Laparoscopic Chromopertubation, Myomectomy with Opening of the Uterine Cavity and Hysteroscopy during the Early Implantation Phase of an Undetected Pregnancy: Delivery of a Child with a Complex Brain Malformation.

A previously infertile woman underwent laparoscopic myomectomy involving opening of the uterine cavity and chromopertubation that showed closed Fallopian tubes during the early implantation stage of an undetected pregnancy. The pregnancy was not terminated, and a child with a complex brain malformation was delivered at 37 weeks of gestation by Cesarean section.

Folate Metabolism and Human Reproduction.

Folate metabolism affects ovarian function, implantation, embryogenesis and the entire process of pregnancy. In addition to its well-established effect on the incidence of neural tube defects, associations have been found between reduced folic acid levels and increased homocysteine concentrations on the one hand, and recurrent spontaneous abortions and other complications of pregnancy on the other. In infertility patients undergoing IVF/ICSI treatment, a clear correlation was found between plasma folate concentrations and the incidence of dichorionic twin pregnancies. In patients supplemented with 0.4 mg/d folic acid undergoing ovarian hyperstimulation and oocyte pick-up, carriers of the MTHFR 677T mutation were found to have lower serum estradiol concentrations at ovulation and fewer oocytes could be retrieved from them. It appears that these negative effects can be compensated for in full by increasing the daily dose of folic acid to at least 0.8 mg. In carriers of the MTHFR 677TT genotype who receive appropriate supplementation, AMH concentrations were found to be significantly increased, which could indicate a compensatory mechanism. AMH concentrations in homozygous carriers of the MTHFR 677TT genotype could even be overestimated, as almost 20 % fewer oocytes are retrieved from these patients per AMH unit compared to MTHFR 677CC wild-type individuals.

Ethical Issues Currently Being Discussed in Relation to Reproductive Medicine and the Laws Governing Reproductive Medicine.

Reproductive medicine laws in Germany currently mean that the relationship status of prospective parents is taken into consideration in decisions on whether their application for assisted reproduction is approved or rejected. In the light of new forms of shared parenthood, we should ask ourselves whether the current regulations are still an appropriate way of guaranteeing the best for the child. Current medical practices and their legal basis will be illustrated using the examples of sperm, egg and embryo donation. From an ethical perspective, the question at stake is to what extent an "Ethics of Parenthood" can make it possible to act responsibly with regard to the changes occurring in forms of shared parenthood. Such an ethics is aimed at supporting parents in realising the reproductive autonomy guaranteed in the German Constitution through social and ethical aspects of the child-parent relationship.

Dopamine in human follicular fluid is associated with cellular uptake and metabolism-dependent generation of reactive oxygen species in granulosa cells: implications for physiology and pathology.

STUDY QUESTION: Is the neurotransmitter dopamine (DA) in the human ovary involved in the generation of reactive oxygen species (ROS)? SUMMARY ANSWER: Human ovarian follicular fluid contains DA, which causes the generation of ROS in cultured human granulosa cells (GCs), and alterations of DA levels in follicular fluid and DA uptake/metabolism in GCs in patients with polycystic ovary syndrome (PCOS) are linked to increased levels of ROS. WHAT IS KNOWN ALREADY: DA is an important neurotransmitter in the brain, and the metabolism of DA results in the generation of ROS. DA was detected in human ovarian homogenates, but whether it is present in follicular fluid and plays a role in the follicle is not known. STUDY DESIGN, SIZE AND DURATION: We used human follicular fluid from patients undergoing in vitro fertilization (IVF), GCs from patients with or without PCOS and also employed mathematical modeling to investigate the presence of DA and its effects on ROS. PARTICIPANTS/MATERIALS, SETTING AND METHODS: DA in follicular fluid and GCs was determined by enzyme-linked immunosorbent assay. GC viability, apoptosis and generation of ROS were monitored in GCs upon addition of DA. Inhibitors of DA uptake and metabolism, an antioxidant and DA receptor agonists, were used to study cellular uptake and the mechanism of DA-induced ROS generation. Human GCs were examined for the presence and abundance of transcripts of the DA transporter (DAT; SLC6A3), the DA-metabolizing enzymes monoamine oxidases A/B (MAO-A/B) and catechol-O-methyltransferase and the vesicular monoamine transporter. A computational model was developed to describe and predict DA-induced ROS generation in human GCs. MAIN RESULTS AND ROLE OF CHANCE: We found DA in follicular fluid of ovulatory follicles of the human ovary and in GCs. DAT and MAO-A/B, which are expressed by GCs, are prerequisites for a DA receptor-independent generation of ROS in GCs. Blockers of DAT and MAO-A/B, as well as an antioxidant, prevented the generation of ROS (P < 0.05). Agonists of DA receptors (D1 and D2) did not induce ROS. DA, in the concentration range found in follicular fluid, did not induce apoptosis of cultured GCs. Computational modeling suggested, however, that ROS levels in GCs depend on the concentrations of DA and on the cellular uptake and metabolism. In PCOS-derived follicular fluid, the levels of DA were higher (P < 0.05) in GCs, the transcript levels of DAT and MAO-A/B in GCs were 2-fold higher (P < 0.05) and the DA-induced ROS levels were found to be more than 4-fold increased (P < 0.05) compared with non-PCOS cells. Furthermore, DA at a high concentration induced apoptosis in PCOS-derived GCs. LIMITATIONS, REASONS FOR CAUTION: While the results in IVF-derived follicular fluid and in GCs reveal for the first time the presence of DA in the human follicular compartment, functions of DA could only be studied in IVF-derived GCs, which can be viewed as a cellular model for the periovulatory follicular phase. The full functional importance of DA-induced ROS in small follicles and other compartments of the ovary, especially in PCOS samples, remains to be shown. WIDER IMPLICATIONS OF THE FINDINGS: The results identify DA as a factor in the human ovary, which, via ROS generation, could play a role in ovarian physiology and pathology. The results obtained in samples from women with PCOS suggest the involvement of DA, acting via ROS, in this condition. STUDY FUNDING/COMPETING INTERESTS: This work was supported by a grant from DFG MA1080/17-3 and in part MA1080/19-1. There are no competing interests.

[Hereditary thrombophilias and placental-mediated pregnancy complications in the II./III. trimester]. / Hereditäre Thrombophilien und Plazenta-medierte Schwangerschaftskomplikationen im II./III. Trimenon.

UNLABELLED: Placental-mediated pregnancy complications (PmC) like preeclampsia, intrauterine growth restriction and placental abruption are common causes of fetal and maternal morbidity and mortality. The high prevalence of hereditary thrombophilias in case-control studies associated with pathological morphological findings of the placenta in these cases gave evidence for the association between hereditary thrombophilias and PmC. However, data from the literature are inconsistent, since subsequent prospective cohort studies could not demonstrate significant associations between inherited thrombophilia and PmC. Because of the multifactorial aetiology of PmC it may be difficult to prove, that hereditary thrombophilias are independent risk factors for PmC. Current guidelines do not recommend screening for inherited thrombophilias in patients with previous PmC. Evidence from current in vitro studies have shown, that heparin has beneficial non-anticoagulatory effects on trophoblast invasion. Retrospective case-control studies and recently published randomised controlled cohort studies have shown, that prophylactic administration of low-molecular-weight heparin (LWH), started in early pregnancy, may lead to a significant reduction in the incidence of PmC in subsequent pregnancies in patients with and without hereditary thrombophilias and previous PmC. CONCLUSION: Large, well-designed multicenter studies are needed to elucidate the role of hereditary thrombophilias in cases of PmC and to confirm the benefit of LWH for subsequent pregnancy outcomes.

Seminal plasma stimulates cytokine production in endometrial epithelial cell cultures independently of the presence of leucocytes.

We examined the effects of normal and leucocyte-positive semen on cytokine expression in endometrial epithelial cell cultures. Cytokines in pooled seminal plasma (SP) samples of men with normal semen parameters without (n = 9) and with leucocytospermia (LCS) (n = 9) were determined. Cultures of epithelial endometrial cells of women (n = 7) in the secretory phase were incubated with 10% SP for 24 h. Cytokines in culture supernatants and mRNA concentrations in the cultured cells were determined. Mean concentrations of interleukin (IL)-1beta and transforming growth factor (TGF)-beta1 were significantly higher (P < 0.05) in culture media with SP of men with normal semen parameters and LCS compared with control. Accordingly, a significant increase (P < 0.05) in mRNA concentrations (amol ml(-1)) of IL-1beta and TGF-beta1 could be detected. The mean TGF-beta1 and IL-1beta concentrations in the culture supernatant with 10% SP from patients with normal semen parameters were 24-fold (P < 0.05) and 2-fold higher respectively when compared with control. The mean protein concentration of TGF-beta1 measured in the supernatant with SP of men with LCS was not significantly reduced as compared with the supernatant with normal SP. In conclusion, our experiments support the concept of an endometrial sensitation effect by semen.

Primary cervical cancer screening by self-sampling of human papillomavirus DNA in internal medicine outpatient clinics.

BACKGROUND: We determined whether testing of self-collected vaginal swabs for human papillomavirus (HPV) DNA can be used to screen for cervical disease within internal medicine outpatient clinics. PATIENTS AND METHODS: In this prospective study, 560 patients visiting two referral outpatient clinics for internal medicine were asked to take an HPV self-sample. Acceptability of self sampling, HPV prevalence and cervical intraepithelial neoplasia (CIN) detection rate were evaluated. RESULTS: 435 women (78%) performed HPV self-sampling; 1.5% considered self-sampling to be difficult. 134 women (31%) tested positive for high-risk type of HPV. There were significant differences between HPV-positive and -negative women with respect to the following: mean age (42 versus 46 years), number of women aged <16 years at first coitus (35% versus 23%) and history of drug abuse (8.3% versus 2.6%). Colposcopy could be performed for 70 HPV positive women: CIN 1-3 was identified in 24%. Two of 52 women with HPV-negative results undergoing colposcopy had biopsy-confirmed CIN 1. Test performance for detection of CIN 2-3 after correction for verification bias: sensitivity, 100%; specificity, 71%; negative predictive value, 100%; positive predictive value, 10%. HPV persistence was associated with a 5.7-fold risk of CIN 2-3 detection at follow-up. CONCLUSIONS: Self-assessment for HPV DNA is an easy, feasible and well-accepted method for HPV testing and for cervical cancer screening in internal medicine outpatient clinics.

Seminal plasma induces mRNA expression of IL-1beta, IL-6 and LIF in endometrial epithelial cells in vitro.

The influence of seminal plasma on the mRNA expression of cytokines in human endometrial epithelial and stromal cells and the cytokine production of spermatozoa were investigated in vitro. Seminal plasma and spermatozoa were collected from healthy volunteers and were screened by enzyme-linked immunosorbent assay for cytokines. Epithelial and stromal cells from fertile women were cultured on matrigel or polystyrol and incubated with pooled seminal plasma or with transforming growth factor beta1 (TGF-beta1), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF), which were found to be significantly concentrated in seminal plasma. Endometrial cytokine expression was analysed by RNase protection assay and supported by RT-PCR. Supernatants of highly purified spermatozoa did not contain detectable levels of IL-1beta, IL-6 and VEGF. Screening of seminal plasma revealed concentrations >10-fold above the serum level for TGF-beta1, IL-8 and VEGF. Incubation of epithelial cells with 0.1, 1 and 10% seminal plasma resulted in concentration-dependant stimulation of IL-1beta, IL-6 and LIF mRNA expression. Maximum stimulation was found in epithelial cells from tissue samples taken in the mid secretory phase. Epithelial mRNA expression of IL-1beta, IL-6 and LIF increased by stimulation with TGF-beta1 and IL-8, but not with VEGF. In conclusion, seminal plasma stimulates expression of pro-inflammatory cytokines in endometrial epithelial cells in vitro. This effect might at least in part be exerted by TGF-beta1 and IL-8, abundantly present in seminal plasma. The in-vivo physiological relevance of these in-vitro studies remains to be determined.

Polymorphisms in the ACE and PAI-1 genes are associated with recurrent spontaneous miscarriages.

BACKGROUND: Successful pregnancies require fine tuning of fibrinolytic activities in order to secure fibrin polymerization and stabilization of the placental basal plate as well as to prevent excess fibrin deposition in placental vessels and intervillous spaces. Fibrinolysis is tightly regulated by plasminogen activator inhibitor-1 (PAI-1). Endothelial PAI-1 synthesis is induced by angiotensin II, which is generated by angiotensin I-converting enzyme (ACE). METHODS: We studied the ACE deletion (D)/insertion (I) polymorphism and the PAI-1 4G/5G polymorphism in women with recurrent spontaneous miscarriages (RM). Both polymorphisms have been shown to be associated with ACE and PAI-1 expression levels respectively. A study group of 184 patients with a history of two or more consecutive unexplained spontaneous miscarriages was compared with a control group of 127 patients with uneventful term deliveries and no history of miscarriages. RESULTS: Our findings show: (i) homozygosity for the D allele of the ACE gene, which results in elevated PAI-1 concentrations and hypofibrinolysis, is associated with an elevated risk of RM; (ii) the combination of the D/D genotype with two 4G alleles of the PAI-1 promoter, which further increases PAI-1 plasma levels, is significantly more frequent in RM patients compared with controls. CONCLUSIONS: Based on these results, we recommend the incorporation of these two polymorphisms into the spectrum of thrombophilic mutations which should be analysed in individuals with recurrent spontaneous miscarriages. Patients homozygous for both the ACE D and PAI-1 4G alleles may benefit from the application of low molecular weight heparin as early as possible in the pregnancy in order to prevent uteroplacental microthromboses.

Endometrial interleukin-6 in vitro is not regulated directly by female steroid hormones, but by pro-inflammatory cytokines and hypoxia.

Endometrial interleukin-6 (IL-6) mRNA has been reported to be suppressed in the mid-secretory phase in patients with recurrent early spontaneous abortions. This prompted our study concerning the regulation of endometrial IL-6 in cell culture models of endometrial epithelial and stromal cells. Steroid-dependent secretion of IL-6 was analysed by 17beta-estradiol (10(-8) mol/l) or progesterone (10(-6) mol/l) treatment and withdrawal (n = 8). Regulation by pro-inflammatory cytokines was studied in co-cultures of endometrial cells with human blood peripheral mononuclear cells (PBMC; n = 5) and by stimulation with IL-1beta, IL-6 and tumour necrosis factor alpha (TNFalpha), secreted by PBMCs at high concentrations. Regulation by hypoxia was assessed by culture of endometrial cells in 2% oxygen for 6 and 24 h (n = 5). IL-6 mRNA and protein levels were analysed by RT-PCR and enzyme-linked immunosorbent assays respectively. Endometrial IL-6 was not directly affected by 17beta-estradiol and/or progesterone. Co-culturing endometrial cells with PBMCs led to an increase of stromal but not epithelial IL-6 mRNA levels. In stromal cells, IL-6 secretion increased 2-10-fold if stimulated with 10 ng/ml recombinant IL-1beta or TNFalpha (P < 0.05). Hypoxia stimulated IL-6 secretion in epithelial cells up to 2-fold and in stromal cells up to 48-fold (P < 0.05). In conclusion, IL-6 expression in stromal and epithelial cells in vitro is regulated differently by pro-inflammatory cytokines and hypoxia. These results suggest a tight and specific network of control for this important cytokine within different endometrial compartments.

Endometrial expression and secretion of interleukin-6 throughout the menstrual cycle.

Previous findings revealing reduced endometrial interleukin-6 (IL-6) mRNA expression in patients with recurrent early abortions gave rise to the analysis of IL-6 synthesis in the human endometrium, and how it relates to concentrations in uterine secretions. Endometrial tissues and uterine secretions were collected from patients undergoing hysterectomy. Expression of IL-6 receptor and gp130 mRNA (n = 28) in total endometrium, of IL-6 mRNA in endometrial epithelial and stromal cells, and CD45-positive leukocytes were investigated by RNAase protection assay throughout the cycle. IL-6 protein was assessed in endometrial tissue by immunohistochemistry (n = 32) and in uterine secretions by enzyme-linked immunosorbent assay (ELISA) (n = 33). IL-6 mRNA was expressed at low levels in the proliferative phase, and expression increased progressively in the secretory phase. The increase was attributed to epithelial and stromal cells and leukocytes. Concentrations of IL-6 protein in endometrial glands and in uterine secretions were low in the proliferative phase, and increased 5-10-fold in the mid- to late secretory phase. mRNA expression of IL-6 receptor and gp130 remained constant in total endometrium throughout the menstrual cycle. High concentrations of IL-6 in the mid-secretory phase, the putative implantation window, and a further increase in the late secretory phase, the premenstrual period, support a role of IL-6 in the regulation of endometrial functions.

Endometrial osteopontin, a ligand of beta3-integrin, is maximally expressed around the time of the "implantation window".

OBJECTIVE: To analyze endometrial mRNA and protein expression of osteopontin and its receptor, beta3-integrin, throughout the menstrual cycle. DESIGN: Study by immunohistochemistry, RNase protection assay, and ELISA. SETTING: Academic research unit. PATIENT(S): Forty-five regularly cycling women without endometrial pathology. INTERVENTION(S): Expression of endometrial osteopontin and beta3-integrin mRNA was analyzed by RNase protection assay in endometrium, endometrial epithelial cells, stromal cells, and endometrial leukocytes (CD45) and by immunohistochemistry in frozen sections of endometrium throughout the menstrual cycle. Concentration of osteopontin was studied in uterine secretions by ELISA. MAIN OUTCOME MEASURE(S): mRNA and protein expression of osteopontin and beta3-integrin. RESULT(S): Osteopontin mRNA and protein was weakly expressed in the proliferative phase and maximally expressed in the mid to late secretory phases in endometrium, endometrial epithelial cells, and endometrial leukocytes and in uterine secretions. Beta3-integrin mRNA and protein were expressed in stromal cells throughout the menstrual cycle and were maximally expressed in epithelial cells in the mid to late secretory phases. CONCLUSION(S): Expression of osteopontin and its receptor, beta3-integrin, in human endometrial glands and osteopontin secretion into the uterine cavity around the time of the "implantation window" suggest a role for these proteins in endometrial function and implantation.

Thrombophilic gene mutations and recurrent spontaneous abortion: prothrombin mutation increases the risk in the first trimester.

PROBLEM: Thrombophilic predisposition may be one of the underlying causes of recurrent spontaneous abortions (RSA). We studied the prevalence of five thrombophilic gene mutations in patients with RSA. METHOD OF STUDY: 102 patients with two or more consecutive abortions and 128 women without miscarriage were analyzed for factor V Leiden mutation (FVL), prothrombin G20210A mutation (PTM), C677T mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, glycoprotein IIIa (GPIIIa) C1565T polymorphism, and beta-fibrinogen G-455A polymorphism by polymerase chain reaction (PCR) techniques. RESULTS: No differences in the prevalence of FVL, MTHFR T/T, GPIIIa and 1-fibrinogen polymorphism were detected. Heterozygous PTM occurred more often in patients with RSA. This effect was significant in a subgroup with abortions exclusively in the first trimester (6.7%, vs. 0.8%, P = 0.027, OR 8.5). CONCLUSIONS: In contrast to the other mutations and polymorphisms, heterozygous PTM is more common in patients with abortions in the first trimester. This might reflect an influence of PTM on pathogenesis of early pregnancy loss.

S680N substitution of the follicle-stimulating hormone receptor is a common polymorphism not associated with spontaneous human twinning.

OBJECTIVE: To determine the influence of the A307/S680 and T307/N680 isoforms of the follicle-stimulating hormone receptor (FSHR) gene on the incidence of spontaneous human twinning. DESIGN: Case-control study. SETTING: Departments of Obstetrics and Gynecology and of Clinical Chemistry-Grosshadern, University Hospital Munich, Germany. PATIENT(S): Fifty-four mothers with dichorionic twin pregnancies and 92 singleton mothers as controls, who had conceived without assisted reproduction. INTERVENTION(S): Exon 10 of the FSHR gene was screened for the G2105A/S680N mutation. MAIN OUTCOME MEASURE(S): Amplification of genomic DNA by the polymerase chain reaction followed by restriction fragment length polymorphism analysis. RESULT(S): Allele frequencies for the G2105A/ S680N substitution of the FSHR in twin mothers were not different from those of controls (genotype (isoform) [twins vs. controls]: G/G (S/S) [24.1% vs. 22.3%]; A/G (N/S) [57.4% vs. 55.4%]; A/A (N/N) [18.5% vs. 22.3%]). Subgroup analysis of women with three or more successful pregnancies gave a similar result (G/G (S/S) [17.7% vs. 13.7%]; A/G (N/S) [64.7% vs. 63.6%]; A/A (N/N) [17.6% vs. 22.7%]). There was no correlation between FSHR isoform and twinning. CONCLUSION(S): The S680N substitution of the follicle-stimulating hormone receptor is a common polymorphism not associated with spontaneous human twinning.

Concentration of granulocyte elastase in seminal plasma is not associated with sperm motility.

Although elastase could affect sperm motility in vitro, secretory leukocytes protease inhibitor (SLPI) prevents sperm from being attacked by elastase. The authors investigated the correlations of elastase level with sperm motility and SLPI level in vivo. Semen samples (n = 116) were collected and centrifuged after semen analysis. Elastase and SLPI levels were determined by an enzyme immunosorbent assay. Samples were classified by elastase levels into low (<250 ng/mL), moderate (250-1,000 ng/mL), and high elastase groups (> or =1,000 ng/mL). Elastase levels (range, 2.8-23,974.4 ng/mL) were not associated with sperm motility. The median SLPI level in the high elastase group was 15,900 ng/mL (range, 2.860-46,900 ng/mL). However, there was no significant correlation between elastase and SLPI levels in seminal plasma. Since SLPI forms a 1:1 complex with elastase, these results suggest that seminal plasma has a sufficient amount of SLPI to protect spermatozoa from elastase.

Decreased suppression of antibody-dependent cellular cytotoxicity by seminal plasma in unexplained infertility.

OBJECTIVE: To determine whether seminal plasma (SP) from unexplained infertile males has different suppressive activity on antibody-dependent cellular cytotoxicity (ADCC) than SP from fertile males or SP from males of couples with known infertility factor. DESIGN: Comparative clinical/experimental study. SETTING: In vitro fertilization program in a university hospital and a hospital research laboratory. PATIENT(S): A total of 245 SP samples from 174 infertile and 16 fertile couples were compared. INTERVENTION(S): SP suppression of ADCC was measured by using human 51chromium-labeled red blood cells (RBC), sensitized with IgG-rabbit-anti-human-RBC as targets and peripheral blood lymphocytes as effector cells. MAIN OUTCOME MEASURE(S): Suppressive activity of each sample was determined by calculating 51Cr-release in the presence and absence of SP. RESULT(S): When analyzed with respect to sperm number, motility, and morphology, suppressive activities of samples with normal semen analyses (n = 142) were significantly higher (x = 37% +/- 14%) than suppressive activities of abnormal samples (n = 103; x = 32% +/- 13%). There was no strong correlation of suppressive activity to other semen parameters. Within the andrologically normal males, SP from the unexplained infertile couples (n = 15) showed significantly lower suppressive activity (x = 24% +/- 11%) compared with the SP from fertile males (n = 16; x = 35% +/- 13%) and from couples with female infertility factor (n = 65; x = 39% +/- 14%). CONCLUSION(S): Loss of suppressive activity is associated with unexplained infertility, even in male patients who previously were considered normal by traditional methods of semen analysis.