Maternal smoking as an independent risk factor for the development of severe retinopathy of prematurity in very preterm infants.

BACKGROUND/OBJECTIVES: Retinopathy of prematurity (ROP) is a severe neonatal complication potentially leading to visual impairment and blindness. Known risk factors include preterm birth, low birth weight and respiratory support. Limited and contradictory data exist on the risk of maternal smoking during pregnancy on the development of ROP. This study aims to investigate smoking as an independent risk factor for the development of severe ROP (≥stage 3). SUBJECTS/METHODS: This is a single centre retrospective case-control study of prospectively collected clinical data of infants born before 32 weeks of gestation between 2001 and 2012 at a tertiary care university hospital. The association between maternal smoking during pregnancy and the development of severe ROP was analyzed by multivariate logistic regression. RESULTS: In total, n = 751 infants born < 32 weeks of gestation were included in this study. In total, 52.9% (n = 397) were diagnosed with ROP and 10.8% (n = 81) developed ROP ≥ stage 3. In total, 8.4% (n = 63) mothers presented with a history of smoking during pregnancy, which was associated to a higher rate of ROP (OR 2.59, 95% CI 1.10-6.12). Low gestational age, low birth weight and prolonged respiratory support were confirmed as independent risk factors for the development of severe ROP. CONCLUSIONS: To date, this is the largest study evaluating the effect of maternal smoking on the development of ROP. Maternal smoking during pregnancy is identified as an independent risk factor for the development of severe ROP in preterm infants born < 32 weeks of gestation.

Two of a kind? Immunological and clinical risk factors differ between recurrent implantation failure and recurrent miscarriage.

Recurrent miscarriage (RM) and recurrent implantation failure (RIF) are unsolved challenges in reproductive medicine. Whether RIF patients share the same risk factors as RM patients is a matter of debate. Besides clinical factors, immune alterations are discussed in both conditions. The scope of this study was to compare the prevalence of clinical and immunological risk factors in a large cohort of RM and RIF patients. Between 11/2011 and 02/2019, 613 RM and 185 RIF patients were included. A screening for anatomical malformations, endocrine, autoimmune, prothrombotic and parental chromosomal disorders was performed. The immune status was assessed using flow cytometry analysis of peripheral lymphocyte subpopulations and uterine natural killer cells (uNK cells) using immunohistochemistry. RM patients showed a higher rate of intrauterine adhesions and elevated antinuclear antibodies ≥ 1:160 (p < 0.05). A higher prevalence of submucous fibroids and increased factor VIII levels were observed in RIF patients (p < 0.05). The prevalence of an antiphospholipid syndrome (APLS) was low and did not differ between the two groups. RIF patients had higher numbers of peripheral regulatory T-cells (p < 0.05). Significant more RIF patients were diagnosed with elevated uNK cells (p < 0.05). Differences in clinical and immunological risk factors of RM and RIF patients reflect different entities. Lower Tregs in RM and higher uNK cells in RIF patients might be related to the previous exposure of the immune system to fetal cells. The low prevalence of an APLS indicates a potential overestimation of this factor in the pathophysiology of RM and RIF.

NK cell subsets in idiopathic recurrent miscarriage and renal transplant patients.

The present review article compares NK cell subsets and cytokine patterns determined in the peripheral blood as well as results of functional in-vitro assays using peripheral NK cells of idiopathic recurrent miscarriage (iRM) patients with corresponding results obtained in female healthy controls and female renal transplant recipients with good long-term graft function. Immune mechanisms, inducing transplant rejection in long-term transplant recipients might also be able to induce rejection of semi-allogeneic fetal cells in patients with iRM. Consequently, the immune status of transplant recipients with good stable long-term graft function should be different from the immune status of iRM patients. iRM patients show a strong persistent cytotoxic NK cell response in the periphery. Simultaneously, immunostimulatory Th1 as well as immunosuppressive Th2 type lymphocytes in the blood are strongly activated but plasma levels of immunosuppressive Th2 type cytokines are abnormally low. In-vitro, unstimulated NK cell cultures of iRM patients show a strong spontaneous TGF-ß1 release in the supernatant but lower TGF-ß1 levels after stimulation with tumor cell line K562, suggesting strong consumption of TGF-ß1 by pre-activated NK cells of iRM patients that might contribute to the low systemic Th2 type plasma levels. iRM patients do not show a systemic switch to a Th2 type cytokine pattern and one might hypothesize that low TGF-ß plasma levels indicate low TGF-ß levels in the micromilieu immediately before fetal rejection. Persistent TGF-ß deficiency implies a persistent unfavorable micromilieu for pregnancy resulting in failing tolerance induction due to lack of TGF-ß, a condition that might contribute to iRM.

Diagnosis and Therapy Before Assisted Reproductive Treatments. Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Register Number 015-085, February 2019) - Part 1, Basic Assessment of the Woman.

Introduction Supporting and counselling couples with fertility issues prior to starting ART is a multidisciplinary diagnostic and therapeutic challenge. The first German/Austrian/Swiss interdisciplinary S2k guideline on "Diagnosis and Therapy Before Assisted Reproductive Treatments (ART)" was published in February 2019. This guideline was developed in the context of the guidelines program of the German Society of Gynecology and Obstetrics (DGGG) in cooperation with the Swiss Society of Gynecology and Obstetrics (SGGG) and the Austrian Society of Gynecology and Obstetrics (OEGGG). Aims One third of the causes of involuntary childlessness are still unclear, even if the woman or man have numerous possible risk factors. Because the topic is still very much taboo, couples may be socially isolated and often only present quite late to a fertility center. At present, there is no standard treatment concept, as currently no standard multidisciplinary procedures exist for the diagnostic workup and treatment of infertility. The aim of this guideline is to provide physicians with evidence-based recommendations for counselling, diagnostic workup and treatment. Methods This S2k guideline was developed on behalf of the Guidelines Commission of the DGGG by representative members from different professional medical organizations and societies using a structured consensus process. Recommendations The first part of this guideline focuses on the basic assessment of affected women, including standard anatomical and endocrinological diagnostic procedures and examinations into any potential infections. Other areas addressed in this guideline are the immunological workup with an evaluation of the patient's vaccination status, an evaluation of psychological factors, and the collection of data relating to other relevant factors affecting infertility. The second part will focus on explanations of diagnostic procedures compiled in collaboration with specialists from other medical specialties such as andrologists, human geneticists and oncologists.

Diagnosis and Treatment Before Assisted Reproductive Treatments. Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Register Number 015-085, February 2019) - Part 2, Hemostaseology, Andrology, Genetics and History of Malignant Disease.

Introduction Supporting and counselling couples with fertility issues prior to starting ART is a multidisciplinary diagnostic and therapeutic challenge. The first German-language interdisciplinary S2k guideline on "Diagnosis and Therapy Before Assisted Reproductive Treatments (ART)" was published in February 2019. The guideline was developed in the context of the guidelines program of the German Society of Gynecology and Obstetrics (DGGG) in cooperation with the Swiss Society of Gynecology and Obstetrics (SGGG) and the Austrian Society of Gynecology and Obstetrics (OEGGG). Aim In one third of cases, the cause of involuntary childlessness remains unclear, even if the woman or man have numerous possible risk factors. Because the topic is still very much taboo, couples may be socially isolated and often only present quite late to a fertility center. There is no standard treatment concept for these patients at present, as there are currently no standard multidisciplinary procedures for the diagnostic workup and treatment of infertility. The aim of this guideline is to provide physicians with evidence-based recommendations for counselling, diagnosis and treatment. Methods This S2k guideline was developed on behalf of the Guidelines Commission of the DGGG by representative members from different professional medical organizations and societies using a structured consensus process. Recommendations This second part of the guideline describes the hematological workup for women as well as additional diagnostic procedures which can be used to investigate couples and which are carried out in cooperation with physicians working in other medical fields such as andrologists, geneticists and oncologists.

Patients with idiopathic recurrent miscarriage have abnormally high TGFß+ blood NK, NKT and T cells in the presence of abnormally low TGFß plasma levels.

BACKGROUND: Previously, we demonstrated up-regulated activated CD4+ and CD8+ T lymphocytes as well as up-regulated cytotoxic NK cells in the blood of patients with idiopathic recurrent miscarriage. In the present study, we tried to identify deficiencies in counter-regulating immune mechanisms of these patients. METHOD: Cytokines were determined in NK cells and in plasma samples of 35 healthy controls, 33 patients with idiopathic recurrent miscarriage, 34 patients with end stage renal disease, 10 transplant patients early and 37 transplant patients late post-transplant using flow-cytometry and luminex. In addition, cytokines were studied in supernatants of cell cultures with peripheral blood mononuclear cells stimulated in-vitro with tumor cell line K562. RESULTS: Patients with idiopathic recurrent miscarriage exhibited the highest absolute cell counts of circulating TGFß1+ NK, NKT and T lymphocytes and the lowest TGFß1 plasma levels of all study groups (for all p < 0.050). In-vitro, peripheral blood mononuclear cells of patients with idiopathic recurrent miscarriage showed high spontaneous TGFß1 production that could not be further increased by stimulation with K562, indicating increased consumption of TGFß1 by activated cells in the cell culture. Moreover, patients with idiopathic recurrent miscarriage had abnormally high IL4+ as well as abnormally high IFNy+ NK cells (p < 0.010) but similar IL10+ NK cell numbers as female healthy controls and showed the lowest plasma levels of IL10, TGFß3, IL1RA, IL1ß, IL5, IL6, IL8, IL17, TNFα, GM-CSF, TPO and VEGF and the highest plasma levels of G-CSF, FGF-basic, CCL3 and CXCL5 as compared to female HC and female transplant recipients (for all p < 0.050). CONCLUSIONS: Patients with idiopathic recurrent miscarriage show an activated immune system that can hardly be stimulated further and cannot be efficiently down-regulated by up-regulated TGFß1+ and IL4+ NK, NKT and T lymphocytes which are present concomitantly in these patients. The strongly decreased TGFß and IL10 plasma levels indicate deficient down-regulation and reflect a dysbalance of the immune system in patients with idiopathic recurrent miscarriage. These findings may be relevant for explaining the pathogenesis of idiopathic recurrent miscarriage.

Tocolysis with the ß2-sympathomimetic fenoterol does not increase the occurrence of infantile hemangioma in preterm and term infants.

PURPOSE: ß2-sympathomimetics are used in obstetrics as tocolytic agents, despite a remarkable profile of side effects. Recently, the ß2-sympathomimetic tocolytic drug hexoprenaline was identified as an independent risk factor for the development of infantile hemangioma (IH) in preterm infants. The aim of this study was to evaluate whether this observed effect was applicable to other ß2-mimetic tocolytic agents like fenoterol. METHODS: Clinical prospectively collected data of all infants born between 2001 and 2012 and admitted to the neonatal intensive care unit (NICU) at Heidelberg University Hospital and respective maternal data were merged. For the current retrospective cohort study, cases (IH) were matched to controls (no IH) at a ratio of 1:4, adjusting for birth weight, gestational age, gender and multiple gestations. Prenatal exposure to fenoterol and perinatal outcome were analyzed in the total cohort and in subgroups. RESULTS: N = 5070 infants were admitted to our neonatal department, out of which n = 172 infants with IH were identified and compared to n = 596 matched controls. Exposure to fenoterol was not associated with a higher rate of IH in the total matched population (OR 0.926, 95% CI 0.619-1.384) or in a subgroup of neonates < 32 weeks of gestation or with a birth weight < 1500 g (OR 1.127, 95% CI 0.709-1.791). In the total matched population, prenatal exposure to glucocorticoids was associated with a reduced occurrence of IH (OR 0.566, 95% CI 0.332-0.964) and neonates with IH showed a prolonged total hospital stay compared to controls (69 vs. 57 days, p = 0.0033). Known risk factors for IH were confirmed by our large study cohort and included female gender, low birth weight, preterm birth and multiple gestations (all p < 0.005). CONCLUSIONS: Exposure to fenoterol during pregnancy does not increase the occurrence of IH. Further studies are needed to explore differences in the risk profiles of different ß2-sympathomimetic tocolytic drugs.

Changes of NK cell subsets with time post-transplant in peripheral blood of renal transplant recipients.

BACKGROUND: There is evidence that NK cells with low cytotoxicity but strong immunoregulatory characteristics contribute to good graft outcome. We attempted to investigate which NK cell subsets increase post-transplant and might affect graft function. METHOD: Lymphocyte and NK cell subsets were determined in whole blood using eight-colour-fluorescence flow cytometry in patients pre-transplant and post-transplant. In total, 31 transplant recipients were studied. RESULTS: When cell numbers were compared in 9 patients pre- and 6 months post-transplant, post-transplant CD56dimCD16+ (p = 0.011) NK cells with the phenotype CD158a+ (p = 0.008), CD158e+ (p = 0.038), NKG2A+ (p = 0.008), NKG2D+ (p = 0.011), IFNyR+ (p = 0.008), perforin+ (p = 0.008), granzymeB+ (p = 0.008), perforin+granzymeB+ (p = 0.008) and perforin-granzymeB- (p = 0.021) were lower than those pre-transplant, indicating a post-transplant reduction of cytotoxic NK cells. In 28 patients NK cell subsets were analyzed with respect to time post-transplant (median 888 days post-transplant). CD56dimCD16+ NK cells co-expressing CD158a (p = 0.014), NKG2D (p = 0.047), IL4R (p = 0.038), IL10R (p = 0.008) and IFNy (p = 0.036) as well as CD56bright NK cells with the phenotype TGFß+ (p = 0.017), TGFR+ (p = 0.035), CD158a+ (p = 0.042) and perforin-granzymeB- (p = 0.048) increased with time post-transplant. CONCLUSION: Post-transplant, cytotoxic NK cells were lower than pre-transplant and remained low, whereas NK cell subsets with potentially immunoregulatory properties increased.

Optimal routes of administration, vehicles and timing of progesterone treatment for inhibition of delivery during pregnancy.

OBJECTIVES: Progestins, notably progesterone (P4) and 17 alpha hydroxyprogesterone caproate, are presently used to treat pregnant women at risk of preterm birth. The aim of this study was to assess the optimal treatment options for progesterone (P4) to delay delivery using a sensitive bioassay for progesterone. STUDY DESIGN: Pregnant rats, known to be highly sensitive to progestins, were treated with P4, including Prochieve® (also known as Crinone®), in various vehicles from day 13 of gestation and in late gestation, days 19 to 22, and delivery times noted. Various routes of administration of P4 and various treatment periods were studied. RESULTS: Use of micronized P4 by rectal, subcutaneous injection (sc) and topical (transdermal) administration in various oils all significantly (P<0.05-<0.001) delay delivery, but vaginal Prochieve® did not. Administration of P4 in late gestation also prevented (P<0.001) delivery even when given 8h before delivery. CONCLUSIONS: Prochieve® possesses little biological activity to suppress delivery in a sensitive bioassay system and suggests that this preparation may be of little value in prevention and inhibition of preterm birth. Further, this study shows: 1) Inhibition of delivery is increased with P4 treatments when given subcutaneously or topically. 2) P4 in fish oil provides the best vehicle for topical treatment and may be an effective treatment of preterm birth. 3) P4 in fish oil also delays delivery even when treatment begins just prior to normal delivery. 4) To prevent preterm birth in pregnant women, randomized controlled studies are needed with a potent progestin using better formulations and routes of administration.

Uterine natural killer cells in patients with idiopathic recurrent miscarriage.

PROBLEM: Uterine natural killer (uNK) cells are major players during implantation and early pregnancy. The aim of our study was to analyze uNK cell concentration in the endometrium of idiopathic recurrent miscarriage (iRM) patients and fertile controls. METHOD OF STUDY: Out of n=130 couples with ≥3 consecutive, clinical RM screened according to a standardized diagnostic protocol, n=58 patients with iRM were identified. Endometrial biopsies were investigated in patients and n=17 fertile women (controls) via immunohistochemistry. RESULTS: Compared to controls, the concentration of uNK cells was significantly higher in iRM patients (257±212 vs. 148±73 uNK cells/mm², P=.04). IRM patients showed a higher prevalence of >300 uNK cells/mm² than controls (34.5% vs. 5.9%, P=.02). In 88% of controls and 62% of iRM patients, uNK cells were detected within the range of 40-300/mm². CONCLUSION: Idiopathic recurrent miscarriage patients showed higher uNK cell levels than controls supporting the possible impact of uNK cells in the pathophysiology of miscarriage. Our cutoff levels might help to select RM patients which may benefit from immunomodulatory treatment.

Assessment of parturition with cervical light-induced fluorescence and uterine electromyography.

Parturition involves increasing compliance (ripening) of the uterine cervix and activation of the myometrium. These processes take place in a different time frame. Softening and shortening of the cervix starts in midpregnancy, while myometrial activation occurs relatively close to delivery. Methods currently available to clinicians to assess cervical and myometrial changes are subjective and inaccurate, which often causes misjudgments with potentially adverse consequences. The inability to reliably diagnose true preterm labor leads to unnecessary treatments, missed opportunities to improve neonatal outcome, and inherently biased research of treatments. At term, the likelihood of cesarean delivery depends on labor management, which in turn depends on accurate assessments of cervical change and myometrial contractility. Studies from our group and others show that noninvasive measurements of light-induced fluorescence (LIF) of cervical collagen and uterine electromyography (EMG) objectively detect changes in the composition of the cervix and myometrial preparedness to labor and are more reliable than clinical observations alone. We present a conceptual model of parturition constructed on cervical LIF and uterine EMG studies. We also explore how these methodologies could be helpful with managing patients experiencing preterm contractions and with optimizing labor management protocols aimed to reduce cesarean section.

A novel optical method to assess cervical changes during pregnancy and use to evaluate the effects of progestins on term and preterm labor.

OBJECTIVE: The purpose of this study was to determine whether optical methods can estimate cervix function during pregnancy and whether progestins modify this process. STUDY DESIGN: Photos of the external cervix of timed-pregnant rats were taken every other day from day 13 until postpartum day 5 after daily treatments with vehicle (controls) or progestin treatments (progesterone, subcutaneously or vaginally; 17-alpha-hydroxyprogesterone caproate [17P] and RU-486 subcutaneously, once on day 16). The surface area of the cervix was estimated from photos. RESULTS: The surface area of cervix increases throughout pregnancy and reverses after delivery in controls. In the progesterone subcutaneously or 17P subcutaneously groups, increases in surface area are lower (17P group until day 19 only; P < .05). Vaginal progesterone does not prevent surface area increases. Only the progesterone subcutaneously blocked delivery. RU-486 increases the surface area of the cervix (P < .05) during preterm delivery. CONCLUSION: An optical method is useful for quantitative assessment of the cervix and evaluation of agents that modify cervical function.

Pharmacologic actions of progestins to inhibit cervical ripening and prevent delivery depend on their properties, the route of administration, and the vehicle.

OBJECTIVE: The purpose of this study was to evaluate cervical changes and delivery at term during pregnancy in rats after various progestin treatments. STUDY DESIGN: Pregnant rats were treated by various routes and vehicles with progesterone, 17-alpha-hydroxyprogesterone caproate (17P), R5020, and RU-486. Delivery time was determined and cervical ripening was assessed in vivo by collagen light-induced fluorescence. RESULTS: The cervix is rigid in the progesterone injection, 17P, and vaginal R5020 groups vs controls. Vaginal progesterone had no effect. RU-486 treatment softened the cervix during preterm delivery. Only subcutaneous injected progesterone, R5020 (subcutaneous and vaginal), and topical progesterone in sesame and fish oil inhibits delivery. Delivery is not changed by subcutaneous injection of 17P, vaginal progesterone, oral progesterone, and topical progesterone in Replens (Crinone; Columbia Labs, Livingston, NJ). CONCLUSION: Inhibition of cervical ripening and delivery by progestins depends on many factors that include their properties, the route of administration, and the vehicle. This study suggests reasons that the present treatments for preterm labor are not efficacious.