Physiological and anatomical aspects of the reproduction of mice with reduced Syndecan-1 expression.

BACKGROUND: Syndecan-1 is a heparan sulfate proteoglycan acting as a co-receptor for cytokines and growth factors mediating developmental, immunological and angiogenic processes. In human, the uteroplacental localization of Syndecan-1 and its reduced expression in pregnancy-associated pathologies, such as the intrauterine growth restriction, suggests an influence of Syndecan-1 in embryo-maternal interactions. The aim of the present study was to identify the effect of a reduced expression of Syndecan-1 on the reproductive phenotype of mice and their progenies. METHODS: Reproductive characteristics have been investigated using animals with reduced Syndecan-1 and their wildtype controls after normal mating and after vice versa embryo transfers. Female mice were used to measure the estrus cycle length and the weight gain during pregnancy, as well as for histological examination of ovaries. Male mice were examined for the concentration, motility, viability and morphology of spermatozoa. Organs like heart, lung, liver, kidney, spleen, brain and ovaries or testes and epididymis of 6-month-old animals were isolated and weighed. Statistical analyses were performed using two-tailed students t-test with P < .05 and P < .02, chi square test (P < .05) and Fisher's Exact Test (P < .05). A linear and a non-linear mixed-effects model were generated to analyze the weight gain of pregnant females and of the progenies. RESULTS: Focusing on the pregnancy outcome, the Syndecan-1 reduced females gave birth to larger litters. However, regarding the survival of the offspring, a higher percentage of pups with less Syndecan-1 died during the first postnatal days. Even though the ovaries and the testes of Syndecan-1 reduced mice showed no histological differences and the ovaries showed a similar number of primary and secondary follicles and corpora lutea, the spermatozoa of Syndecan-1 reduced males showed more tail and midpiece deficiencies. Concerning the postnatal and juvenile development the pups with reduced Syndecan-1 expression remained lighter and smaller regardless whether carried by mothers with reduced Syndecan-1 or wildtype foster mothers. With respect to anatomical differences kidneys of both genders as well as testes and epididymis of male mice with reduced syndecan-1 expression weighed less compared to controls. CONCLUSIONS: These data reveal that the effects of Syndecan-1 reduction are rather genotype- than parental-dependent.

Fertility protection: complications of surgery and results of removal and transplantation of ovarian tissue.

Fertility-preserving measures are becoming important for patients receiving oncological treatment. One method involves cryopreservation of ovarian tissue and transplanting it when treatment is completed. We report complications resulting from surgical and fertility medicine, and the results of procedures for the removal and transplantation of ovarian tissue carried out within the FertiProtekt network. A survey using a structured questionnaire was conducted among the FertiProtekt network centres between November 2015 and June 2016. The analysis included surgical techniques used to remove and transplant ovarian tissue, surgical complications and results. Laparoscopic removal and transplantation of ovarian tissue have a low risk of complications. Surgical complications occurred in three of the network's 1373 ovarian tissue removals (n = 1302) and transplantations (n = 71); two complications (0.2%) occurred during removal and one during transplantation. Menstruation resumed in 47 out of 58 women (81%) who underwent ovarian tissue transplantation. Hormonal activity occurred in 63.2% of transplantations with a follow-up of 6 months or over. Sixteen pregnancies occurred in 14 patients, with nine births. The risks and complications of removal and transplantation of ovarian tissue are similar to those of standard laparoscopy. These procedures are becoming standard for fertility protection in cancer patients.

hCG stimulates angiogenic signals in lymphatic endothelial and circulating angiogenic cells.

Human chorionic gonadotropin (hCG) has long been associated with the initiation and maintenance of pregnancy, where angiogenesis plays an important role. However, the function of hCG in angiogenesis and the recruitment of vascular active cells are not fully understood. In this study, the role of hCG and its receptor in circulating angiogenic and human endothelial cells, including lymphatic, uterine microvascular, and umbilical vein endothelial cells, was examined. Immunohistochemistry and immunoblot analysis were used to detect LH/hCG receptor expression and the expression of hCG-induced angiogenic molecules. HIF-1α was determined via ELISA and downstream molecules, such as CXCL12 and CXCR4, via real-time PCR. Chemotaxis was analyzed using Boyden chambers. Our results show that the LH/hCG receptor was present in all tested cells. Furthermore, hCG was able to stimulate LH/hCG-receptor-specific migration in a dose-dependent fashion and induce key angiogenic molecules, including HIF-1α, CXCL12, and CXCR4. In conclusion, our findings underscore the importance of hCG as one of the first angiogenic molecules produced by the conceptus. hCG itself alters endothelial motility, recruitment, and expression of pro-angiogenic molecules and may therefore play an important role in vascular adaption during implantation and early placental formation.

Interferon stimulated gene 15 expression at the human embryo-maternal interface.

PURPOSE: In early pregnancy the dialogue between maternal endometrium and embryo is a key process in establishing a receptive decidua and placental network. Decidual ISG15 induction is thought to promote pregnancy maintenance and development. ISG15 is involved in RNA splicing, cytoskeletal organization, stress response and further intracellular processes. METHODS: ISG15 expression was examined immunohistologically in paraffin-embedded human placental and decidual tissue samples of all pregnancy trimesters on adjacent sections (first trimester n = 5, second n = 5, third n = 3). Samples were processed using a protocol applying a rabbit polyclonal ISG15 antibody. A mouse monoclonal cytokeratin seven antibody was utilized to identify the different placental departments and decidual glands. Staining results and anatomical features were evaluated blindly with strict rating criteria. RESULTS: ISG15 expression was identified in first and second trimester tissue samples. ISG15 localized especially to the extravillous cytotrophoblasts in the maternal wall and in maternal blood vessel. Expression was detected in cytotrophoblast progenitor cells in the placental villi and the cell column with a maximum in the first trimester. The syncytial layer stained positive in first and second trimester samples. Third trimester samples showed no expression of ISG15 at all. CONCLUSIONS: ISG15 abundance in the human placenta is an interesting finding, with implications for placental development, fetal growth and potential defense mechanism against infections. The maximal expression of ISG15 in the first and second trimester of pregnancy suggests that ISG function is needed when placental and embryo development is enormous and embryo susceptibility to external influences is high.

A possible ambivalent role for relaxin in human myometrial and decidual cells in vitro.

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.

The effect of relaxin on the oxytocin receptor in human uterine smooth muscle cells.

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.

Maternal IVS1-401 T allele of the estrogen receptor alpha is an independent predictor of late fetal loss.

OBJECTIVE: To investigate whether sequence variants in the gene encoding for estrogen receptor alpha (ER-alpha) are risk determinants for fetal loss. DESIGN: Case-control study. SETTING: University medical center. PATIENT(S): One hundred four women with a history of fetal loss and 277 healthy women with at least one previous pregnancy and no previous fetal loss. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The IVS1-401C/T polymorphism of the human ER-alpha, the G1691A mutation of the factor V gene (factor V Leiden), the G20210A mutation of the prothrombin gene, and the C677T polymorphism of the methylenetetrahydrofolate-reductase (MTHFR) gene were determined by polymerase chain reaction. RESULT(S): In the subgroup analysis of women with at least one late miscarriage (n = 70), the prevalences of the ER-alpha IVS1-401 T allele (T/T vs. C/C, odds ratio [OR]: 2.85, P=.018; T/T + C/T vs. C/C, OR: 2.28, P=.043) and of heterozygous factor V Leiden (OR, 3.2; P=.002) were significantly higher among women with late fetal loss than among healthy women. Carriers of both risk determinants have an at-least additive increase in risk for late abortions (OR, 7.0; P=.0004). The population of all late abortions that would be attributable to the genetic variants (population attributable risk) was 13.9% for factor V Leiden and 49.2% for the ER-alpha IVS1-401 T allele. CONCLUSION(S): Women with the IVS1-401 T allele of the ER-alpha and/or factor V Leiden are at increased risk for late fetal loss.

The polymorphism of platelet membrane integrin alpha2beta1 (alpha2807TT) is associated with premature onset of fetal loss.

Inherited thrombophilia could increase susceptibility to adverse pregnancy outcomes such as fetal loss. We determined the G1691A mutation of the factorV gene (FVL), the G20210A mutation of the prothrombin gene, the C677T polymorphism of the methylenetetrahydrofolate-reductase (MTHFR) gene, the HPA-1 polymorphism of the beta3 subunit of the platelet integrin alphaIIbbeta3 and the C807T polymorphism of the alpha2 subunit of integrin alpha2beta1 in 104 women with fetal loss and 277 normal women. In a subgroup analysis of women with recurrent early fetal loss (n=34), the prevalence of the genetic markers did not differ significantly between the women with early fetal loss and the normal women. However, in this subgroup of patients the onset of fetal loss was significantly earlier in women with the alpha2807TT genotype (7.1 +/- 1.9 vs. 8.8 +/- 1.5 weeks, p=0.001). No such significant difference was observed in carriers of the other genetic markers. In the subgroup analysis of women with late fetal loss (n=70), only the prevalence of heterozygous FVL was significantly associated with late fetal loss (odds ratio 3.2, p=0.002). There was no significant association of any genetic risk factor with premature fetal loss in the subgroup analysis of women with at least one late miscarriage. This study demonstrates a significant association of the alpha2807TT genotype of the platelet membrane integrin alpha2beta1 with premature onset of early fetal loss. It appears that this risk factor does not induce the pathomechanism, but modulates the course of fetal loss. Furthermore, our study confirms the association of FVL with late fetal loss.

Regulation of embryonic implantation.

The preimplantation embryo produces several factors during its development to signal its presence to the maternal organism. This paper will focus on the role of two distinctive cytokine and growth factor systems (interleukin-1 (IL-1) system and the vascular endothelial growth factor (VEGF) system) during early embryonic development and implantation. IL-1 receptor is expressed in the endometrium of various species and antagonising the biological effects of IL-1 leads to implantation failure in mice. We could show that this is due to an endometrial, not an embryonic effect. Furthermore, we could detect the expression of all components of the IL-1 system in preimplantation embryos from mice and humans. We could show a possible influence of IL-1 on other systems involved in embryonic implantation, including invasion (MMPs/TIMPs) and angiogenesis (VEGF), therefore suggesting a role of this cytokine family during early embryonic development. Immediately after contact to the endometrium, the embryo must induce angiogenesis to ensure its survival, VEGF is a potent angiogenetic growth factor. We have shown a cyclic regulation of the soluble VEGF-receptor, sflt, in human endometrium and have detected the expression of the transmembraneous VEGF-receptors, Flt-1 and kinase insert domain containing receptor (KDR) throughout the menstrual cycle. Furthermore, we have shown that the VEGF gene is one of the earliest genes activated during human preimplantation embryo development, giving rise to the assumption that VEGF is crucial for embryonic development.

Expression and function of 3beta hydroxisteroid dehydrogenase (3beta HSD) type II and corticosteroid binding globulin (CBG) in granulosa cells from ovaries of women with and without endometriosis.

PURPOSE: To investigate the secretion of progesterone (P4) and corticosteroid binding globulin (CBG) by granulosa luteal cells (GC) as well as the mRNA levels of CBG and 3beta hydroxisteroid dehydrogenase (3beta HSD), in women with and without endometriosis in vivo and in vitro. METHODS: Prospective study in a private, university-affiliated assisted reproduction unit, including women with severe endometriosis (n = 14) or without the disease (n = 20) undergoing in vitro fertilization/intracytoplasmic sperm injection and embryo transfer. GC were obtained from each follicle aspirated, pooled for each patient, and follicular and blood contaminating leukocytes depleted through immunomagnetic purification. Secreted P4 and CBG, and mRNA for both 3beta HSD and CBG were determined in vivo and in vitro using RIA and reverse transcription followed by competitive polymerase chain reaction (cRT-PCR). RESULTS: The pattern of expression of 3beta HSD and CBG mRNAs in vivo and in vitro was similar in both groups. Also, GC from patients with endometriosis produced equal amounts of P4 and CBG than controls without the disease, either in freshly isolated cells or in 24-h cultures. CONCLUSIONS: The GC function in terms of 3beta HSD and CBG mRNA expression and P4/CBG secretion does not seem to be altered in patients with endometriosis in comparison with those without this condition.