A comparison of metabolic health parameters in ICSI-conceived and naturally conceived adolescents.

RESEARCH QUESTION: Are there differences in the cardiometabolic health of ICSI-conceived adolescents compared with a control group, taking parental risk factors into account? DESIGN: ICSI-conceived adolescents (n = 272), their mothers (n = 273) and naturally conceived control adolescents (n = 273) and their mothers (n = 273) provided a blood test and answered a health-related questionnaire. The adolescents also attended a physical examination. RESULTS: ICSI-conceived males showed significantly higher mean weight (72.6 ± 15.1 versus 67.7 ± 12.6 kg, P = 0.005), body mass index (BMI) (22.2 ± 3.7 versus 21.0 ± 3.2 kg/m2, P = 0.007) and waist circumference (79.1 ± 11.6 versus 74.5 ± 8.7 cm, P < 0.001). The mean values for weight and BMI were also significantly higher in the ICSI parents. In the ICSI-conceived females significant differences in high-density lipoprotein cholesterol (1.5 ± 0.3 versus 1.6 ± 0.3 mmol/l, P = 0.033) and triglyceride values (1.1 ± 0.5 versus 1.0 ± 0.4 mmol/l, P = 0.013) were observed. ICSI mothers also had significantly higher triglycerides (P = 0.002), higher glutamate pyruvate transaminase/alanine aminotransferase (P < 0.001) and higher alkaline phosphatase values (P < 0.001). CONCLUSIONS: Increased values for weight were found in the male and differences in lipid parameters in the female ICSI-conceived adolescents, which were reflected in the values of their parents. Adjustment for parental risk factors generally attenuated the differences between the ICSI and the control groups, but did not completely remove them. Whether these observed differences are clinically relevant for the future health of the participants requires further study. To increase knowledge in this area, future studies should also include parental data.

Serum estradiol and progesterone in the mid-luteal phase predict clinical pregnancy outcome in IVF/ICSI cycles.

In this prospective study, we tested the hypothesis if E2 and P serum levels significantly differ during the luteal phase following in vitro-fertilization/intracytoplasmic sperm injection (IVF/ICSI) therapy in conception (CC) versus non-conception (NC) cycles, and their potential in the prediction of pregnancy at the earliest point in time. Serum was sampled from the day of embryo transfer (ET) and throughout the luteal phase until ET + 14 from patients consecutively enrolling for IVF/ICSI therapy. The luteal phase was supported by vaginal P suppositories only, clinical pregnancies were detected by ultrasound and followed up until the 20th week. Overall pregnancy rate was 30.9% constituting the two study groups of CC (n = 22) and NC cycles (n = 49). Significantly, higher E2 (3326 ± 804 versus 1072 ± 233 pmol/l, p = 0.014) and P (244 ± 68 versus 73 ± 10 nmol/l, p = 0.023) were present in CC versus NC from as early as ET + 7. In the CC group, patients with ongoing pregnancies (CC-OG) as compared with miscarriages (CC-MC) had significantly higher E2 and P from ET + 7, predicting ongoing pregnancy in receiver operator characteristics analysis.

FSH prevents depletion of the resting follicle pool by promoting follicular number and morphology in fresh and cryopreserved primate ovarian tissues following xenografting.

BACKGROUND: Cryopreservation and transplantation of ovarian tissue is one option for re-establishing ovarian function, but optimal conditions for graft sustainment and follicular survival are still considered experimental. The present study aims to analyze the effect of FSH treatment on the resting follicle pool in fresh and cryopreserved primate ovarian tissues following xenografting. METHODS: Ovarian tissues from adult marmosets were grafted freshly or following cryopreservation to ovarectomized nude mice treated with FSH 25 IU twice daily post transplantation or left untreated as controls. Grafts were retrieved 2 or 4 weeks after transplantation to evaluate the number and morphological appearance of follicles. RESULTS: Early start of FSH treatment within 1 week following transplantation partly prevents primordial follicle loss in fresh and frozen-thawed tissues, whereas after a 3 weeks time interval this effect is present only in fresh tissues. A similar positive effect of early, but not later FSH treatment on primary follicles is seen in fresh tissues compared to only marginal effects in frozen-thawed tissues. The percentage of morphologically normal follicles is generally increased in FSH treated tissues, whereas the percentage of primary follicles over all primordial and primary follicles is increased by FSH only in freshly-grafted tissues. CONCLUSIONS: FSH treatment alleviates depletion of the resting follicle pool and promotes normal follicular morphology both in freshly and frozen-thawed grafted tissues. In previously cryopreserved tissues, applying to most of the tissues intended for clinical use in fertility preservation attempts, its positive effect on primordial follicle numbers and potential graft sustainment is dependent on an early start of treatment within one week of transplantation.

An integrated view on the luteal phase: diagnosis and treatment in subfertility.

The term 'luteal phase deficiency' was first coined more than 60 years ago, and, since then, it has been suggested as a clinical entity per se and an aetiological factor for subfertility, implantation failure and recurrent miscarriage. Despite the existing recommendations for rational work-up in subfertility, luteal phase evaluation and progesterone therapy alone is still common in daily practice. This review comprises results from a Pubmed literature search with the terms 'luteal phase' and 'subfertility', focussing on clinical situations not primarily related to assisted reproduction techniques. Additional data from the experimental studies published in the past 10 years on follicular maturation, oocyte developmental competence and the ovulatory cascade are integrated into the clinical continuum of dysfunctional ovulation, menstrual cycle irregularity and impaired corpus luteum function. As reliable diagnostic tools for adequate luteal function are missing, the presence of clinical symptoms such as cycle irregularity or premenstrual spotting is indicative and should initiate early follicular phase diagnostic work-up. New evidence on the interdependence of oocyte and follicular maturation and resulting developmental competence of the embryo further support the use of ovarian stimulation as the first-line therapeutic option in different subsets of patients with subfertility including luteal phase deficiency.

Effects of the FSH receptor gene polymorphism p.N680S on cAMP and steroid production in cultured primary human granulosa cells.

The study was designed to evaluate in vitro the cellular mechanisms of the single nucleotide polymorphism (SNP) p.N680S of the FSH receptor gene (FSHR) in human granulosa cells (GC) and included patients homozygous for the FSHR SNP (NN/SS) undergoing ovarian stimulation. GC were isolated during oocyte retrieval and cultured for 1­7 days. Basal oestradiol and progesterone concentrations were measured after short-term culture. The kinetics of cAMP, oestradiol and progesterone concentrations in response to various amounts of FSH were analysed in a 6­7 day culture. Basal oestradiol, but not progesterone, concentrations on day 1 of GC culture, were significantly higher in NN compared with SS (P = 0.045), but non-responsive to FSH stimulation. Immunofluorescence microscopy demonstrated the re-appearance of FSHR expression with increasing days in culture. Upon stimulation with FSH, GC cultured for 6­7 days displayed a dose-dependent increase of cAMP, oestradiol and progesterone but no difference in the EC50 values between both variants. Primary long-term GC cultures are a suitable system to study the effects of FSH in vitro. However, the experiments suggest that factors down-stream of progesterone production or external to GC might be involved in the clinically observed differences in an FSHR variant-mediated response to FSH.

Advanced follicle development in xenografted prepubertal ovarian tissue: the common marmoset as a nonhuman primate model for ovarian tissue transplantation.

OBJECTIVE: To establish a nonhuman primate model addressing follicular development in cryopreserved prepubertal ovarian tissue after xenografting. DESIGN: Experimental study. SETTING: Academic research center. ANIMAL(S): Ovarian tissue from female prepubertal common marmoset (Callytrix jacchus jacchus) grafted into immunodeficient nude mice (Crl:NU-FoxnI(nu)). INTERVENTION(S): Removal and subsequent cryopreservation of ovarian tissues with dimethyl sulfoxide, followed by grafting to subcutaneous sites of ovariectomized and intact nude mice. MAIN OUTCOME MEASURE(S): Histologic evaluation for the mean number of total and morphologically normal follicles in each class. RESULT(S): The mean number of unadvanced follicles in frozen-thawed grafted ovarian tissues was reduced compared with pregraft controls, but the prevalence of normal follicular morphology was either slightly increased (primordial follicles) or unchanged (primary follicles). Previous ovariectomy in graft recipients increased total follicle numbers without effect on normal follicular morphology and shifted the ratio of primordial to primary follicles toward an increase in primary follicles, indicating activation of follicular maturation. CONCLUSION(S): The marmoset is a suitable primate model for studies on the subsequent use of cryopreserved ovarian tissue, demonstrating graft sustainment and the development of follicles from prepubertal ovarian tissue in immunodeficient hosts up to secondary and preantral stages.

Androgens and insulin--two key players in polycystic ovary syndrome. Recent concepts in the pathophysiology and genetics of polycystic ovary syndrome.

Androgens and insulin are endocrine key players in the pathophysiology of polycystic ovary syndrome (PCOS), a heterogenic condition of unexplained etiology and a suspected genetic background. Androgens mediate the clinical phenotype of the disease. Therefore,all criteria of the recent PCOS consensus definition are based on their biological effects. Insulin resistance, followed by compensatory hyperinsulinemia, is frequently found in patients with PCOS. Insulin resistance is correlated with a risk of metabolic complications of PCOS, and recent research has focused on possible long-term health consequences of the syndrome. Newest molecular genetic findings at the receptor level of both androgens and insulin support their pivotal role in PCOS. These results could help to better characterize the heterogenic disorder, enabling a refinement of existing individualized therapeutic strategies.

Preterm birth but not mode of delivery is associated with an increased risk of developing inflammatory bowel disease later in life.

BACKGROUND: Exposure to bacterial antigens and other environmental factors in combination with a genetic susceptibility have been implicated in the etiology of inflammatory bowel disease (IBD). As certain perinatal circumstances, e.g., delivery by cesarean section, predispose to a different intestinal colonizations the aim of this analysis was to define a potential influence on the development of IBD in later life. METHODS: In a case-control study design, birth data were recorded from patients diagnosed with IBD (Crohn's disease [CD], n = 1,096; ulcerative colitis [UC], n = 763) and healthy controls ([C], n = 878) by a self-administered questionnaire. RESULTS: Preterm birth (CD: odds ratio [OR] 1.5 [95% confidence interval 1.1-2.0], UC: OR 1.3 [0.9-1.9]), mother's disease during pregnancy (CD: OR 1.9 [1.3-2.9], UC: OR 1.6 [1.0-2.4]), and disease in the first year of life (CD: OR 2.2 [1.6-2.9], UC: OR 1.7 [1.3-2.3]) are associated with the development of IBD in later life. No significant associations were found for the mode of delivery and breast feeding. In a logistic regression analysis female sex, smoking, appendectomy, maternal IBD, and disease in the first year of life were independently associated with CD. Female sex, appendectomy, and disease in the first year of life were independently associated with UC. CONCLUSIONS: Preterm birth and other perinatal circumstances are associated with the development of IBD, of which disease in the first year of life is an independent risk factor in multivariate analysis.

Anti-Müllerian hormone and anti-Müllerian hormone type II receptor polymorphisms are associated with follicular phase estradiol levels in normo-ovulatory women.

BACKGROUND: In mice, anti-Müllerian hormone (AMH) inhibits primordial follicle recruitment and decreases FSH sensitivity. Little is known about the role of AMH in human ovarian physiology. We hypothesize that in women AMH has a similar role in ovarian function as in mice and investigated this using a genetic approach. METHODS: The association of the AMH Ile(49)Ser and the AMH type II receptor (AMHR2) -482 A > G polymorphisms with menstrual cycle characteristics was studied in a Dutch (n = 32) and a German (n = 21) cohort of normo-ovulatory women. RESULTS: Carriers of the AMH Ser(49) allele had higher serum estradiol (E(2)) levels on menstrual cycle day 3 when compared with non-carriers in the Dutch cohort (P = 0.012) and in the combined Dutch and German cohort (P = 0.03). Carriers of the AMHR2 -482G allele also had higher follicular phase E(2) levels when compared with non-carriers in the Dutch cohort (P = 0.028), the German cohort (P = 0.048) and hence also the combined cohort (P = 0.012). Women carrying both AMH Ser(49) and AMHR2 -482G alleles had highest E(2) levels (P = 0.001). For both polymorphisms no association with serum AMH or FSH levels was observed. CONCLUSIONS: Polymorphisms in the AMH and AMHR2 genes are associated with follicular phase E(2) levels, suggesting a role for AMH in the regulation of FSH sensitivity in the human ovary.

Relationship of cytoplasmic droplets to motility, migration in mucus, and volume regulation of human spermatozoa.

A significantly greater percentage of motile than immotile spermatozoa bore droplets at the osmolality of semen and cervical mucus. The percentage of spermatozoa with droplets was not significantly correlated with the osmolality of semen or the extent of cell swelling in response to quinine in hypotonic medium. Droplets appeared slightly more frequently (ie, were more obvious) in the presence of quinine, which blocks regulatory volume decrease, indicating that they are the major site of volume expansion. There was no selection for or against droplet-bearing spermatozoa migrating through viscous surrogate mucus at high or low osmolality. Sperm swelling in response to quinine at mucus osmolality was significantly greater in fathers than in patients whose partners had no fertility problem. Therefore, cytoplasmic droplets are not deleterious to sperm motility and may be related to physiological volume regulation, which may be predictive of some forms of human male infertility.

Metformin alters insulin signaling and viability of human granulosa cells.

OBJECTIVE: To study whether insulin signaling pathways in the ovary are altered by metformin. DESIGN: In vitro human granulosa cell culture system. SETTING: Academic research environment. PATIENT(S): Infertility patients undergoing oocyte retrieval for IVF/ICSI. MAIN OUTCOME MEASURE(S): Cell viability and phosphorylated protein kinase B (PKB/AKT) and p44/42 mitogen-activated protein kinase (MAPK) expression of human primary and HGL5 granulosa cells. RESULT(S): Basal cell viability of primary granulosa cells was significantly increased relative to control by metformin preincubation, without an additional stimulatory effect of insulin or IGF. Phosphorylated AKT expression in lysates of the human granulosa cell line HGL5 was significantly increased in contrast to decreased phosphorylated MAPK expression by metformin preincubation. CONCLUSION(S): Besides systemic effects, the ovulation inducing action of metformin may at least partially be due to direct effects on insulin signaling intermediates and follicular growth patterns in the ovary.

Differences in serum LH and FSH levels using depot or daily GnRH agonists in controlled ovarian stimulation: influence on ovarian response and outcome of ART.

PURPOSE: To study effects of endogenous LH levels on ovarian response and outcome in ART cycles a controlled study was performed with two patient groups differing in the intensity of pituitary downregulation. METHODS: Group I (n = 27) received 3.75 mg of the GnRH agonist triptorelin acetate depot, group II (n = 54) was given 0.1 mg triptorelin acetate daily, followed by ovarian stimulation with recombinant FSH. RESULTS: After downregulation serum LH and FSH levels were significantly lower in group I. Patients of group I needed significantly higher FSH doses to achieve comparable levels of serum estradiol and preovulatory follicles. The number of retrieved oocytes and transferable embryos was lower in group I. CONCLUSION: Patients with profound endogenous LH suppression by depot GnRH agonists show higher FSH stimulation dose requirements and lower oocyte number and fertilization rate, indicating a need for minimal LH activity in folliculogenesis and oocyte development.

Single-nucleotide polymorphisms in the promoter region influence the expression of the human follicle-stimulating hormone receptor.

OBJECTIVE: To characterize novel single-nucleotide polymorphisms (SNPs) in the human FSH receptor (FSHR) promoter region. DESIGN: Retrospective and basic research study. SETTING: University hospital. PATIENTS: Women (202 from Germany and 55 from Indonesia) with male or tubal factor infertility undergoing controlled ovarian stimulation for IVF treatment. INTERVENTIONS: None. MAIN OUTCOME MEASURE(S): Frequency, distribution, and correlation with clinical data of the SNPs. Dual luciferase assays and electrophoretic mobility shift assays (EMSA). RESULT(S): We identified two SNPs and three mutations in the promoter region of the human FSHR which could be allocated to positions -29, -37, -114, -123, and -138 upstream of the translational initiation codon. One SNP showed a high incidence (-29: 44%, n = 202), but no correlation with basal FSH serum levels or ovarian response with the SNP at position -29 was found. Luciferase reporter assays, using pGL3 vector constructs, showed that mutations at positions -37 and -138 lead to significantly higher promoter activity. EMSA indicate that putative binding sites for transcription factors are affected by the SNPs. CONCLUSIONS: The newly identified SNPs do not seem to influence clinical parameters substantially, but modulate expression of the FSHR via changes in transcription factor binding sites.

Significance of a common single nucleotide polymorphism in exon 10 of the follicle-stimulating hormone (FSH) receptor gene for the ovarian response to FSH: a pharmacogenetic approach to controlled ovarian hyperstimulation.

The p.N680S sequence variation of the follicle-stimulating hormone (FSH) receptor gene was previously shown to influence the ovarian response to FSH in normo-ovulatory women undergoing controlled ovarian hyperstimulation. In this prospective, randomized, controlled study, we tested whether the same daily dose of FSH results in lower levels of oestradiol in women homozygous for the p.N680S sequence variation, and whether the difference can be overcome by higher FSH doses. Women undergoing controlled ovarian hyperstimulation for in vitro fertilization or intracytoplasmic sperm injection and homozygous for the wild-type or for the p.N680S FSH receptor were randomly assigned to group I (Ser/Ser, n=24), receiving an FSH dose of 150 U/day, or group II (Ser/Ser, n=25), receiving an FSH dose of 225 U/day. In group III (Asn/Asn, n=44), the FSH dose was 150 U/day. Age and basal FSH levels were not different between groups. At ovulation induction, total FSH doses were comparable in group I (1631+/-96 U) and group III (1640+/-57 U) but significantly higher in group II (2421+/-112 U) (P<0.001). Peak oestradiol levels on the day of human chorionic gonadotrophin (hCG) administration were significantly lower in group I (5680+/-675 pmol/l) compared to group III (8679+/-804 pmol/l) (P=0.028). Increasing the FSH dose from 150 to 225 U/day overcame the lower oestradiol response in women with Ser/Ser (group II, 7804+/-983 pmol/l). In women undergoing controlled ovarian hyperstimulation, the p.N680S sequence variation results in lower oestradiol levels following FSH stimulation. This lower FSH receptor sensitivity can be overcome by higher FSH doses.

Lipoprotein (a) and other prothrombotic risk factors in Caucasian women with unexplained recurrent miscarriage. Results of a multicentre case-control study.

From 1998 to 2003, 133 Caucasian women aged 17-40 years (median 29 years) suffering from unexplained recurrent miscarriage (uRM) were consecutively enrolled. In patients and 133 age-matched healthy controls prothrombotic risk factors (factor V (FV) G1691A, factor II (FII) G20210A, MTHFR T677T, 4G/5G plasminogen activator inhibitor (PAI)-1, lipoprotein (Lp) (a), protein C (PC), protein S (PS), antithrombin (AT), antiphospholipid/anticardiolipin (APA/ACA) antibodies) as well as associated environmental conditions (smoking and obesity) were investigated. 70 (52.6%) of the patients had at least one prothrombotic risk factor compared with 26 control women (19.5%; p<0.0001). Body mass index (BMI; p=0.78) and smoking habits (p=0.44) did not differ significantly between the groups investigated. Upon univariate analysis the heterozygous FV mutation, Lp(a) > 30 mg/dL, increased APA/ACA and BMI > 25 kg/m(2) in combination with a prothrombotic risk factor were found to be significantly associated with uRM. In multivariate analysis, increased Lp(a) (odds ratio (OR): 4.7/95% confidence interval (CI): 2.0-10.7), the FV mutation (OR:3.8/CI:1.4-10.7), and increased APA/ACA (OR: 4.5/CI: 1.1-17.7) had independent associations with uRM.

Overexpression of Endothelin-A-receptor in breast cancer: regulation by estradiol and cobalt-chloride induced hypoxia.

Previous studies have demonstrated the potential significance of Endothelin (ET)-1 and its receptors, ETAR and ETBR, in the development and progression of breast cancer. The objective of this study was to assess the expression levels and potential regulation of the "ET axis" in human non-neoplastic and neoplastic breast tissue as well as in various human breast cancer cell lines. Expression of ET-1, ETAR and ETBR was evaluated in 31 neoplastic and 7 non-neoplastic breast tissue samples and in six human breast cancer cell lines using conventional and quantitative real-time RT-PCR, Western blotting and immunohistochemistry. The effects of 17beta-estradiol (E2) and cobalt-chloride (CoCl2) treatment on ET-1, ETAR and ETBR expression were studied in vitro. ETAR mRNA expression levels were found to be statistically significantly higher in breast cancer specimens than in non-neoplastic breast tissue (p<0.001). For ET-1 and ETBR mRNA expression, no significant difference was observed between the two groups. All cell lines exhibited expression of ET-1 and ETAR mRNA, whereas none showed significant ETBR mRNA expression. We observed a strong and reproducible induction of ETAR mRNA and protein expression by E2 and CoCl2 in MDA-MB-468 and BT-474 cells and in MDA-MB-453 and SK-BR-3 cells with a maximum increase after 8 and 16 h of treatment, respectively, while MCF-7 and HBL-100 cells showed a constitutive expression pattern. The present data suggest a novel mechanism in the regulation of ETAR expression in breast cancer. Based on these findings, a combination of ETAR-antagonists with adjuvant endocrine treatment seems to be a reasonable therapeutic strategy.

Association of inhibin B serum levels with parameters of follicular response in a randomized controlled trial comparing gnRH agonist versus antagonist protocols for ovarian hyperstimulation.

PURPOSE: To study the association of inhibin B with ovarian response to FSH stimulation, applying either GnRH agonist or antagonist. METHODS: In a prospective randomized controlled trial, 46 patients undergoing COH received either triptorelin (group I, n = 15) or ganirelix (group II, n = 31). Parameters of follicular response and inhibin B serum levels were assessed. RESULTS: Inhibin B before FSH stimulation was significantly lower in group I than group II. The FSH stimulation phase was significantly longer in group I than group II, and the total FSH dose was significantly higher with a comparable number of retrieved oocytes. Day 1 inhibin B in group I, but not group II, was significantly correlated with the number of large ovarian follicles and retrieved oocytes. In group II, but not group I, inhibin B on day 1 was inversely correlated with the daily and total FSH dose as well as FSH stimulation duration. CONCLUSIONS: The association of inhibin B serum levels with parameters of follicular response in COH is different in patients assigned to GnRH agonist vs. antagonist treatment protocols.

Pregnancy-associated plasma protein-a production in rat granulosa cells: stimulation by follicle-stimulating hormone and inhibition by the oocyte-derived bone morphogenetic protein-15.

Pregnancy-associated plasma protein-A (PAPP-A) is the major IGF binding protein-4 (IGFBP-4) protease in follicular fluid, consistent with its proposed role in folliculogenesis. Despite growing interest, almost nothing is known about how PAPP-A expression is regulated in any tissue. Here we show that FSH and oocytes regulate PAPP-A expression in granulosa cells (GCs). By in situ hybridization, ovary PAPP-A mRNA was markedly increased by pregnant mare serum gonadotropin treatment, and the message was localized to the membrana GCs but not cumulus GCs (CGCs) of dominant follicles. To explore the mechanism, we used primary cultures of rat GCs. Control (untreated) cells produced little or no PAPP-A spontaneously. Conversely, FSH markedly stimulated PAPP-A mRNA and protein in a dose- and time-dependent fashion. Interestingly, PAPP-A expression in isolated CGCs was also strongly induced by FSH, and the induction was inhibited by added oocytes. To investigate the nature of the inhibition, we tested the effect of oocyte-derived bone morphogenetic protein-15 (BMP-15). BMP-15 alone had no effect on basal levels of PAPP-A expression by cultures of membrana GCs or CGCs. However, BMP-15 markedly inhibited the FSH stimulation of PAPP-A production in a dose-dependent manner. The cleavage of IGFBP-4 by conditioned media from FSH-treated GCs was completely inhibited by anti-PAPP-A antibody, indicating the IGFBP-4 protease secreted by GCs is PAPP-A. These results demonstrate stimulatory and inhibitory roles for FSH and BMP-15, respectively, in regulating PAPP-A production by GCs. We propose that FSH and oocyte-derived BMP-15 form a controlling network that ensures the spatiotemporal pattern of GC PAPP-A expression in the dominant follicle.