Progesterone and 17-hydroxy-progesterone concentrations in follicular fluid and serum reflect their production in granulosa and theca cells.

RESEARCH QUESTION: How is the production of progesterone (P4) and 17-hydroxy-P4 (17-OH-P4) regulated between theca cells and granulosa cells during the follicular phase, during ovulation and after transformation into a corpus luteum? DESIGN: Three cohorts were examined: (i) 31 women undergoing natural and stimulated cycles, with serum hormone measurements taken every 3 days; (ii) 50 women undergoing ovarian stimulation, with hormone concentrations in serum and follicular fluid assessed at five time points during final follicle maturation; and (iii) 12 women undergoing fertility preservation, with hormone concentrations evaluated via the follicular fluid of small antral follicles. RESULTS: In the early follicular phase, theca cells primarily synthesized 17-OH-P4 while granulosa cells produced limited P4, maintaining the P4:17-OH-P4 ratio <1. As follicles reached follicle selection at a diameter of approximately 10 mm, P4 synthesis in granulosa cells was up-regulated, but P4 was mainly accumulated in follicular fluid. During final maturation, enhanced activity of the enzyme HSD3B2 in granulosa cells enhanced P4 production, with the P4:17-OH-P4 ratio increasing to >1. The concentration of 17-OH-P4 in the luteal phase was similar to that in the follicular phase, but P4 production increased in the luteal phase, yielding a P4:17-OH-P4 ratio significantly >1. CONCLUSIONS: The P4:17-OH-P4 ratio reflects the activity of granulosa cells and theca cells during the follicular phase and following luteinization in the corpus luteum. Managing the function of granulosa cells is key for reducing the concentration of P4 during ovarian stimulation, but the concerted action of FSH and LH on granulosa cells during the second half of the follicular phase makes this complex.

A distinctive epigenetic ageing profile in human granulosa cells.

STUDY QUESTION: Does women's age affect the DNA methylation (DNAm) profile differently in mural granulosa cells (MGCs) from other somatic cells? SUMMARY ANSWER: Accumulation of epimutations by age and a higher number of age-related differentially methylated regions (DMR) in MGCs were found compared to leukocytes from the same woman, suggesting that the MGCs have a distinctive epigenetic profile. WHAT IS KNOWN ALREADY: The mechanisms underlying the decline in women's fertility from the mid-30s remain to be fully elucidated. The DNAm age of many healthy tissues changes predictably with and follows chronological age, but DNAm age in some reproductive tissues has been shown to depart from chronological age (older: endometrium; younger: cumulus cells, spermatozoa). STUDY DESIGN, SIZE, DURATION: This study is a multicenter cohort study based on retrospective analysis of prospectively collected data and material derived from healthy women undergoing IVF or ICSI treatment following ovarian stimulation with antagonist protocol. One hundred and nineteen women were included from September 2016 to June 2018 from four clinics in Denmark and Sweden. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples were obtained from 118 healthy women with varying ovarian reserve status. MGCs were collected from 63 of the 119 women by isolation from pooled follicles immediately after oocyte retrieval. DNA from leukocytes and MGCs was extracted and analysed with a genome-wide methylation array. Data from the methylation array were processed using the ENmix package. Subsequently, DNAm age was calculated using established and tailored age predictors and DMRs were analysed with the DMRcate package. MAIN RESULTS AND ROLE OF CHANCE: Using established age predictors, DNAm age in MGCs was found to be considerable younger and constant (average: 2.7 years) compared to chronological age (average: 33.9 years). A Granulosa Cell clock able to predict the age of both MGCs (average: 32.4 years) and leukocytes (average: 38.8 years) was successfully developed. MGCs differed from leukocytes in having a higher number of epimutations (P = 0.003) but predicted telomere lengths unaffected by age (Pearson's correlation coefficient = -0.1, P = 0.47). DMRs associated with age (age-DMRs) were identified in MGCs (n = 335) and in leukocytes (n = 1) with a significant enrichment in MGCs for genes involved in RNA processing (45 genes, P = 3.96 × 10-08) and gene expression (152 genes, P = 2.3 × 10-06). The top age-DMRs included the metastable epiallele VTRNA2-1, the DNAm regulator ZFP57 and the anti-Müllerian hormone (AMH) gene. The apparent discordance between different epigenetic measures of age in MGCs suggests that they reflect difference stages in the MGC life cycle. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: No gene expression data were available to associate with the epigenetic findings. The MGCs are collected during ovarian stimulation, which may influence DNAm; however, no correlation between FSH dose and number of epimutations was found. WIDER IMPLICATIONS OF THE FINDINGS: Our findings underline that the somatic compartment of the follicle follows a different methylation trajectory with age than other somatic cells. The higher number of epimutations and age-DMRs in MGCs suggest that their function is affected by age. STUDY FUNDING/COMPETING INTEREST(S): This project is part of ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS, the Danish National Research Foundation and the European Research Council. The authors declare no conflict of interest.

Response to ovulation trigger is correlated to late follicular phase progesterone levels: A hypothesis explaining reduced reproductive outcomes caused by increased late follicular progesterone rise.

STUDY QUESTION: Is there an association between progesterone (P4) levels on the day of hCG or GnRH trigger and on the day of oocyte retrieval in IVF/ICSI cycles? SUMMARY ANSWER: A significant positive correlation between P4 levels on the day of trigger and the day of oocyte retrieval is seen; HCG trigger induces a steeper P4 increase than GnRHa trigger. WHAT IS KNOWN ALREADY: FSH induces LH receptor (LHR) expression on granulosa cells, and LHR produces progesterone when exposed to LH-like activity. FSH per se also to some extent induces P4 secretion. Late follicular phase progesterone rise has been associated with reduced reproductive outcomes. STUDY DESIGN, SIZE, DURATION: This study is based on data from a previously published RCT conducted from 2009 to 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 384 participants were enrolled; 199 received 5000 IU hCG and 185 received buserelin 0.5 mg for triggering ovulation. P4 was measured on the day of ovulation induction and on the day of oocyte retrieval. FSH consumption and number of retrieved follicles were recorded. MAIN RESULTS AND THE ROLE OF CHANCE: A significant linear relationship between P4 on the day of ovulation induction and oocyte retrieval was seen in the hCG trigger group (P < 0.00001) as well as in the GnRHa trigger group (P < 0.00001). The P4 ratio (the increase in P4 between ovulation induction and oocyte retrieval) was significantly higher in the group of patients with <5 follicles compared to those with 5-15 and >15 follicles (P < 0.0001). The FSH consumption per follicle was significantly higher in the group of patients with <5 follicles compared to those with 5-15 and >15 follicles (P < 0.0001). LIMITATIONS, REASONS FOR CAUTION: Although the study demonstrates a significant correlation between P4 levels before and after ovulation trigger, it does not demonstrate a causal relation to the number of LHRs present on granulosa cells. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study support the proposed hypothesis that follicles exposed to high levels of FSH during ovarian stimulation will respond with an inappropriately high LHR expression. This in turn causes a high P4 output in response to the trigger. This study further expands our understanding of the underlying mechanisms affecting reproductive outcomes in relation to ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S): The authors received no specific funding for this work and disclose no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Within-day energy deficiency and reproductive function in female endurance athletes.

We aimed to estimate and compare within-day energy balance (WDEB) in athletes with eumenorrhea and menstrual dysfunction (MD) with similar 24-hour energy availability/energy balance (EA/EB). Furthermore, to investigate whether within-day energy deficiency is associated with resting metabolic rate (RMR), body composition, S-cortisol, estradiol, T3 , and fasting blood glucose. We reanalyzed 7-day dietary intake and energy expenditure data in 25 elite endurance athletes with eumenorrhea (n = 10) and MD (n = 15) from a group of 45 subjects where those with disordered eating behaviors (n = 11), MD not related to low EA (n = 5), and low dietary record validity (n = 4) had been excluded. Besides gynecological examination and disordered eating evaluation, the protocol included RMR measurement; assessment of body composition by dual-energy X-ray absorptiometry, blood plasma analysis, and calculation of WDEB in 1-hour intervals. Subjects with MD spent more hours in a catabolic state compared to eumenorrheic athletes; WDEB < 0 kcal: 23.0 hour (20.8-23.4) vs 21.1 hour (4.7-22.3), P = .048; WDEB < -300 kcal: 21.8 hour (17.8-22.4) vs 17.6 hour (3.9-20.9), P = .043, although similar 24-hour EA: 35.6 (11.6) vs 41.3 (12.7) kcal/kg FFM/d, (P = .269), and EB: -659 (551) vs -313 (596) kcal/d, (P = .160). Hours with WDEB <0 kcal and <-300 kcal were inversely associated with RMRratio (r = -.487, P = .013, r = -.472, P = .018), and estradiol (r = -.433, P = .034, r = -.516, P = .009), and positively associated with cortisol (r = .442, P = .027, r = .463, P = .019). In conclusion, although similar 24-hour EA/EB, the reanalysis revealed that MD athletes spent more time in a catabolic state compared to eumenorrheic athletes. Within-day energy deficiency was associated with clinical markers of metabolic disturbances.

ESC expert statement on the effects on mood of the natural cycle and progestin-only contraceptives.

Hormonal fluctuations during the natural cycle, as well as progestins used for hormonal contraception, can exert effects on mood especially in vulnerable women. Negative effects of levonorgestrel-releasing intrauterine contraception on mood are rare.

Combined assessment of polymorphisms in the LHCGR and FSHR genes predict chance of pregnancy after in vitro fertilization.

STUDY QUESTION: Can gonadotrophin receptor variants separately or in combination, be used for the prediction of pregnancy chances in in vitro fertilization (IVF) trials? SUMMARY ANSWER: The luteinizing hormone/human chorionic gonadotrophin receptor (LHCGR) variant N312S and the follicle-stimulating hormone receptor (FSHR) variant N680S can be utilized for the prediction of pregnancy chances in women undergoing IVF. WHAT IS KNOWN ALREADY: The FSHR N680S polymorphism has been shown to affect the ovarian response in response to gonadotrophin treatment, while no information is currently available regarding variants of the LHCGR in this context. STUDY DESIGN, SIZE, DURATION: Cross-sectional study, duration from September 2010 to February 2015. Women undergoing IVF were consecutively enrolled and genetic variants compared between those who became pregnant and those who did not. The study was subsequently replicated in an independent sample. Granulosa cells from a subset of women were investigated regarding functionality of the genetic variants. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women undergoing IVF (n = 384) were enrolled in the study and genotyped. Clinical variables were retrieved from medical records. For replication, an additional group of n = 233 women was utilized. Granulosa cells from n = 135 women were isolated by flow cytometry, stimulated with Follitropin alpha or Menotropin, and the downstream targets 3',5'-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) measured with enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE: Women homozygous for serine (S) in both polymorphisms displayed higher pregnancy rates than women homozygous asparagine (N) (OR = 14.4, 95% CI: [1.65, 126], P = 0.016). Higher pregnancy rates were also evident for women carrying LHCGR S312, regardless of FSHR variant (OR = 1.61, 95% CI: [1.13, 2.29], P = 0.008). These women required higher doses of FSH for follicle recruitment than women homozygous N (161 versus 148 IU, P = 0.030). When combining the study cohort with the replication cohort (n = 606), even stronger associations with pregnancy rates were noted for the combined genotypes (OR = 11.5, 95% CI: [1.86, 71.0], P = 0.009) and for women carrying LHCGR S312 (OR = 1.49, 95% CI: [1.14, 1.96], P = 0.004). A linear significant trend with pregnancy rate and increasing number of G alleles was also evident in the merged study population (OR = 1.34, 95% CI: [1.10, 1.64], P = 0.004). A lower cAMP response in granulosa cells was noted following Follitropin alpha stimulation for women homozygous N in both polymorphisms, compared with women with other genotypes (0.901 pmol cAMP/mg total protein versus 2.19 pmol cAMP/mg total protein, P = 0.035). LIMITATIONS, REASONS FOR CAUTION: Due to racial differences in LHCGR genotype distribution, these results may not be applicable for all populations. WIDER IMPLICATIONS OF THE FINDINGS: Despite that >250 000 cycles of gonadotrophin stimulations are performed annually worldwide prior to IVF, it has not been possible to predict neither the pregnancy outcome, nor the response to the hormone with accuracy. If LHCGR and FSHR variants are recognized as biomarkers for chance of pregnancy, more individualized and thereby more efficient treatment modalities can be developed. STUDY FUNDING, COMPETING INTERESTS: This work was supported by Interreg IV A, EU (grant 167158) and ALF governments grant (F2014/354). Merck-Serono (Darmstadt, Germany) supported the enrollment of the subjects. The authors declare no conflict of interest.

Low-energy density and high fiber intake are dietary concerns in female endurance athletes.

Low or reduced energy availability (LEA) is linked to functional hypothalamic oligomenorrhea/amenorrhea (FHA), which is frequently reported in weight-sensitive sports. This makes LEA a major nutritional concern for female athletes. The aim of this study was to describe dietary characteristics of athletes with LEA and/or FHA. Endurance athletes (n = 45) were recruited from national teams and competitive clubs. Protocols included gynecological examination, body composition, eating disorder evaluation, and 7-day dietary intake and EA assessment. Athletes with disordered eating behavior/eating disorders (n = 11), menstrual dysfunction other than FHA (n = 5), and low dietary record validity (n = 4) were excluded. Remaining subjects (n = 25) were characterized by EA [optimal: ≥ 45 kcal (188 kJ)/kg fat-free mass (FFM)/day (n = 11), LEA: < 45 kcal (188 kJ)/kg FFM/day (n = 14)] and reproductive function [eumenorrhea (EUM; n = 10), FHA (n = 15)]. There was no difference in EA between FHA and EUM subjects. However, FHA and LEA subjects shared the same dietary characteristics of lower energy density (ED) [(P = 0.012; P = 0.020), respectively], and fat content [(P = 0.047; P = 0.027), respectively]. Furthermore, FHA subjects had a lower intake of carbohydrate-rich foods (P = 0.019), higher fiber content (P < 0.001), and drive for thinness score (P = 0.003). Conclusively, low ED together with high fiber content may constitute targets for dietary intervention in order to prevent and treat LEA and FHA in female athletes.

Energy availability and the female athlete triad in elite endurance athletes.

The female athlete triad (Triad), links low energy availability (EA), with menstrual dysfunction (MD), and impaired bone health. The aims of this study were to examine associations between EA/MD and energy metabolism and the prevalence of Triad-associated conditions in endurance athletes. Forty women [26.2 ± 5.5 years, body mass index (BMI) 20.6 ± 2.0 kg/m(2), body fat 20.0 ± 3.0%], exercising 11.4 ± 4.5 h/week, were recruited from national teams and competitive clubs. Protocol included gynecological examination; assessment of bone health; indirect respiratory calorimetry; diet and exercise measured 7 days to assess EA; eating disorder (ED) examination; blood analysis. Subjects with low/reduced EA (< 45 kcal/kg FFM/day), had lower resting metabolic rate (RMR) compared with those with optimal EA [28.4 ± 2.0 kcal/kg fat-free mass (FFM)/day vs 30.5 ± 2.2 kcal/kg FFM/day, P < 0.01], as did subjects with MD compared with eumenorrheic subjects (28.6 ± 2.4 kcal/kg FFM/day vs 30.2 ± 1.8 kcal/kg FFM/day, P < 0.05). 63% had low/reduced EA, 25% ED, 60% MD, 45% impaired bone health, and 23% had all three Triad conditions. 53% had low RMR, 25% hypercholesterolemia, and 38% hypoglycemia. Conclusively, athletes with low/reduced EA and/or MD had lowered RMR. Triad-associated conditions were common in this group of athletes, despite a normal BMI range. The high prevalence of ED, MD, and impaired bone health emphasizes the importance of prevention, early detection, and treatment of energy deficiency.

Polycystic ovary syndrome and low-grade inflammation with special reference to YKL-40.

OBJECTIVE: To evaluate the plasma level of YKL-40 in a Danish polycystic ovary syndrome (PCOS) population and to investigate whether YKL-40 is associated with CVD risk factors such as waist circumference, body mass index (BMI), insulin resistance (IR), fasting glucose, fasting insulin, blood lipids and CRP. DESIGN: Cross-sectional study. SETTING: Gynecological clinics at three Danish University Hospitals. PATIENTS: One hundred seventy-one premenopausal women with PCOS recruited consecutively from April 2010 to February 2012. PCOS was diagnosed according to the Rotterdam criteria. MAIN OUTCOME MEASURES: Plasma level of YKL-40 in four phenotypes of PCOS defined by BMI and IR. RESULTS: No statistically significant difference was observed in the plasma level of YKL-40 across the four BMI/IR-phenotypes. Positive associations were observed between YKL-40 and BMI, total and free testosterone, triglycerides, and CRP. Total and free testosterone were independent predictors of YKL-40. CONCLUSION: YKL-40, the marker of low-grade inflammation is not increased in women with PCOS.

Polycystic ovary syndrome: infertility, cardiovascular, metabolic and obstetrical risks, laboratory and clinical outcomes -- the PICOLO Study.

OBJECTIVES: The primary objective of this multicenter study is to evaluate the relative impact of insulin resistance (IR) and body mass index (BMI) in women with polycystic ovary syndrome (PCOS) on (1) Key hemodynamic/thrombogenic variables, (2) Oocyte quality and early embryo development, (3) Fetal growth, placental function and adverse obstetric outcome. SECONDARY OBJECTIVE: To establish a PCOS database and biobank facilitating future basic and interventional research related to PCOS. DESIGN: A cross-sectional and longitudinal cohort study at four University Hospitals in Denmark. POPULATION INCLUSION: About 200 women fulfilling the Rotterdam Criteria and 100 women without PCOS recruited from 2010 to 2012. METHODS: The impact of PCOS, as well as the impact of IR and BMI on the hormonal, metabolic and hemostatic key variables will be analyzed combining conventional, molecular techniques and selected gene analysis. Oocytes will be characterized by gene expression of granulosa and cumulus cells and the early embryo development will be followed by time lapse microscopy. Fetal growth will be assessed by repeated ultrasound measurements, and the pregnancy outcome compared to maternal and fetal biochemical markers of growth and inflammation and clinical pregnancy complications. MAIN OUTCOME MEASURES: Metabolic and hemostatic risk-biomarkers, oocyte and embryo quality, adverse pregnancy outcome, fetal growth and placental function in women with PCOS.

Cigarette smoking during early pregnancy reduces the number of embryonic germ and somatic cells.

BACKGROUND: Cigarette smoking during pregnancy is associated with negative reproductive consequences for male fetuses in adult life such as reduced testicular volume and sperm concentration. The present study evaluates the number of germ and somatic cells present in human embryonic first-trimester gonads in relation to maternal smoking. METHODS: The study includes 24 human first-trimester testes, aged 37-68 days post-conception, obtained from women undergoing legal termination of pregnancy. A questionnaire was used to obtain information about smoking and drinking habits during pregnancy. Validated stereological methods were used to estimate gonadal cell numbers in histological sections. Results were also evaluated in the context of previously published data on ovaries from our laboratory. RESULTS: A significant reduction in the number of germ cells by 55% [95% confidence interval (CI) 74-21% reduction, P = 0.004] and somatic cells by 37% (95% CI 59-3%, P = 0.023) was observed in testes prenatally exposed to maternal cigarette smoking, compared with unexposed. The effect of maternal smoking was dose-dependent being higher in the heavy smokers and remained consistent after adjusting for possible confounders such as alcohol and coffee consumption (P = 0.002). The number of germ cells in embryonic gonads, irrespective of gender, was also significantly reduced by 41% (95% CI 58-19%, P = 0.001) in exposed versus non-exposed embryonic gonads. CONCLUSIONS: Prenatal exposure to maternal cigarette smoke reduces the number of germ and somatic cells in embryonic male and female gonads. This effect may have long-term consequences on the future fertility of exposed offspring. These findings may provide one potential cause of the reduced fertility observed during recent years.

Hormone therapy affects plasma measures of factor VII-activating protease in younger postmenopausal women.

OBJECTIVES: Current reviews indicate that hormone therapy (HT) has a protective role in coronary heart disease (CHD) in younger postmenopausal women, whereas HT contributes to CHD in older women. Factor VII-activating protease (FSAP) is a serine protease that accumulates in unstable atherosclerotic plaques. FSAP is presumably involved in plaque stability and rupture. Reduced plasma concentration of FSAP may be associated with the development and expression of atherosclerosis and may thus contribute to precipitation of CHD. Here we address the potential influence of various HT regimens on plasma measures of FSAP in postmenopausal women treated for 1 year with different HT formulations or no HT. METHODS: Six groups of postmenopausal women (n = 139) were allocated to five different HT modalities or no HT. Samples were collected at baseline and after 12 months of treatment. Prototype assays were used for the determination of FSAP antigen and FSAP activity. RESULTS: The FSAP measures were comparable at baseline. No significant changes were observed in the control group after 12 months. HT in general induced a significant increase in FSAP antigen (7.7 microg/ml at baseline and 8.0 microg/ml after 12 months, p = 0.05), FSAP activity (1.54 PEU/ml at baseline and 1.68 PEU/ml after 12 months, p < 0.001) and FSAP ratio (202 mPEU/microg at baseline and 210 mPEU/microg after 12 months, p = 0.01). CONCLUSIONS: HT increases the plasma measures of FSAP. This increase may contribute to the protective effect on CHD induced by HT in younger postmenopausal women.

Contraceptive use and behavior in the 21st century: a comprehensive study across five European countries.

OBJECTIVE: The choice of currently available contraceptive methods has increased considerably in recent years, offering women of reproductive age a variety of different methods dependent on their needs and lifestyle. In order to determine the pattern of use of current methods in contraception, a survey was conducted in a large population of women drawn from five European countries (France, Germany, Italy, Spain and the United Kingdom). METHOD: More than 12,000 randomly selected women, aged 15-49 years, were interviewed using a standardized questionnaire which addressed the use of current methods of contraception. The responses were analyzed for the total study population, and, where appropriate, by country and age. RESULTS: An oral contraceptive (OC) was confirmed as the most widely used method of contraception for women in the European study population, with an estimated 22 million users in the five countries. Women using an OC reported very high levels of satisfaction (>90%). Male and female sterilization were the main methods of contraception in women aged 40 years and older. One-half of the women had undergone their sterilization before the age of 35 years. More than 50% of the women who had undergone sterilization had not been adequately informed and counselled about alternative reversible contraceptive options. No method of contraception was being used currently by 23% of the European study population, and unreliable methods of contraception (including cap/diaphragm, chemical, and natural and withdrawal methods) were being used by a further 6% of the population. Although valid reasons (e.g. not in a sexual relationship, wish to become pregnant) were given by many women who were not using contraception, there still remains a large number of women who need counselling regarding the importance of using reliable contraceptive methods. The number of women aged 15-49 years in the five European countries who are considered at risk of an unwanted pregnancy is estimated to be 4.7 million (6.5%). CONCLUSIONS: Differences in the use pattern of contraceptive methods were demonstrated that emphasize the social and cultural differences between the countries. The findings in the current study can be used as a baseline from which to monitor trends in contraceptive use and behavior in subsequent studies.

Age determination enhanced by embryonic foot bud and foot plate measurements in relation to Carnegie stages, and the influence of maternal cigarette smoking.

BACKGROUND: Reliable age determination of first-trimester human embryos and fetuses is an important parameter for clinical use and basic science. Age determination by ultrasound or morphometric parameters of embryos 4-6 weeks post conception (p.c.) have been questioned, and more accurate methods are required. Data on whether and how maternal smoking and alcohol consumption influence embryonic and fetal foot growth is also lacking. METHODS: Embryonic tissue from 102 first-trimester legal abortions (aged 35-69 days p.c.) were collected. All women answered a questionnaire concerning smoking and drinking habits, and delivered a urine sample for cotinine analysis. Embryonic age was evaluated by vaginal ultrasound measurements and by post-termination foot length and compared with the Carnegie stages. RESULTS: Foot bud and foot plate were defined and measured as foot length in embryos aged 35-47 days p.c. (range 0.8-2.1 mm). In embryos and fetuses aged 41-69 days p.c., heel-toe length was measured (range 2.5-7.5 mm). We found a significant linear correlation between foot length and age. Morphology of the feet was compared visually with the Carnegie collection, and we found that the mean ages of the two collections correlated well. Foot length was independent of gender, Environmental Tobacco Smoke, maternal smoking and alcohol consumption. CONCLUSION: Foot length correlated linearly to embryonic and foetal age, and was unaffected by gender, ETS, maternal smoking and alcohol consumption.

The number of oogonia and somatic cells in the human female embryo and fetus in relation to whether or not exposed to maternal cigarette smoking.

BACKGROUND: Prenatal exposure to maternal cigarette smoking or compounds of cigarette smoke is associated with serious reproductive hazards such as apoptotic death of oogonia in murine offspring and decreased fecundability in human offspring. The present study addresses potential effects of in utero exposure to cigarette smoking. METHODS: Twenty-nine human first-trimester ovaries from legal abortions [aged 38-64 days post-conception (p.c.)] were collected. Mothers filled out a questionnaire about their smoking habits and delivered a urine sample for cotinine analysis. The ovarian cell numbers were estimated using stereological methods. RESULTS: A non-linear correlation between the numbers of oogonia and somatic cells in relation to age of the embryo/fetus was shown in 28 ovaries, including the first estimates performed in ovaries younger than 47 days p.c. Prenatal exposure to smoke showed a significant decrease in the number of somatic cells (P < or = 0.01). The number of oogonia was not significantly associated with prenatal exposure to maternal smoking (P < or = 0.09). The ratio between the two cell types decreased considerably from 1:45 to 1:23 from 38 to 46 days p.c. and was not affected by smoking. CONCLUSIONS: Oogonia proliferate and/or invade the developing ovary at a much faster relative rate than somatic cells. In utero exposure to maternal smoking significantly reduces the number of somatic cells from Days 38 to 64 p.c. Since oocytes cannot survive without being enclosed by somatic cells in a follicle, reduction in the somatic cells number may have long-range consequences on the number of oocytes available in adult life and on the future fertility of female offspring exposed to smoking in utero.

The effect of continuous combined conjugated equine estrogen plus medroxyprogesterone acetate and tibolone on cardiovascular metabolic risk factors.

OBJECTIVES: Hormone treatment (HT) after the menopause affects lipid and carbohydrate metabolism and inflammation and may modify risk factors relevant for the clinical expression of the metabolic syndrome and cardiovascular disease. Tibolone has pharmacodynamic properties different from other hormone preparations. Here, we compare the effect of combined HT and tibolone on metabolic risk markers for the development of cardiovascular disease. METHODS: Postmenopausal women were randomly assigned to 1.25 or 2.5 mg/day of tibolone or oral continuous combined conjugated equine estrogen plus medroxyprogesterone acetate (CEE/MPA). Cardiovascular risk factors were determined at baseline and after 12 months of treatment. RESULTS: Body mass index and blood pressure were unaffected by the HT. HOMA-IR decreased in the CEE/MPA group (3.69 vs. 3.38; p = 0.02). Treatment with tibolone increased tissue-type plasminogen activator activity (0.87 IU/ml vs. 1.21 IU/ml; p = 0.005) and C-reactive protein (0.83 mg/l vs. 1.88 mg/l; p < 0.001), and decreased plasminogen activator inhibitor activity (6.9 IU/ml vs. 2.0 IU/ml; p < 0.001) and triglycerides (0.99 vs. 0.87 mmol/l; p = 0.004). Both treatments decreased total cholesterol significantly. CONCLUSIONS: CEE/MPA and tibolone have comparable effects on most metabolic risk factors investigated. The effect of tibolone on fibrinolysis and triglycerides suggests that tibolone has a favorable pharmacological profile on these risk factors when compared to CEE/MPA.

Ovulation inhibition by estetrol in an in vivo model.

Background Currently, the synthetic steroid ethinylestradiol is the preferred estrogen in combined oral contraceptives. The aim of the present study was to evaluate the effectiveness of the natural steroid estetrol (E4) as an ovulation inhibitor in rats when compared to ethinylestradiol. Study design Regularly cycling female rats were treated orally twice daily for four consecutive days, starting on the day of estrus, with E4 (0.03, 0.1, 0.3, 1.0 or 3.0 mg/kg), ethinylestradiol (0.0003, 0.001, 0.003, 0.01 or 0.03 mg/kg) or vehicle control (eight animals per group). In a second experiment conducted under the same experimental protocol, 2.0 mg/kg of E4 was administered as a single daily dose or as a dose of 1.0 mg/kg twice daily. In both studies, the primary endpoint was the number of ovulated oocytes in the genital tract. Results Estetrol at the twice-daily dose of 0.3 mg/kg and above inhibited ovulation. This effect was statistically significant (p < 0.05). The comparator, ethinylestradiol, significantly inhibited ovulation (p < 0.05) at the highest dose (0.03 mg/kg) administered twice daily. An E4 dose of 2.0 mg/kg administered once daily for four consecutive days inhibited ovulation in four of eight rats. In contrast, when the same dose was administered in two separate doses, that is 1.0 mg/kg twice daily, ovulation was inhibited in eight of eight rats. The ED50 for the ethinylestradiol and the E4 dose-response curves shows that ethinylestradiol is 18 times more potent than E4. Conclusion Twice-daily administration of E4 effectively inhibits ovulation in cycling rats. The effect is dose-dependent. The relative potency of E4 is about 18 times less compared to that of ethinylestradiol. We conclude that, based on these data, combined with available pharmacological and clinical data on the safety and efficacy of E4, the human fetal estrogenic steroid estetrol is a potential candidate to replace ethinylestradiol in combined oral contraceptives.

Semen quality associated with subsequent hospitalizations - Can the effect be explained by socio-economic status and lifestyle factors?

Semen quality is suggested to be a universal biomarker for future health. Previous studies have mostly been registry based excluding the possibility to address the importance of lifestyle, fertility status, health and socio-economic status. We aimed to investigate whether the association between semen quality and subsequent risk of hospitalization could be explained by differences in occupation, education, fertility, cryptorchidism, BMI or smoking; 1423 men with first semen sample at Fertility Clinic, Frederiksberg Hospital, Denmark, from 1977 to 2010 responded to a questionnaire in 2012 about current health, lifestyle, educational level and occupation. They were followed in the Danish National Patient Registry to first-time hospitalizations using ICD-8 and ICD-10 classification. Data were analysed by Cox proportional hazard regression models to adjust for the possible confounding factors. We found a significant higher risk of being hospitalized with decreasing sperm concentrations (0-15 mill/mL: HR1.78, 95% CI:1.51-2.09; 16-50 mill/mL: HR 1.37 95% CI: 1.17-1.60; 51-100 mill/mL: HR1.25 95% CI: 1.07-1.45). Same significant association of being hospitalized with decreasing total sperm counts was seen. The dose-response increase in risk in hospitalization with decreasing sperm concentration and total sperm count remained constant after further individual adjustment for occupation, marital status, fertility, cryptorchidism, BMI or smoking. The association between semen quality and subsequent morbidity was not explained by differences in lifestyle, behavioural or fertility status. We were unable to adjust for all possible confounders simultaneously due to limited sample size, and reverse causation is a possible explanation as information about education and lifestyle was obtained after semen analysis and hospitalizations occurred and may have changed as consequence of both. Semen quality may be a universal biomarker for future health not explained by lifestyle and socio-economic status, but this needs to be addressed further in future studies.

Significant reduction of the antiatherogenic effect of estrogen by long-term inhibition of nitric oxide synthesis in cholesterol-clamped rabbits.

The purpose of this study was to investigate whether endothelium-derived nitric oxide (NO) is involved in the plasma lipid-independent antiatherogenic effect of estrogen and levormeloxifene, a partial estrogen receptor agonist. 85 rabbits were ovariectomized and balloon-injured in the middle thoracic aorta. The rabbits were fed a cholesterol-enriched diet supplemented with 17beta-estradiol, levormeloxifene, or placebo, either alone, or together with 160 microg/ml NG-nitro- -arginine methyl ester (-NAME), an NO synthase inhibitor, in their drinking water for 12 wk. Plasma cholesterol was maintained at 25-30 mmol/liter by individualized cholesterol feeding. In the undamaged aorta, the extent of atherosclerosis in the estrogen group was only one-third that in the placebo group. Simultaneous administration of -NAME, however, significantly reduced the antiatherogenic effect of estrogen (P < 0.01). There was no significant difference between the placebo group given -NAME and the group treated with placebo alone. At the previously endothelium-denuded site, estrogen had no effect on atherosclerosis development, whereas -NAME combined with estrogen significantly increased atherogenesis (P < 0.05). The effects of levormeloxifene were almost similar to those of estrogen. Active vascular concentrations of -NAME were demonstrated in an additional study, in which maximal aortic/coronary endothelium-dependent relaxation was significantly inhibited in rabbits given -NAME. Thus, in this study a considerable part of the plasma lipid-independent antiatherogenic effect of estrogen was mediated through its effect on endothelial NO in cholesterol-fed rabbits. The results for levormeloxifene suggest a common mechanism of action for estrogen and partial estrogen receptor agonists on atherogenesis.