid. Levels of these cytokines were similar in FF obtained from follicles that resulted in fertilized oocytes and those that did not. EGF levels were significantly lower in patients establishing a pregnancy as compared to patients achieving no pregnancy (P < .007). LIF levels were similar in both groups of patients., Conclusion: EGF appears to be associated with IVF outcome.

For the specific analysis of endothelial NO synthase (eNOS) function in the coronary vasculature, we generated a mouse homozygous for a defective eNOS gene (eNOS-/-). Western blot as well as immunohistochemical staining revealed the absence of eNOS protein in eNOS-/- mice. Aortic endothelial cells derived from eNOS-/- mice displayed only background levels of NOx formation compared with wild-type (WT) cells (88 versus 1990 pmol NOx x h-1/mg protein-1). eNOS-/- mice were hypertensive (mean arterial pressure, 135 +/- 15 versus 107 +/- 8 mm Hg in WT) without the development of cardiac hypertrophy. Coronary hemodynamics, analyzed in Langendorff-perfused hearts, showed no differences either in basal coronary flow or in maximal and repayment flow of reactive hyperemia. Acute NOS inhibition with Nomega-nitro-L-arginine methyl ester (L-NAME) in WT hearts substantially reduced basal flow and reactive hyperemia. The coronary response to acetylcholine (ACh) (500 nmol/L) was biphasic: An initial vasoconstriction (flow, -35%) in WT hearts was followed by sustained vasodilation (+190%). L-NAME significantly reduced vasodilation in WT hearts (+125%) but did not alter the initial vasoconstriction. In eNOS-/- hearts, the initial vasoconstriction was augmented (-70%), whereas the ACh-induced vasodilation was not affected. Inhibition of cyclooxygenase with diclofenac converted the ACh-induced vasodilation into vasoconstriction (-49% decrease of basal flow). This effect was even more pronounced in eNOS-/- hearts (-71%). Our results demonstrate that (1) acute inhibition of eNOS reveals a role for NO in setting the basal coronary vascular tone as well as participation in reactive hyperemia and the response to ACh; (2) chronic inhibition of NO formation in eNOS-/- mutant mice induces no changes in basal coronary flow and reactive hyperemia, suggesting the activation of important compensatory mechanisms; and (3) prostaglandins are the main mediators of the ACh-induced vasodilation in both WT and eNOS-/- mice.

The germline transmission (g.l.t.) of gene trap or gene targeted mutations by ES-cell-derived chimaeric mice is a crucial step in the generation of stable transgenic lines. The wild-type ES cell lines CJ7, D3 and R1 of different passage numbers and their transfected clone-descendants generated in gene targeting or gene trap experiments were tested for their ability to colonize the germline. The maximal g.l.t. age for wild-type ES cells was equal to passage 26 and for transfected clones was equivalent to passage 32 of parental lines. It is shown that wild-type ES cells of less than a passage 15 should be used for effective production of transgenic g.l.t. clones. A simple system is outlined to evaluate the probability of g.l.t. on the basis of the chimaeric progeny obtained.