HLA-DRB1 polymorphism in recurrent pregnancy loss: New evidence for an association to HLA-DRB1*07.

Many cases of recurrent pregnancy loss (RPL) defined as ≥3 consecutive pregnancy losses are suggested to be caused by an aberrant maternal immune response against the fetus or trophoblast. Human leukocyte antigen (HLA)-DRB1 and -DQB1 polymorphisms are associated with most autoimmune disorders and studies of HLA-DBB1 polymorphism in RPL patients are thus relevant. In previous studies, the HLA-DRB1*03 allele was found with increased prevalence in RPL patients. We wanted to clarify whether HLA-DRB1 alleles indeed were associated with RPL among women of Caucasian descent. A total of 1078 women with unexplained RPL and 2066 bone marrow donors were HLA-DRB1-typed and subsets were also HLA-DQB1 typed. All patients were initially HLA-DRB1-typed by DNA-based low-resolution techniques and subsets of patients and all controls were typed by high-resolution techniques. Among patients, the HLA-DRB1*07 allele frequency was significantly increased compared with controls; OR 1.29 (95 % CI 1.09-1.52), p < 0.0025; after correction for multiple comparisons pc = 0.031. The HLA-DRB1*07/*07 genotype was highly increased in patients with RPL compared with controls: OR 2.27 (1.31-3.93), p = 0.0027. The frequency of the HLA-DRB1*07 phenotype in RPL patients had increased significantly (p = 0.002) in three studies from our group published 1994-2021. The allele frequency of HLA-DRB1*03 was not increased in RPL patients compared with controls; OR 0.96 (0.83-1.12). In conclusion, the previous association between HLA-DRB1*03 and RPL could not be confirmed in our study whereas an association to HLA-DRB1*07 was detected for the first time. Since the latter association is a new finding, it should be confirmed in future studies.

Genome-Wide Study Links PNPLA3 Variant With Elevated Hepatic Transaminase After Acute Lymphoblastic Leukemia Therapy.

Remission induction therapy for acute lymphoblastic leukemia (ALL) includes medications that may cause hepatotoxicity, including asparaginase. We used a genome-wide association study to identify loci associated with elevated alanine transaminase (ALT) levels after induction therapy in children with ALL enrolled on St. Jude Children's Research Hospital (SJCRH) protocols. Germline DNA was genotyped using arrays and exome sequencing. Adjusting for age, body mass index, ancestry, asparaginase preparation, and dosage, the PNPLA3 rs738409 (C>G) I148M variant, previously associated with fatty liver disease risk, had the strongest genetic association with ALT (P = 2.5 × 10-8 ). The PNPLA3 rs738409 variant explained 3.8% of the variability in ALT, and partly explained race-related differences in ALT. The PNPLA3 rs738409 association was replicated in an independent cohort of 2,285 patients treated on Children's Oncology Group protocol AALL0232 (P = 0.024). This is an example of a pharmacogenetic variant overlapping with a disease risk variant.

Recurrent pregnancy loss: what is the impact of consecutive versus non-consecutive losses?

STUDY QUESTION: Is there a different prognostic impact for consecutive and non-consecutive early pregnancy losses in women with secondary recurrent pregnancy loss (RPL)? SUMMARY ANSWER: Only consecutive early pregnancy losses after the last birth have a statistically significant negative prognostic impact in women with secondary RPL. WHAT IS KNOWN ALREADY: The risk of a new pregnancy loss increases with the number of previous pregnancy losses in patients with RPL. Second trimester losses seem to exhibit a stronger negative impact than early losses. It is unknown whether the sequence of pregnancy losses plays a role for the prognosis in patients with a prior birth. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study of pregnancy outcome in patients with unexplained secondary RPL included in three previously published, Danish double-blinded placebo-controlled trials of intravenous immunoglobulin (IvIg) conducted from 1991 to 2014. No other treatments were given. Patients with documented explained pregnancy losses (ectopic pregnancies and aneuploid miscarriages) were excluded. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the 168 patients included in the trials, 127 had secondary RPL and experienced a subsequent live birth or unexplained pregnancy loss in the first pregnancy after giving informed consent to participate in the trials (the index pregnancy). Data analyzed by multivariate analysis included the independent variables age, the number of early pregnancy losses before and after the last birth, respectively and a second trimester pregnancy loss before or after the last birth, respectively. The outcome variable was unexplained loss in the index pregnancy. MAIN RESULTS AND THE ROLE OF CHANCE: In patients with secondary RPL, both a late and each early loss before the last birth did not significantly influence the risk of a new pregnancy loss in the index pregnancy: incidence rate ratio (IRR) 1.31 (95% CI 0.62-2.77) and IRR 0.88 (95% CI 0.70-1.11), respectively. In contrast, the impact on risk of pregnancy loss conferred by a late and by each early pregnancy loss occurring after the birth was significant: IRR 2.15 (95% CI 1.57-2.94, P < 0.0001) and IRR 1.14 (95% CI 1.04-1.24, P = 0.002), respectively. LIMITATIONS, REASONS FOR CAUTION: Of the patients, 48% were treated with IvIg, which could influence the results. However, allocation to IvIg was random and prognostic variables were equally distributed in IvIg and placebo-treated patients. WIDER IMPLICATIONS OF THE FINDINGS: A birth in women with secondary RPL eradicates the negative prognostic impact of previous pregnancy losses and this finding is important for our understanding of the pathogenesis. It indicates that only consecutive pregnancy losses should count in the definition of RPL. STUDY FUNDING/COMPETING INTERESTS: There was no particular funding for this study. The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable for two of the included randomized controlled trials. For the last trial: Clinical.Gov NCT00722475.

Family intentions and personal considerations on postponing childbearing in childless cohabiting and single women aged 35-43 seeking fertility assessment and counselling.

STUDY QUESTION: What characterizes childless women aged 35 years and above seeking fertility assessment and counselling in relation to their reproduction and are there significant differences between single and cohabiting women? SUMMARY ANSWER: Despite the women's advanced age and knowledge of the age-related decline in fecundity, 70% of the single women sought fertility assessment and counselling to gain knowledge regarding the possibility of postponing pregnancy. WHAT IS KNOWN ALREADY: Recent studies have indicated an increasing demand for ovarian reserve testing in women without any known fertility problem to obtain knowledge on their reproductive lifespan and pro-fertility advice. Women postpone their first pregnancy, and maternal age at first birth has increased in western societies over the past two to four decades. Postponed childbearing implies a higher rate of involuntary childlessness, smaller families than desired and declining fertility rates. STUDY DESIGN, SIZE, AND DURATION: Baseline data from a cross-sectional cohort study of 340 women aged 35-43 years examined at the Fertility Assessment and Counselling (FAC) Clinic at Copenhagen University Hospital from 2011 to 2014. The FAC Clinic was initiated to provide individual fertility assessment and counselling. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eligible women were childless and at least 35 years of age. All completed a web-based questionnaire before and after the consultation including socio-demographic, reproductive, medical, lifestyle and behavioural factors. Consultation by a fertility specialist included transvaginal ultrasound, full reproductive history and AMH measurement. MAIN RESULTS AND THE ROLE OF CHANCE: The study comprised 140 cohabiting and 200 single women. The majority (82%) were well-educated and in employment. Their mean age was 37.4 years. Nonetheless, the main reasons for attending were to obtain knowledge regarding the possibility of postponing pregnancy (63%) and a concern about their fecundity (52%). The majority in both groups (60%) wished for two or more children. The women listed their ideal age at birth of first child and last child as 33 (±4.7) years and 39 (±3.5) years, respectively. Of the single women, 70% would accept use of sperm donation compared with 25% of the cohabiting women (P < 0.001). In general, 45% considered oocyte vitrification for social reasons, yet only 15% were positive towards oocyte donation. The two groups were comparable regarding lifestyle factors, number of previous sexual partners, pregnancies, and ovarian reserve parameters. LIMITATIONS, REASONS FOR CAUTION: The women in the present study were conscious of the risk of infertility with increasing age and attended the FAC Clinic due to a concern about their remaining reproductive lifespan, which in combination with their high educational level could impair the generalizability to the background population. WIDER IMPLICATIONS OF THE FINDINGS: The results indicate that in general women overestimate their own reproductive capacity and underestimate the risk of future childlessness with the continuous postponement of pregnancies.

Ovarian reserve assessment in users of oral contraception seeking fertility advice on their reproductive lifespan.

STUDY QUESTION: To what extent does oral contraception (OC) impair ovarian reserve parameters in women who seek fertility assessment and counselling to get advice on whether their remaining reproductive lifespan is reduced? SUMMARY ANSWER: Ovarian reserve parameters defined by anti-Müllerian hormone (AMH), antral follicle count (AFC) and ovarian volume were found to be significantly decreased by 19% (95% CI 9.1-29.3%), 18% (95% CI 11.2-24.8%) and 50% (95% CI 45.1-53.7%) among OC users compared with non-users. WHAT IS KNOWN ALREADY: AMH and AFC have proved to be reliable predictors of ovarian ageing. In women, AMH declines with age and data suggest a relationship with remaining reproductive lifespan and age at menopause. OC may alter parameters related to ovarian reserve assessment but the extent of the reduction is uncertain. STUDY DESIGN, SIZE, DURATION: A cross-sectional study of 887 women aged 19-46 attending the Fertility Assessment and Counselling Clinic (FACC) from 2011 to 2014 comparing ovarian reserve parameters in OC users with non-OC users. PARTICIPANTS/MATERIALS, SETTING, METHODS: The FAC Clinic was initiated to provide individual fertility assessment and counselling. All women were examined on a random cycle day by a fertility specialist. Consultation included; transvaginal ultrasound (AFC, ovarian volume, pathology), a full reproductive history and AMH measurement. Women were grouped into non-users and users of OC (all combinations of estrogen-progestin products and the contraceptive vaginal ring). Non-users included women with an intrauterine device (IUD) or no hormonal contraception. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 887 women, 244 (27.5%) used OC. In a linear regression analyses adjusted for age, ovarian volume was 50% lower (95% CI 45.1-53.7%), AMH was 19% lower (95% CI 9.1-29.3%), and AFC was 18% lower (95% CI 11.2-24.8%) in OC users compared with non-users. Comparison of AMH at values of <10 pmol/l OC was found to have a significant negative influence on AMH (OR 1.6, 95% CI 1.1; 2.4, P = 0.03). Furthermore, we found a significant decrease in antral follicles sized 5-7 mm (P < 0.001) and antral follicles sized 8-10 mm (P < 0.001) but an increase in antral follicles sized 2-4 mm (P = 0.008) among OC users. The two groups (OC users versus non-users) were comparable regarding age, BMI, smoking and maternal age at menopause. LIMITATIONS, REASON FOR CAUTION: The study population comprised women attending the FAC Clinic. Recruitment was based on self-referral, which could imply a potential selection bias. Ovarian reserve was examined at a random cycle day. However, both AMH and AFC can be assessed independently of the menstrual cycle. The accuracy in predicting residual reproductive lifespan is still needed in both users and non-users of OC. WIDER IMPLICATIONS OF THE FINDINGS: OC has a major impact on the ovarian volume, and a moderate impact on AFC and AMH with a shift towards the smaller sized antral follicle subclasses. The most evident reduction occurs in the antral follicles of 5-7 and 8-10 mm with the highest number of AMH secreting granulosa cells. It is essential to be aware of the impact of OC use on ovarian reserve parameters when guiding OC users on their fertility status and reproductive lifespan. STUDY FUNDING/COMPETING INTERESTS: The FAC Clinic was established in 2011 as part of the ReproHigh collaboration. This study received funding through the Capital Region Research Fund and by EU-regional funding. There are no competing interests. TRIAL REGISTRATION NUMBER: The biobank connected to FAC Clinic is approved by the Scientific Ethical Committee (H-1-2011-081).

Intravenous immunoglobulin treatment for secondary recurrent miscarriage: a randomised, double-blind, placebo-controlled trial.

OBJECTIVE: To determine whether infusions with intravenous immunoglobulin (IVIg) during early pregnancy increase live birth rate in women with secondary recurrent miscarriage compared with placebo. DESIGN: A single-centre, randomised, double-blind, placebo-controlled trial. SETTING: A tertiary centre for recurrent miscarriage in Copenhagen, Denmark. POPULATION: A group of 82 women with unexplained secondary recurrent miscarriage and at least four miscarriages. METHODS: Women were randomly assigned to repeated infusions with IVIg or placebo (albumin) from the time of positive pregnancy test to gestational week 15 or pregnancy loss. MAIN OUTCOME MEASURE: Primary outcome was birth with neonatal survival in all randomised women. RESULTS: In the intention-to-treat analyses, live birth rates were 23/42 (54.8%) in the IVIg and 20/40 (50.0%) in the placebo group, relative risk 1.11 (95% CI 0.70-1.74). In a per protocol analysis, almost identical results were found. The median gestational length at delivery was higher in the IVIg than the placebo group (282 versus 272 days, P = 0.02) but the mean birthweight was not significantly increased. CONCLUSIONS: In this trial, which is the largest so far, IVIg did not increase the live birth rate in patients with secondary recurrent miscarriage and the treatment cannot be recommended in clinical practice.

A 10-year follow up of reproductive function in women treated for childhood cancer.

Previously, this study group found that female childhood cancer survivors could be at risk of early cessation of fertility. The aim of the present study was to evaluate reproductive function in the same group of survivors 10 years after the initial study. Of the original cohort of 100, 71 were re-examined. Thirty-six survivors reported regular menstrual cycles. When they were compared with 210 controls, they differed significantly in antral follicle count (AFC) (median 15 versus 18, P=0.047) but not in anti-Müllerian hormone (AMH) (median 13.0 versus 17.8 pmol/l). Survivors cured with minimal gonadotoxic treatment had significantly higher AMH and AFC compared with survivors cured with either potentially gonadotoxic treatment or treatment including alkylating chemotherapy and ovarian irradiation (20.0, 5.8 and <3 pmol/l, P<0.001; and 15, 9 and 2, P=0.03, respectively). Thirty-eight survivors had achieved at least one live birth. Complicated second-trimester abortions (n=4) were observed primarily in survivors cured with radiotherapy affecting pelvic organs. In conclusion, childhood cancer survivors have signs of diminished ovarian reserve. However, if the ovarian function is preserved in the early to mid-twenties, it is likely to persist until the mid-thirties, giving a good chance of childbearing.

Ovarian antral follicle subclasses and anti-mullerian hormone during normal reproductive aging.

CONTEXT: The interindividual variation in the age-related decline of ovarian follicles is wide. Hence, it is important to identify reliable, sensitive, and specific markers to assess the ovarian reserve of the individual woman. OBJECTIVE: The aim of this study was to characterize the relation between age and ovarian reserve parameters in a population of healthy women with regular menstrual cycle. DESIGN AND SETTING: We conducted a prospective, population-based, cross-sectional study. PARTICIPANTS: A total of 366 health care workers aged 21-41 years employed at a University Hospital were included. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURES: Serum anti-Müllerian hormone (AMH) concentration, antral follicle count (AFC), antral follicle size categories (small: 2-4 mm; intermediate: 5-7 mm; and large: 8-10 mm), and ovarian volume were measured. RESULTS: Serum AMH level declined by 5.6% per year (95% confidence interval 3.7-7.4%, P < .001), AFC (2-10 mm) declined by 4.4% per year (3.2-5.7%, P < .001), and ovarian volume declined by 1.1% per year (0.2-2.0, P = .002), respectively. The mean proportion of small follicles decreased with age (P = .04), the proportion of intermediate follicles displayed no significant change with age (P = .58), and the mean proportion of large follicles increased with age (P < .001). The prevalence of large follicles increased with decreasing serum AMH concentration [odds ratio 1.04 per 1 pmol/L (1.02-1.06), P < .001, area under the curve 0.66], and with decreasing total AFC [odds ratio 1.04 per follicle (1.02-1.05), P < .001, area under the curve 0.62]. CONCLUSION: Chronological age was inversely related to serum AMH concentration, total AFC, and ovarian volume. Subclasses of AFC sized 2-4 and 5-7 mm decreased with increasing age, whereas AFC sized 8-10 mm increased with increasing age. Within AFC, a shift toward larger follicles with increasing age was observed. The occurrence of large follicles was more strongly related to biological age in terms of AMH and AFC than chronological age.

Maternal menopause as a predictor of anti-Mullerian hormone level and antral follicle count in daughters during reproductive age.

STUDY QUESTION: Is the ovarian reserve in a woman at a given age associated with her mother's age at menopause? SUMMARY ANSWER: We demonstrated a significant, positive association between age at maternal menopause and serum anti-Müllerian hormone (AMH) levels and antral follicle count (AFC) in daughters. The rate of decline in serum-AMH level and AFC is also associated with age at maternal menopause. WHAT IS KNOWN AND WHAT THIS PAPER ADDS: The association between menopausal age in mothers and daughters has been established through several epidemiological studies. This paper shows that early maternal menopause is related to an advanced depletion of the ovarian reserve and that late maternal menopause is related to a delayed depletion. STUDY DESIGN AND SIZE: Cross-sectional data were obtained from a prospective cohort study of 863 women. The study comprised 527 participants from this prospective cohort whose mothers' age at natural menopause was known. PARTICIPANTS, SETTING AND METHODS: Participants were recruited from female health care workers aged 20-40 years employed at Copenhagen University Hospital, Rigshospitalet, and were enrolled in the study between September 2008 and February 2010. The response rate was 52.1%. Endocrine and ovarian parameters related to reproductive ageing (AMH and AFC) were assessed by serum AMH analyses and transvaginal ovarian sonography on cycle Day 2-5. Data on reproductive history, including age at natural maternal menopause, were obtained through an internet-based questionnaire. We used an analysis of covariance model with serum-AMH and AFC as outcomes, age as the quantitative predictor and onset of maternal menopause as the categorical predictor, with further adjustments for BMI, use of oral contraceptives, participants' smoking habits and prenatal smoking exposure. MAIN FINDINGS: We found a significant effect of age at maternal menopause on both serum AMH levels (P < 0.001) and AFC (P = 0.005). Median serum-AMH concentration declined by 8.6% per year [95% confidence interval (CI): 6.4-10.8%, P < 0.001] in the group with early maternal menopausal age (≤ 45 years), by 6.8% per year (95% CI: 5.0-8.6%, P < 0.001) in the group with normal maternal menopausal age (46-54 years) and by 4.2% per year (95% CI: 2.0-6.4%, P < 0.001) in the group with late maternal menopausal age (≥ 55 years). Median AFC declined by 5.8% per year (95% CI: 4.0-7.5%, P < 0.001) in the group with early maternal menopausal age (≤ 45 years), by 4.7% per year (95% CI: 3.3-6.1%, P < 0.001) in the group with normal maternal menopausal age (46-54 years) and by 3.2% per year (95% CI: 1.4-4.9%, P < 0.001) in the group with late maternal age (≥ 55 years) at menopause. BIAS, LIMITATIONS AND GENERALIZABILITY: Information on 'age at maternal menopause' was obtained retrospectively and may be prone to recall bias and digit preference. The study population consisted of health care workers, which implies a potential selection bias. Finally, the cross-sectional nature of the data limits the generalizability. STUDY FUNDING/POTENTIAL COMPETING INTERESTS: This study was co-financed by PhD scholarships where funding was covered by the Danish Agency for Science, Technology and Innovation, Copenhagen Graduate School of Health Science (CGSHS) and the Fertility Clinic at Copenhagen University Hospital, Rigshopitalet. No competing interests are declared.

Ovarian reserve parameters: a comparison between users and non-users of hormonal contraception.

It remains controversial whether anti-Müllerian hormone (AMH) concentration is influenced by hormonal contraception. This study quantified the effect of hormonal contraception on both endocrine and sonographic ovarian reserve markers in 228 users and 504 non-users of hormonal contraception. On day 2-5 of the menstrual cycle or during withdrawal bleeding, blood sampling and transvaginal sonography was performed. After adjusting for age, ovarian reserve parameters were lower among users than among non-users of hormonal contraception: serum AMH concentration by 29.8% (95% CI 19.9 to 38.5%), antral follicle count (AFC) by 30.4% (95% CI 23.6 to 36.7%) and ovarian volume by 42.2% (95% CI 37.8 to 46.3%). AFC in all follicle size categories (small, 2-4 mm; intermediate, 5-7 mm; large, 8-10 mm) was lower in users than in non-users of hormonal contraception. A negatively linear association was observed between duration of hormonal-contraception use and ovarian reserve parameters. No dose-response relation was found between the dose of ethinyloestradiol and AMH or AFC. This study indicates that ovarian reserve markers are lower in women using sex steroids for contraception. Thus, AMH concentration and AFC may not retain their accuracy as predictors of ovarian reserve in women using hormonal contraception. Serum anti-Müllerian hormone (AMH) concentration is an indirect marker of the number of small follicles in the ovary and thereby the ovarian reserve. The AMH concentration is now widely used as one of the markers of the ovarian reserve in ovarian hormonal stimulation regimens. Hence the AMH concentration in a patient is used to decide the dose of the ovarian hormonal stimulation prior to IVF treatment. In some infertile patients, hormonal contraception is used prior to ovarian hormonal stimulation and therefore it is important to clarify whether serum AMH concentration is influenced by the use of sex steroids. The aim of this study was to quantify the potential effect of hormonal contraception on the ovarian function by hormonal analyses and ovarian ultrasound examination. Examinations were performed in the early phase of the menstrual cycle or the hormone-free interval of hormonal contraception. We compared the AMH concentration, the antral follicle count (AFC) and the ovarian volume in 228 users versus 504 non-users of hormonal contraception. Users of hormonal contraception had 29.8% lower AMH concentration, 30.4% lower AFC and 42.2% lower ovarian volume than non-users. These findings were more pronounced with increasing duration of hormonal contraception. No dose-response relation was found between the dose of ethinylestradiol and the impact on serum AMH and AFC. The study indicates that ovarian reserve markers are lower in women using sex steroids for contraception. Thus, serum AMH concentration and AFC may not retain their accuracy as predictors of the ovarian reserve in women using hormonal contraception.

Risk of ovarian failure and fertility preserving methods in girls and adolescents with a malignant disease.

Girls and young women suffering from a malignant disease that requires treatment with chemo- and/or radiotherapy are at risk of losing fertility. The most significant risk factors are age and type of treatment given. Preserving fertility is of high priority to both the young patient and her parents. This article reviews the effect of chemo- and radiotherapy on gonadal function, and thus fertility, and offers different fertility preserving methods based on the literature. Cryopreservation of ovarian tissue is a possible way of preserving fertility in this group of patients in the future.

Synaptotagmin C2A loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis.

Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca(2+)-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-triggered vesicle exocytosis and that Ca(2+)-dependent membrane binding by itself is insufficient to trigger fusion. A structure-based model of the SNARE-binding surface of C2A provided a new view of how Ca(2+)-dependent SNARE and membrane binding occur simultaneously.

Effect of tricyclodecan-9-yl potassium xanthate (D609) on phospholipid metabolism and cell death during oxygen-glucose deprivation in PC12 cells.

Alterations in lipid metabolism play an integral role in neuronal death in cerebral ischemia. Here we used an in vitro model, oxygen-glucose deprivation (OGD) of rat pheochromocytoma (PC12) cells, and analyzed changes in phosphatidylcholine (PC) and sphingomyelin (SM) metabolism. OGD (4-8 h) of PC12 cells triggered a dramatic reduction in PC and SM levels, and a significant increase in ceramide. OGD also caused increases in phosphatidylcholine-phospholipase C (PC-PLC) and phospholipase D (PLD) activities and PLD2 protein expression, and reduction in cytidine triphosphate:phosphocholine cytidylyltransferase-alpha (CCTalpha, the rate-limiting enzyme in PC synthesis) protein expression and activity. Phospholipase A2 activity and expression were unaltered during OGD. Increased neutral sphingomyelinase activity during OGD could account for SM loss and increased ceramide. Surprisingly, treatment with PC-PLC inhibitor tricyclodecan-9-yl potassium xanthate (D609) aggravated cell death in PC12 cells during OGD. D609 was cytotoxic only during OGD; cell death could be prevented by inclusion of sera, glucose or oxygen. During OGD, D609 caused further loss of PC and SM, depletion of 1,2-diacylglycerol (DAG), increase in ceramide and free fatty acids (FFA), cytochrome c release from mitochondria, increases in intracellular Ca2+ ([Ca2+]i), poly-ADP ribose polymerase (PARP) cleavage and phosphatidylserine externalization, indicative of apoptotic cell death. Exogenous PC during OGD in PC12 cells with D609 attenuated PC, SM loss, restored DAG, attenuated ceramide levels, decreased cytochrome c release, PARP cleavage, annexin V binding, attenuated the increase in [Ca2+]i, FFA release, and significantly increased cell viability. Exogenous PC may have elicited these effects by restoring membrane PC levels. A tentative scheme depicting the mechanism of action of D609 (inhibiting PC-PLC, SM synthase, PC synthesis at the CDP-choline-1,2-diacylglycerol phosphocholine transferase (CPT) step and causing mitochondrial dysfunction) has been proposed based on our observations and literature.

Diminished ovarian reserve in female childhood cancer survivors with regular menstrual cycles and basal FSH <10 IU/l.

BACKGROUND: Approximately 70% of children treated for cancer are cured, however the treatment often impairs reproductive function. The aim of the present study was to assess ovarian reserve in women with an apparently normal ovarian function, who were cured of cancer in childhood. METHODS: Twenty-one female survivors with regular menstrual cycles and basal FSH <10 IU/l were included. The control group included 21 healthy age-matched regularly cycling women. On cycle day 2-5, ovarian volume and number of small antral follicles (2-5 mm) were evaluated with transvaginal ultrasonography. Repeated sonography, urinary LH testing, and endocrine assessments were performed during the actual cycle. Cycle length was calculated. RESULTS: The female survivors had significantly smaller volume per ovary (4.9 versus 6.8 cm(3); P < 0.01), a lower number of small antral follicles per ovary (4.5 versus 8.0; P < 0.01), and lower total number of follicles per ovary (8 versus 11, P < 0.01). Hormonal profiles were similar, but the mean cycle length of the female survivors was significantly shorter than in the control group (28.3 versus 31.0 days; P < 0.05). CONCLUSIONS: Childhood cancer survivors with regular menstrual cycles and basal FSH <10 IU/l seem to have a diminished ovarian reserve. Consequently, they may have a shortened reproductive span and an early menopause.

G protein betagamma subunit-mediated presynaptic inhibition: regulation of exocytotic fusion downstream of Ca2+ entry.

The nervous system can modulate neurotransmitter release by neurotransmitter activation of heterotrimeric GTP-binding protein (G protein)-coupled receptors. We found that microinjection of G protein betagamma subunits (Gbetagamma) mimics serotonin's inhibitory effect on neurotransmission. Release of free Gbetagamma was critical for this effect because a Gbetagamma scavenger blocked serotonin's effect. Gbetagamma had no effect on fast, action potential-evoked intracellular Ca2+ release that triggered neurotransmission. Inhibition of neurotransmitter release by serotonin was still seen after blockade of all classical Gbetagamma effector pathways. Thus, Gbetagamma blocked neurotransmitter release downstream of Ca2+ entry and may directly target the exocytotic fusion machinery at the presynaptic terminal.

[Pregnancy associated pelvic pain. I: Prevalence and risk factors]. / Graviditetsrelaterede baekkensmerter. I: Praevalens og risikofaktorer.

With this study, we wanted to determine the incidence of symptom-giving pelvic girdle relaxation during pregnancy and the prevalence post partum, identify predisposing factors, and determine the frequency of sick leave. A total of 1600 pregnant women entered the study. The incidence during pregnancy was 14%, the prevalence two, six, and twelve months post partum was 5%, 4%, and 2%, respectively. Multivariate analysis indicated that the most important predisposing factor was pelvic pain in a previous pregnancy. Other factors were uncomfortable working conditions, lack of exercise, and previous low back and low abdominal pain. At least 37% of the women with symptom-giving pelvic girdle relaxation were on sick leave during pregnancy, on average for twelve weeks. Symptom-giving pelvic girdle relaxation is a considerable problem both in pregnancy and post partum. The occupational risk can possibly be prevented. The syndrome has a great social impact because of the high frequency of sick live.

[Pregnancy associated pelvic pain. II: Symptoms and clinical findings]. / Graviditetsrelaterede baekkensmerter. II: Symptomer og kliniske fund.

Pelvic pain in pregnancy appears to be a problem on the increase. This study was undertaken to describe and analyse the relationship between subjective symptoms, daily disability, and clinical findings in women with symptom-giving pelvic girdle relaxation in pregnancy. Out of 1600 pregnant women, 238 had pelvic pain. After a clinical examination 11 women were excluded due to low back pain. The rest, 227 women, was considered to have symptom-giving pelvic girdle relaxation during pregnancy. Symptom-giving pelvic girdle relaxation in pregnancy seriously interferes with many activities of daily living such as housekeeping, walking, working, and sexual life. The women's statements of pelvic pain are well correlated to the number of positive clinical tests. Symptom-giving pelvic girdle relaxation in pregnancy causes considerable disabilities concerning daily activities.

Differential requirement for classic and novel PKC isoforms in respiratory burst and phagocytosis in RAW 264.7 cells.

The binding of Ab (IgG)-opsonized particles by FcgammaRs on macrophages results in phagocytosis of the particles and generation of a respiratory burst. Both IgG-stimulated phagocytosis and respiratory burst involve activation of protein kinase C (PKC). However, the specific PKC isoforms required for these responses have yet to be identified. We have studied the involvement of PKC isoforms in IgG-mediated phagocytosis and respiratory burst in the mouse macrophage-like cell line, RAW 264.7. Like primary monocyte/macrophages, their IgG-mediated phagocytosis was calcium independent and diacylglycerol sensitive, consistent with novel PKC activation. Respiratory burst in these cells was Ca2+ dependent and inhibited by staurosporine and calphostin C as well as by the classic PKC-selective inhibitors Gö 6976 and CGP 41251, suggesting that classic PKC is required. In contrast, phagocytosis was blocked by general PKC inhibitors but not by the classic PKC-specific drugs. RAW 264.7 cells expressed PKCs alpha, betaI, delta, epsilon, and zeta. Subcellular fractionation demonstrated that PKCs alpha, delta, and epsilon translocate to membranes during phagocytosis. In Ca2+-depleted cells, only novel PKCs delta and epsilon increased in membranes, and the time course of their translocation was consistent with phagosome formation. Confocal microscopy of cells transfected with green fluorescent protein-conjugated PKC alpha or epsilon confirmed that these isoforms translocated to the forming phagosome in Ca-replete cells, but only PKC epsilon colocalized with phagosomes in Ca2+-depleted cells. Taken together, these results suggest that the classic PKC alpha mediates IgG-stimulated respiratory burst in macrophages, whereas the novel PKCs delta and/or epsilon are necessary for phagocytosis.

Oocyte donation in women cured of cancer with bone marrow transplantation including total body irradiation in adolescence.

Female survivors of cancer in childhood and adolescence who have been treated with bone marrow transplantation including total body irradiation (TBI) are at high risk of developing ovarian follicular depletion and infertility. The lack of oocytes may be compensated for by oocyte donation but these patients also seem to have a uterine factor. Even though oestrogen replacement therapy is given, the growth of the uterus during adolescence is impaired. To our knowledge there have been no earlier reports of live births after oocyte donation in such patients. We report three cases of oocyte donation in women who, at a young age, were cured of haematological malignancies with bone marrow transplantation including TBI. In adolescence they developed ovarian failure and uterine volumes were assessed by ultrasonography. One woman with a uterus of almost normal size delivered a healthy child in the 37th week of gestation. Another woman with severely diminished uterine volume miscarried in the 17th week of gestation. The third woman has not yet conceived. Pregnancy achieved by oocyte donation is possible despite TBI in adolescence. However, the uterine factor is a concern and complications during pregnancy and preterm birth may be expected in these patients.

The C terminus of SNAP25 is essential for Ca(2+)-dependent binding of synaptotagmin to SNARE complexes.

The plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) and the vesicle SNARE protein vesicle-associated membrane protein (VAMP) are essential for a late Ca(2+)-dependent step in regulated exocytosis, but their precise roles and regulation by Ca(2+) are poorly understood. Botulinum neurotoxin (BoNT) E, a protease that cleaves SNAP25 at Arg(180)-Ile(181), completely inhibits this late step in PC12 cell membranes, whereas BoNT A, which cleaves SNAP25 at Gln(197)-Arg(198), is only partially inhibitory. The difference in toxin effectiveness was found to result from a reversal of BoNT A but not BoNT E inhibition by elevated Ca(2+) concentrations. BoNT A treatment essentially increased the Ca(2+) concentration required to activate exocytosis, which suggested a role for the C terminus of SNAP25 in the Ca(2+) regulation of exocytosis. Synaptotagmin, a proposed Ca(2+) sensor for exocytosis, was found to bind SNAP25 in a Ca(2+)-stimulated manner. Ca(2+)-dependent binding was abolished by BoNT E treatment, whereas BoNT A treatment increased the Ca(2+) concentration required for binding. The C terminus of SNAP25 was also essential for Ca(2+)-dependent synaptotagmin binding to SNAP25. syntaxin and SNAP25.syntaxin.VAMP SNARE complexes. These results clarify classical observations on the Ca(2+) reversal of BoNT A inhibition of neurosecretion, and they suggest that an essential role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca(2+)-dependent interactions between synaptotagmin and SNARE protein complexes.