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Recent studies suggest that migraine may be associated with gastrointestinal (GI) disorders, including irritable bowel syndrome (IBS), inflammatory bowel syndrome, and celiac disease. Here, an overview of the associations between migraine and GI disorders is presented, as well as possible mechanistic links and clinical implications. People who regularly experience GI symptoms have a higher prevalence of headaches, with a stronger association with increasing headache frequency. Children with a mother with a history of migraine are more likely to have infantile colic. Children with migraine are more likely to have experienced infantile colic compared to controls. Several studies demonstrated significant associations between migraine and celiac disease, inflammatory bowel disease, and IBS. Possible underlying mechanisms of migraine and GI diseases could be increased gut permeability and inflammation. Therefore, it would be worthwhile to investigate these mechanisms further in migraine patients. These mechanisms also give a rationale to investigate the effects of the use of pre- and probiotics in migraine patients.

Objective: To review the literature and present original data to answer the question of whether in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) is associated with an increase in imprinted diseases in offspring. If the answer is positive, to investigate whether there is a causal relationship between IVF or ICSI and the imprinted diseases., Design: Review study., Result(s): Eight epidemiologic studies were suitable to calculate the weighted relative risk for the birth of a child with Beckwith-Wiedemann syndrome following IVF or ICSI compared with the risk in the normal population. This relative risk was 5.2 (95% CI 1.6-7.4). In one study the relative risk was corrected for parents' fertility problems and no significant association was found. Data on the Silver-Russell syndrome are too sparse to draw conclusions, but a positive association with IVF or ICSI treatment is probable. No significant associations were found between the incidences of the Angelman and Prader-Willi syndromes and IVF or ICSI treatments. Children with Prader-Willi syndrome or Angelman syndrome are more likely to be born to parents with fertility problems. All retinoblastomas in children born after IVF or ICSI could be explained by de novo mutations in the RB1 gene and were not associated with imprinted genes. Imprinted diseases result from methylation errors already present in sperms or oocytes. There is no proof of a causal relationship between imprinted diseases and IVF or ICSI treatments., Conclusion(s): Imprinting disorders are more prevalent after human IVF or ICSI. Future studies should correct for fertility problems in the affected and comparison groups. It is highly improbable that assisted reproduction technologies cause imprinted diseases in humans., (Copyright © 2013. Published by Elsevier Inc.)

Background: Little is known about post-natal growth in IVF offspring and the effects of rates of early post-natal growth on blood pressure and body fat composition during childhood and adolescence., Methods: The follow-up study comprised 233 IVF children aged 8-18 years and 233 spontaneously conceived controls born to subfertile parents. Growth data from birth to 4 years of age, available for 392 children (n = 193 IVF, n = 199 control), were used to study early post-natal growth. Furthermore, early post-natal growth velocity (weight gain) was related to blood pressure and skinfold measurements at follow-up., Results: We found significantly lower weight, height and BMI standard deviation scores (SDSs) at 3 months, and weight SDS at 6 months of age in IVF children compared with controls. Likewise, IVF children demonstrated a greater gain in weight SDS (P < 0.001), height SDS (P = 0.013) and BMI SDS (P = 0.029) during late infancy (3 months to 1 year) versus controls. Weight gain during early childhood (1-3 years) was related to blood pressure in IVF children (P = 0.014 systolic, 0.04 diastolic) but not in controls. Growth during late infancy was not related to skinfold thickness in IVF children, unlike controls (P = 0.002 peripheral sum, 0.003 total sum). Growth during early childhood was related to skinfold thickness in both IVF and controls (P = 0.005 and 0.01 peripheral sum and P = 0.003 and 0.005 total sum, respectively)., Conclusions: Late infancy growth velocity of IVF children was significantly higher compared with controls. Nevertheless, early childhood growth instead of infancy growth seemed to predict cardiovascular risk factors in IVF children. Further research is needed to confirm these findings and to follow-up growth and development of IVF children into adulthood.

Background: Previous studies demonstrated a link between adverse conditions during prenatal life and the development of diseases in adult life. It is still unclear whether IVF conception could permanently affect early prenatal development in humans, with post-natal health consequences. The objective of the present study is to examine pubertal development in 8-18-year-old IVF singletons and controls born from subfertile parents who attended one Dutch fertility clinic were included., Methods: IVF singletons and controls born from subfertile parents who attended one clinic in the Dutch OMEGA study were included. Pubertal stage by Tanner's classification, age at menarche and menstrual cycle characteristics were studied in the total population (n = 233: 115 IVF-conceived boys and 118 IVF-conceived girls, each with age-matched comparison groups). Bone age and sex hormone levels were examined in two distinct pubertal subpopulations., Results: Pubertal stage and age at menarche were not significantly different between IVF and control children. In the pubertal subpopulation, a higher bone age-chronological age (BA-CA) ratio and a larger BA-CA difference were observed in IVF-conceived girls compared with controls (1.04 +/- 0.07 versus 1.02 +/- 0.08, P = 0.022; 0.54 +/- 0.82 versus 0.18 +/- 1.00 year, P = 0.021, respectively). Furthermore, dehydroepiandrosterone sulphate (DHEAS) and LH levels were significantly higher in IVF-conceived girls than in control subjects (2.5 versus 1.9 micromol/l, P = 0.017, and 1.5 versus 0.6 U/l, P = 0.031, respectively)., Conclusions: Bone age appeared to be advanced in pubertal IVF-conceived girls, but not in boys, compared with controls. Increased DHEAS and LH concentrations were found among IVF girls.

Objective: To evaluate growth and development of children born after IVF treatment., Design: Literature review., Conclusion(s): At present there is substantial evidence that children born after IVF are at increased risk for adverse perinatal outcome, congenital malformations, and rare epigenetic defects. It is still unclear whether observed health problems originate from the IVF procedure itself or the underlying subfertility problems of the parents. Current follow-up studies regarding postnatal growth and morbidity rates are scarce with conflicting results and other areas of long-term research in children born after IVF are still in its infancy. The importance of the worldwide continuing monitoring of children born after IVF to investigate potential long-term consequences including the development of cardiovascular diseases is therefore highlighted.

Context: Increasing evidence suggests that adverse conditions during early prenatal life are associated with cardiometabolic dysfunction in postnatal life. In vitro fertilization (IVF) conception may be an early prenatal life event with long-term health consequences., Objective: Our objective was to investigate several cardiometabolic measures in 8- to 18-yr-old IVF singletons and spontaneously conceived controls born from subfertile parents., Design and Setting: This follow-up study was conducted at the VU University Medical Center, Amsterdam, The Netherlands., Participants: Blood pressure was examined in 225 IVF-conceived children and 225 age- and gender-matched spontaneously conceived control children. Several indicators of insulin resistance were studied in a pubertal subpopulation (131 IVF children and 131 controls)., Main Outcome Measures: Blood pressure, fasting glucose, and fasting insulin were determined., Results: Systolic and diastolic blood pressure levels were higher in IVF children than controls (109 +/- 11 vs. 105 +/- 10 mm Hg, P < 0.001; and 61 +/- 7 vs. 59 +/- 7 mm Hg, P < 0.001, respectively). Children born after IVF were also more likely to be in the highest systolic and diastolic blood pressure quartiles (odds ratio = 2.1, 95% confidence interval 1.4, 3.3; odds ratio = 1.9, 95% confidence interval 1.2, 3.0, respectively). Furthermore, higher fasting glucose levels were observed in pubertal IVF children (5.0 +/- 0.4 vs. 4.8 +/- 0.4 mmol/liter in controls; P = 0.005). Blood pressure and fasting glucose differences could not be explained by current body size, birth weight, and other early life factors or by parental characteristics, including subfertility cause., Conclusions: These findings highlight the importance of continued cardiometabolic monitoring of IVF-conceived children and might contribute to current knowledge about periconceptional influences and their consequences in later life.

Background: Evaluation of relationships between assisted reproduction technologies (ART), fertility problems and disorders caused by disturbed genetic imprinting such as Angelman syndrome (AS) and Beckwith-Wiedemann syndrome (BWS)., Methods: A nation-wide questionnaire survey was performed regarding ART in families with a child with AS, BWS or Prader-Willi syndrome (PWS) including questions on fertility. Molecular data on the genetic disorder in affected children were gathered., Results: Of the 220 affected children in this study, 14 (6.4%) were born following any form of ART compared with 83 818 (2.1%) in the Dutch population. Of AS, PWS or BWS children 15 (6.8%) were born after a fertility problem (Time To Pregnancy > 12 months, no forms of ART) compared to 141,340 (3.5%) in the Dutch population. Maternal age in the individual syndromes was higher than in the Dutch population. Families with affected children were three times more likely to experience fertility problems than the general population. All three syndromes were also individually associated with increased fertility problems in the families., Conclusions: After correction for the increased fertility problems of the parents, there is no increased incidence of ART related birth of AS, PWS or BWS children. ART does not seem to have a direct effect on the increase of imprinted diseases.

Context: Increasing evidence suggests that adverse conditions during prenatal life are associated with the development of chronic diseases in adult life. It is still unclear whether in vitro fertilization (IVF) conception could affect the vulnerable developmental processes in humans occurring during early prenatal development with long-term perturbations of developmental pathways., Objective: Our objective was to examine body composition in 8- to 18-yr-old IVF singletons and spontaneously conceived controls born from subfertile parents., Design and Setting: This follow-up study was conducted at the VU University Medical Center in Amsterdam, The Netherlands., Participants: Participants included 233 IVF children (139 pubertal children) and 233 age- and gender-matched control children (143 pubertal children)., Main Outcome Measures: Body composition measures were assessed by anthropometry and dual-energy x-ray absorptiometry in the pubertal subpopulation., Results: IVF children had a significantly lower subscapular-triceps skinfold ratio and a significantly higher sum of peripheral skinfolds, peripheral body mass, and percentage of peripheral body fat as compared with controls. Although not reaching statistical significance, both dual-energy x-ray absorptiometry and skinfold measurements suggested that total body fat in IVF children is increased. Neither current and early risk factors nor parental factors, such as subfertility cause, could explain the differences in peripheral fat assessed by anthropometry between IVF children and controls. No differences in bone mineral composition between IVF children and controls were found., Conclusions: Our observations indicate that body fat composition in IVF children is disturbed. Follow-up of IVF children to monitor body fat pattern and potentially related health problems from adolescence into adulthood is of great importance.

Current research aims at reducing the number of sperm per insemination dose thereby making measurement of sperm concentration in raw semen and the production of uniform insemination doses much more crucial. The present study evaluated the determination of sperm concentration using FACSCount AF System (FACS), Improved Neubauer hemocytometer (HEMO), Corning 254 photometer (Photo C254), SpermVision CASA System (SpermVision), UltiMate CASA System (UltiMate) and NucleoCounter SP-100 (SP-100). The instruments were evaluated with respect to repeatability and to establishing the regression curve towards both HEMO and FACS. Repeatability for the instruments was 2.7, 7.1, 10.4, 8.1, 5.4 and 3.1% for FACS, HEMO, Photo C254, SpermVision, UltiMate and SP-100, respectively. Correlation between instruments was highest between FACS and SP-100. This was made possible due to the high repeatability for both instruments. The agreement between the instruments and HEMO as the gold standard was lower than expected as the largest difference in estimation of concentration was -25 to +50%. The largest percentage difference was observed for measurements of dilute semen. It was clear that percentage difference between instruments depended on sperm concentration. In comparison to the gold standard, agreement was highest between SpermVision and HEMO for dilute semen, but for concentrated semen, agreement was highest between SP-100 and HEMO. However, the agreement between HEMO and all other instruments was not as good as expected. The reason may lie within the presence of agglutinated sperm, preventing proper HEMO counts.

Background: The high iatrogenic multiple pregnancy rate associated with intrauterine insemination (IUI) in hyperstimulated cycles is becoming less acceptable. Therefore we investigated data from an earlier prospective trial with regard to the specific question of whether the application of mild hyperstimulation in IUI cycles could be an alternative strategy for obtaining acceptable pregnancy rates while preventing a high multiple pregnancy rate, compared with natural cycles for IUI., Methods: Pregnancy outcome of 310 natural and 334 mildly hyperstimulated cycles for IUI in 171 couples with unexplained or mild male factor subfertility was analysed on a patient level with random coefficient models., Results: Pregnancy rates were similar: 35 and 39.8% per couple in the natural and mildly hyperstimulated cycles respectively (P = 0.60). Multiple pregnancies, all twin pregnancies, were conceived significantly more frequently in the mild hyperstimulation group (27% of the pregnancies) than in the natural cycle group (4% of the pregnancies) (P = 0.01). All multiple pregnancies in the hyperstimulation group were conceived in multifollicular cycles. Multifollicular development was strongly associated with the application of mild hyperstimulation only (odds ratio 21.14, 95% confidence interval 8.15-54.79)., Conclusion: The application of a mild hyperstimulation protocol as an alternative to a standard hyperstimulation protocol for IUI does not result in higher pregnancy rates than IUI in the natural cycle, while at the same time multiple pregnancies cannot be avoided. Therefore, there is no place for the use of gonadotrophins in IUI treatment.

Background: With the occasional reports of unexpectedly poor ovarian response to controlled ovarian hyperstimulation (COH) for IVF in young normally cyclic women in mind, we studied age-related ovarian response to COH in a group of women who underwent standard IVF., Methods: Ovarian response to COH was defined as the number of follicles > or = 14 mm on the day of hCG administration. Ovarian response to COH was analysed by multiple regression analysis with woman's age and basal FSH concentration as explanatory variables in a prospective cohort of patients with idiopathic and mild male factor subfertility (n = 85), and additionally in a large retrospective cohort of women with unexplained, mild male and tubal subfertility (n = 1155), with age as explanatory variable., Results: Ovarian response to COH was associated significantly with age (P < 0.001) and basal FSH concentration (P = 0.002). However, in women with idiopathic or mild male subfertility, in both cohorts the relationship took the form of an inverted U-shape with both older and--surprisingly--young women having a reduced ovarian response (P < 0.001). Maximum ovarian response was around the age of 28 years. In women with tubal infertility, there was only a linear decline of ovarian response with age., Conclusion: It is hypothesized that diminished ovarian response to COH in IVF is the very first sign of ovarian ageing in young women diagnosed with idiopathic and mild male subfertility.

Capillary loaded chambers are frequently used for semen analysis. Poiseuille flow of specimen into these chambers causes migration of suspended particles or cells in a direction transverse to the flow, which results in their preferential accumulation in the Segre-Silberberg (SS) planes. This SS effect depends on the transverse velocity gradient in the laminar flow. For semen analysis in thin capillary-loaded slides, the SS effect can lead to erroneous estimation of sample sperm-cell concentration. To better understand chamber flow dynamics and SS effect significance, we assessed flow uniformity, inflow cell velocity, and results of concentration measurements under different flow conditions for latex bead and porcine and human sperm suspensions. Overall, a concentration peak was present at the meniscus, which continued through chamber loading. High-velocity SS preferred planes, which channeled particles toward the meniscus, were located at the fractional positions of beta = .27 and beta = .73, where beta is the distance from wall to plane normalized to the chamber depth. In computer-automated semen analysis, a standard 20-microm x 18-mm x 6-mm chamber is commonly used, and these studies supported our previously published fluid-flow theory for this type of chamber. Conversely, the SS effect does not appear to have time to develop in the 100-microm-depth hemacytometer, which is deeper than the standard slide, has lower transverse velocity gradient, and consequently does not exhibit concentration variation due to the SS effect. These findings provide further support that hemacytometry, when performed properly, remains the gold standard. Applicability of our findings to routine semen analyses was then tested in 2 studies performed with independent boar studs. These studies compared diluted boar semen concentrations estimated by standard hemacytometry and in capillary-loaded 20-microm slides, using a computer-automated semen-analysis system designed to compensate for the SS effect. Good numerical agreement for sperm concentration with a high degree of correlation (r(2) = .936) was found between the 2 techniques. These findings reaffirm the need to critically assess new technologies for accuracy, repeatability, and precision.

Accurate determination of sperm concentration in fluid suspension is a critical component in a semen analysis. Inaccurate estimations can lead to misinterpretation of the spermiogram and, in the case of livestock production, can lead to faulty insemination doses, which can adversely affect stud power, fertility, fecundity, and cost effectiveness of breeding programs. Capillary-loaded slides, like the hemacytometer, have been the standard for calibration of other concentration estimation modalities such as photometry, Coulter counter, flow cytometry, and computer-automated semen analysis (CASA). Single-use capillary-loaded slides, much smaller than the hemacytometer, are frequently used by many of the current CASA systems. As the use of CASA increases, more field reports are suggesting differences between CASA results and hemacytometry. In this article, we establish that these differences are, in large part, due to the Segre-Silberberg effect, which occurs during Poiseuille flow in high-gradient fluid flow in thin capillary-loaded slides. We develop the theory of this phenomenon and derive the scaling and significance of the effect. Finally, we graphically provide a means for predicting the necessary compensation factor when using capillary-loaded slides to determine sperm concentration.

Background: For some years, there has been a debate as to the place of the crossover trial in assisted reproduction technology (ART). We aimed to investigate whether crossover and parallel designs result in different estimates of treatment effects., Methods: We carried out computer simulation of cohorts of patients undergoing either intra-uterine insemination (IUI) or IVF under both parallel and crossover designs, under scenarios involving censoring and carryover effects. Results of the simulation were analysed using a maximum likelihood approach., Results: No relevant difference was found between the designs. The crossover design resulted in slightly more pregnancies than the parallel design. Carryover effects may slightly distort the estimates of treatment effects. Crossover and parallel designs will produce essentially the same statistical estimates of treatment effect and percentage of pregnancies. The crossover design is an acceptable design in infertility research provided the data are analysed correctly.

Background: Changes in concentration of seminal plasma alpha(1)-acid glycoprotein (AGP) have been studied in detail before. However, the source of high levels of AGP as well as the glycosylation of seminal plasma AGP has not been elucidated yet., Methods: The glycosylation of AGP was studied by crossed affinity immunoelectrophoresis using fucose-specific lectins and immunostaining. Glycan structure and monosaccharide analyses were performed by high pH anion exchange chromatography with pulsed amperometric detection. Fucosyltransferases were analyzed for activity and their substrate specificity was determined., Results: Two types of fucosylation were detected; Lewis(x) and Lewis(a). Lewis(a) groups were only present on AGP of individuals with a high concentration and were completely absent when the AGP concentration in seminal plasma was low. Lewis(a) expression coincides with a higher degree of branching of the glycans and a relative increased alpha4-fucosyltransferase activity. The molecular weight of all seminal plasma AGP was slightly higher than of blood plasma AGP (approx. 47 vs. 41-43 kDa)., Conclusions: The results indicate that AGP in seminal fluid most likely originates from the prostate and that it is either alpha3- or alpha4-fucosylated., (Copyright 2002 Wiley-Liss, Inc.)

Background: The aim was to assess whether the type of embryo transfer set used for embryo transfer affects the ongoing pregnancy rate in IVF., Methods: The TDT set was compared with the K-soft 5000 in a large, prospective, randomized study. Patients were randomized moments before transfer by drawing a consecutively numbered, sealed, opaque envelope indicating the catheter to be used., Results: 2059 embryo transfers in 1296 patients were analysed. The ongoing pregnancy rate was significantly higher in the K-soft group. If the first transfer of a patient (n = 1296) within this study period was analysed, the ongoing pregnancy rates were 27.1 versus 20.5% (P = 0.006). If the analysis is limited to patients that underwent their very first transfer ever (n = 607), the ongoing pregnancy rates were 30.3 versus 20.0% (P = 0.003) in favour of the K-soft., Conclusion: We conclude from these data that the type of embryo transfer set used for embryo transfer does affect the ongoing pregnancy rate and that the impact of the variable transfer catheter on the ongoing pregnancy rate increases when the a priori chance of pregnancy increases.

Background: Estimating the probability of pregnancy leading to delivery and the influence of clinical factors on that probability is of fundamental importance in the treatment counselling of infertile couples. A variety of statistical techniques have been used to analyse fertility data, many borrowed from survival analysis., Methods and Results: We propose an alternative method of analysis which is based on a discrete time Markov chain approach, with states 'pregnancy (leading to a delivery)', 'not pregnant', and 'censored' and in which the transition probabilities are dependent both on the clinical characteristics of the patient and the treatment given., Conclusions: We believe that the method of analysis presented here may be preferable to standard analyses in that it better reflects the clinical situation, it is a truly discrete time analysis applied to a discrete time situation, it explicitly models the censoring process (a process which in itself provides information of interest to the physician) and can be readily extended to a variety of clinical situations.

Objective: To predict the chance of total fertilization failure (TFF) before the day of ovum pickup with known semen and female variables., Design: A statistical model was constructed to predict TFF by retrospective analysis (2,366 couples) and subsequently tested on a new IVF population (917 couples)., Setting: Academic tertiary referral center., Patient(s): Three thousand three hundred eighty-three couples who underwent an IVF-ET treatment., Intervention(s): None., Main Outcome Measure(s): The ability to predict the probability of TFF in IVF., Result(s): Two variables-postwash total progressively motile sperm cell count (postwash TPMC) and number of follicles-were found to be significant. Taking a probability of 25% as an acceptable risk of TFF, we calculated that a postwash TPMC of <1.1 x 10(6) cells results in a risk of TFF of >25%. Low responders (<4 follicles) needed a postwash TPMC of >2.2 x 10(6) cells to reduce the risk of TFF to <25%. High responders (>15 follicles) needed only 0.35 x 10(6) postwash progressively motile spermatozoa., Conclusion(s): When postwash TPMC and number of follicles are known and an unacceptable TFF outcome is expected, one can propose an ICSI procedure a few days before the day of ovum pickup.

Assisted reproduction is used to resolve infertility problems in human and in breeding programs to generate livestock. Except for gestation length and birth weight, perinatal outcome of children conceived by In Vitro Fertilization is similar to that of spontaneously conceived children. However, large offspring syndrome observed after In Vitro Production in livestock is quite alarming. The distinct parts of assisted reproduction (oocyte maturation, fertilization and culture) have been found to contribute to abnormal fetal growth and development. Genomic imprinting is suggested to be involved in the induction of the aberrant phenotypes observed after assisted reproduction. Furthermore, current knowledge on postnatal health of offspring conceived by assisted reproduction and speculations on potential longterm effects of In Vitro Fertilization will be described.

Background: The accuracy by which a number of newly described semen variables can predict either total fertilization failure (TFF) or pregnancy outcome in IVF, has not previously been investigated. The study aim was, therefore, to determine prospectively the predictive value of these variables., Methods: The semen variables investigated were the post-wash total progressively motile sperm cell count (TPMC(post-wash)), the acrosome index (AI), 'cytoplasmic residues' and normal sperm morphology, evaluated according to the strict criteria ('strict criteria'), as well as the fast and slow total radical trapping antioxidant potential ('fast TRAP' and 'slow TRAP' respectively)., Results: The study group (n = 87) showed a mean (+/- SD) number of 10.2 +/- SD retrieved oocytes, 12.6% TFF, a mean fertilization rate of 59.7% and a pregnancy rate of 19.5% (17/87). TFF was significantly predicted by TPMC(post-wash), 'strict criteria', AI and 'cytoplasmic residues' (all P < 0.05). The outcome after embryo transfer was significantly predicted by AI and 'fast TRAP'. Semen samples with an AI <5% and a 'fast TRAP' <1.14 mmol/l in particular did not result in any pregnancies after IVF-embryo transfer., Conclusions: Of all the measured and calculated semen variables, TPMC(post-wash) was the best predictor of TFF, whilst AI and 'fast TRAP' were the best predictors of pregnancy after IVF.

Semen samples of 24 patients were analysed. Volumes were measured and the numbers of progressively motile (PMS), motile (MS) and nonmotile spermatozoa (NMS) were determined. These 24 samples appeared to show a large variation in motility percentages and numbers. Spermatozoa of these semen samples were isolated from the seminal plasma and exposed to induced radical oxygen stress imposed by iron/ascorbate. Lipid peroxidation (LPO) was quantified as thiobarbituric acid reactive material. The contributions of PMS, MS and NMS were also estimated. It was found that the PMS did not contribute to the formation of lipid peroxides. The cellular radical defence system of PMS may offer them adequate protection against the harsh conditions of radical oxygen stress. Stepwise regression analyses showed that only the population of NMS contributed significantly to the explanation of the variance in LPO production (R2 = 0.56, P < 0.001). Pre-existing membrane lipid peroxides were not detected in spermatozoa. It is therefore suggested that LPO takes place only after radical oxygen stress has exhausted the cellular defence system. LPO is not the initial, but one of the later, events leading to the death of spermatozoa. It is concluded that the population of progressively motile spermatozoa in semen samples does not contribute to the production of thiobarbituric acid reactive substances as induced by in vitro radical oxygen stress.

Total radical-trapping antioxidant potential (TRAP) measurements of human seminal plasma (N = 25) were performed by using a post-addition assay based on trapping 2,2' Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radicals. This method enables the antioxidant capacity of human seminal plasma and its constituents to be quantified. The standard procedure consisted of determination of the Trolox equivalent antioxidant capacity (TEAC) after incubating the test sample in the ABTS radical solution for 10 seconds (fast TRAP) and 300 s (total TRAP). Interestingly, seminal plasma showed a fast TRAP and a high slow TRAP (Total TRAP - Fast TRAP). The final total TRAP of seminal plasma is about 10 times higher than that of blood plasma. Various components of seminal plasma contribute to its fast TRAP; 37% can be attributed to vitamin C, uric acid, and tyrosine; proteins and polyphenolic compounds contribute a further 57%. In contrast, the slow TRAP was attributed to vitamin C (1%), uric acid (2%), and tyrosine (15%) and to proteins and polyphenolic compounds (33%). It was not possible to account for the remaining 49%. Neither known putative antioxidants, such as spermine, pyruvate, and taurine, nor other seminal compounds, such as carnitine, sialic acid, fructose, spermidine, glycerophosphorylcholine, and hyaluronic acid, contributed to any significant radical-trapping activity at a standard concentration of 1 mM. Of the amino acids, only tyrosine possessed a slow TRAP, and it is present at a high concentration in seminal plasma. Glutathione and hypotaurine show high fast and slow TRAPs, respectively. However, because of their low concentration in seminal plasma, their contribution to the TRAP is negligible. In conclusion, seminal plasma possesses a high antioxidant buffer capacity that protects spermatozoa from oxidative stress. Moreover, these findings suggest that the fast and slow TRAPs may have an important role as infertility markers and treatment targets in future antioxidant therapies.

Gonadotrophin-releasing hormone agonists (GnRHa) are routinely used in IVF programmes to prevent an unwanted LH surge and consequent ovulation. Despite its widespread use in IVF, a convincing dose recommendation for GnRHa in IVF does not exist. In our opinion, the lowest possible dose of GnRHa should be used. Thus, we performed a prospective, randomized, double-blind, placebo-controlled study to determine the minimal daily dose of triptorelin acetate needed to suppress a premature LH surge during IVF treatment in a long protocol. A total of 240 women (60 in each group) was randomized to either placebo or to one of three doses of triptorelin, i.e. 15, 50 or 100 microg daily. Ovarian stimulation was performed with two or three ampoules of FSH daily. A premature LH surge occurred in 23% of placebo-treated patients, but in none of the triptorelin acetate-treated patients. There were significantly more oocytes and embryos in the 50 and 100 microg triptorelin groups. There was no dose relationship in rates of either implantation, pregnancy, ongoing pregnancy, live birth or baby take-home. In this study we showed that daily administration of 15 microg triptorelin is sufficient to prevent a premature LH surge, and that 50 microg is equivalent to 100 microg in terms of IVF results.

In singleton pregnancies after IVF a high rate of preterm deliveries and an increased rate of small-for-gestational age (SGA) children in comparison to the general parturient population have been reported. However, due to differences between IVF mothers and their peers who conceived naturally, careful selection of the control group is necessary to assess whether IVF pregnancies really carry increased risks of adverse outcome. In our study 307 IVF pregnancies were compared with 307 control pregnancies after elaborate matching for an extensive number of maternal characteristics, as well as for the hospital that provided the obstetric care. Four Dutch university hospitals contributed to the study. In cases with spontaneous onset of labour, gestational age at delivery was 3 days shorter in the IVF group (275 versus 278 days, P = 0.05). The proportion of SGA was higher in the IVF group (16.2 versus 7.9%, P < 0.001). The combination of these two results denotes a distinct difference between IVF and control pregnancies. Placental weight was comparable in both groups.

A novel post-addition method, based on the trapping of ABTS-radicals, is applied for studying the total antioxidant capacity of seminal plasma. A remarkable profile is observed, in which seminal plasma quenches radicals in a continuous, relatively slow fashion. Five putative antioxidants present in seminal plasma were studied using the same assay. Some of the compounds such as ascorbic acid, alpha-tocopherol and uric acid exert immediate, fast radical trapping, whereas hypotaurine and tyrosine give rise to the same slow radical trapping curve as seminal plasma. Due to this slow, continuous radical trapping, quantification of the total antioxidant capacity (expressed as trolox equivalent antioxidant capacity, TEAC) strongly depends on the chosen time point after onset of radical trapping. When determined during the slow antioxidant trapping phase, tyrosine has a powerful antioxidant capacity, which in combination with its relatively high plasma concentration makes it an important contributor to the total antioxidant capacity of seminal plasma.

In singleton pregnancies after in-vitro fertilization (IVF), increased rates of obstetric and perinatal complications have been reported. Studies that compared IVF twin pregnancies with spontaneously conceived twins have yielded conflicting results. We compared 96 IVF twin pregnancies to 96 controls after elaborate matching. The design of our study precluded matching by zygosity. The monozygosity rate was higher in the control group and this implies that beforehand the risk for a less favourable outcome in the control group was higher than in the IVF group. However, the average birthweight of the IVF children was less than that of children in the control group (P = 0.04). This was not due to more intrauterine growth retardation in the IVF group. The mean gestational age at birth was 5 days shorter in IVF than control pregnancies, and although this difference was not significant it might explain the lower birthweight in the IVF group. The discordance rate in the IVF group was significantly increased. We found no difference in perinatal mortality and morbidity. We conclude that this study provides further evidence for a different outcome of IVF twin pregnancies in comparison with spontaneously conceived twin pregnancies.

The results of in-vitro fertilization in natural cycles (NIVF) in women with tubal infertility at our department are presented. The study had a prospective design. We needed 75 cycles in 50 patients to obtain one oocyte from each patient. Successful oocyte recovery rate was 67% per started cycle and 82% per oocyte retrieval. Thirty-five embryos were transferred and resulted in four ongoing pregnancies (5.3% per cycle, 6.5% per oocyte retrieval, 11.4% per embryo transfer and 11.4% per embryo). Six patients who participated in the study made a second attempt at NIVF. Five of them conceived of which four were ongoing. Cumulative ongoing pregnancy rates are 9. 8% per cycle, 11.9% per oocyte retrieval, 19.5% per embryo transfer and 19.5% per embryo. We conclude that NIVF is an easy, cheap and realistic method to obtain a pregnancy for patients with tubal infertility.

Background: Couples affected by idiopathic subfertility or male subfertility have an estimated spontaneous conception rate of about 2% per cycle. Although various infertility treatments are available, counselling of a couple in their choice of treatment is difficult because of the lack of consistent data from good-quality comparative studies. We compared the results of treatment with intrauterine insemination (IUI) with those of in-vitro fertilisation (IVF), and did a cost-effectiveness analysis., Methods: In a prospective, randomised, parallel trial, 258 couples with idiopathic subfertility or male subfertility were treated for a maximum of six cycles of either IUI in the spontaneous cycle (IUI alone), IUI after mild ovarian hyperstimulation, or IVF. The primary endpoint was a pregnancy resulting in at least one livebirth after treatment. Cost-effectiveness based on real costs was studied by Markov chain analysis., Findings: 86 couples were assigned IUI alone, 85 IUI plus ovarian hyperstimulation, and 87 IVF. Ten couples dropped out before treatment began. Although the pregnancy rate per cycle was higher in the IVF group than in the IUI groups (12.2% vs 7.4% and 8.7%, respectively; p=0.09), the cumulative pregnancy rate for IVF was not significantly better than that for IUI. Couples in the IVF group were more likely than those in the IUI groups to give up treatment before their maximum of six attempts (37 [42%] drop-outs vs 13 [15%] and 14 [16%], respectively; p<0.01). The woman's age was the only factor that influenced a couple's chance of success. IUI was a more cost-effective treatment than IVF (costs per pregnancy resulting in at least one livebirth 8423-10661 Dutch guilders [US$4511-5710] for IUI vs 27409 Dutch guilders [US$14679] for IVF)., Interpretation: Couples with idiopathic or male subfertility should be counselled that IUI offers the same likelihood of successful pregnancy as IVF, and is a more cost-effective approach. IUI in the spontaneous cycle carries fewer health risks than does IUI after mild hormonal stimulation and is therefore the first-choice treatment.

The aim of this study was to find the minimal effective daily s.c. dose of the gonadotrophin-releasing hormone (GnRH) agonist, triptorelin acetate, that suppresses the GnRH-induced release of luteinizing hormone (LH) at time of human chorionic gonadotrophin (HCG) injection and thereby prevents spontaneous LH surges during in-vitro fertilization (IVF) stimulation cycles. Therefore, a double-blind, prospective and randomized titration study was performed. A total of 48 IVF patients were divided into four groups of 12 patients. Each group received a different dose of triptorelin acetate, namely 5, 15, 50 or 100 microg s.c. daily. Standard ovarian stimulation was carried out using urinary follicle stimulating hormone (FSH) preparations. A 500 microg GnRH test was performed 90 min before the HCG injection in order to measure the degree of pituitary desensitization. Spontaneous LH surges were not detected in any of the groups, although three patients in the 5 microg group had ovulated at the time of ovum retrieval. The pituitary LH response to the GnRH test at time of HCG, expressed as area under the curve (AUC), appeared to be dose-dependent. Thus, a daily s.c. dose of 100 microg triptorelin acetate appears to be too high, since adequate desensitization of the pituitary (i.e. no spontaneous LH surge) can be achieved with doses as low as 15 and 50 microg.

In the spontaneous menstrual cycle, the mid-cycle gonadotrophin surge causes maturation of the cumulus-oocyte complex, mucification of cumulus cells and expansion of the cumulus oophorus, resumption of meiosis and maturation of the cytoplasm of the oocyte. Whether this is an effect purely of luteinizing hormone (LH) or whether follicle stimulating hormone (FSH) also plays a role is unknown. The effect of an artificially induced FSH surge at the time of human chorionic gonadotrophin (HCG) injection on maturation of the cumulus-oocyte complex was investigated in a prospective randomized double-blind trial. Twelve patients underwent controlled ovarian hyperstimulation [long gonadotrophin-releasing hormone agonist (GnRHa)/human menopausal gonadotrophin (HMG) protocol] for in-vitro fertilization (IVF) treatment. At the time of HCG administration, six patients received a bolus injection of FSH (450 IU i.m.); the other six patients received a placebo. The peak plasma concentrations of FSH of the experimental group were compared with the peak values of FSH obtained at the mid-cycle gonadotrophin surge of the natural cycle of a group of 12 volunteers to validate the bolus injection of FSH. Maturation of the cumulus-oocyte complex was quantified by measuring the expansion of the cumulus, by the fertilization rate and the implantation rate. The quality of the embryos was scored according the average morphology score. The bolus injection of FSH mimicked the mid-cycle gonadotrophin surge. The mean peak value of FSH (12.9 IU/l) in the experimental group was fully comparable with the mean peak value of FSH (10.0 IU/l) of the mid-cycle gonadotrophin surge in the natural cycle. No effect of a bolus injection of FSH on the maturation of the cumulus-oocyte complex or any other outcome variable was found. It is not advantageous to combine the final HCG injection with a bolus injection of FSH in GnRHa/HMG stimulated cycles.

Objective: To determine the relationships between sperm acrosin activity, sperm morphology evaluated according to strict criteria, visually observed acrosomal morphology, and IVF rates., Design: Prospective analytic study. Acrosin activity was determined on all semen samples together with a standard semen analysis. Emphasis was placed on sperm morphology and especially a novel criterion viz acrosome morphology (acrosome index) as recorded with bright field microscopy., Setting: University-based tertiary care center., Patients: Thirty-three couples undergoing IVF or GIFT with two or more metaphase II ova inseminated in vitro., Main Outcome Measure: In vitro fertilization rates of inseminated ova., Results: Strong correlations were found between acrosome index, normal sperm morphology, and IVF rates. An acrosome index cutoff value could be established at > 10% normal acrosomes for IVF rates of > or = 50% (sensitivity and specificity = 100%) and an acrosin activity cutoff value at > 18 microIU/10(6) sperm. A multiple linear regression analysis showed that the acrosome index and acrosin activity added a significant contribution to the explanation of the variation in the fertilization rates., Conclusions: A strong positive correlation was found between acrosome index and IVF rates. Although the numbers of the study are small, the results indicate that the acrosome index possibly may be regarded as an additional tool in the prediction of IVF outcome and especially may be of value in the group of men with severe teratozoospermia, i.e., < or = 4% morphologically normal spermatozoa.

Because the process of conception is affected by many variables, a multiple logistic regression analysis was performed to assess (i) the impact and relative weight of both patient and embryo variables and (ii) their possible effects on the probability of a vital pregnancy after embryo transfer. A statistical model was constructed predicting the probability of pregnancy after embryo transfer. The variables that contributed significantly to the predictive value of the model were the age of the patient, the cause of infertility, the number of embryos transferred and the average morphology score of the transferred embryos. Embryo variables appeared to have a significant but modest value in predicting the probability of pregnancy after embryo transfer. Other variables, such as the thickness of the endometrium, were found to have no prognostic value. Moreover, we found that their effect could be explained by the variables already included in the model.

A urinary luteinizing hormone (LH) test (LH Color, Organon, Oss, The Netherlands), was used to time intrauterine insemination in 177 cycles. Morning and evening urine samples were tested. In 58 women (33%) the test was positive in the morning urine sample. Fifteen of these patients were inseminated 8-10 h thereafter and one patient (6.7%) conceived. The remaining 43 women were inseminated the following day, 25-31 h after LH detection, and seven pregnancies (16.3%) ensued. In 119 cycles showing a positive urinary test in the evening sample, insemination was performed the next day, between 17 and 23 h after the LH surge, and 18 patients (15.1%) became pregnant. Statistical analysis showed no significant differences in pregnancy rates between the three different schedules, or in the time of insemination between conceptional and non-conceptional cycles within each group. Most ovulations occurred between 16 and 28 h after the positive test was observed. These findings suggest that while the lifespan of the gametes allows a relatively long period for fertilization, from 8 to 31 h after urinary LH surge detection, better results may be expected when inseminating about 24 (+/- 6) h after the positive test.

In this study, intrauterine insemination (IUI) was timed either after the detection of a urinary luteinizing hormone (LH) surge at home by the patient (group A), or following a positive LH test as interpreted by the gynaecologist (group B). Afterwards, samples tested by the patient were retested by the gynaecologist and vice versa. The gynaecologist also rechecked his own findings and the results were correlated with ultrasound data and charts of basal body temperature. Forty-seven cycles were evaluated. The patient's and the gynaecologist's readings agreed (+/- 12 h) in 42 of the cases (89%), and in five cycles (11%) a difference greater than 24 h was found. The intra-observer variation in the gynaecologist's results was +/- 12 h in four cycles (8.5%). These findings suggest that the LH test can be used as a reliable home device for the prediction of pending ovulation and timing of IUI.

Forty-eight patients in a programme of intrauterine insemination (IUI) were randomized in a cross-over study. All were stimulated with clomiphene citrate (CC) and inseminated either after follicular rupture induced by human chorionic gonadotrophin (HCG) or after a spontaneous urinary luteinizing hormone (LH) surge. The HCG was administered when follicles of 18-22 mm in diameter were observed on ultrasound and IUI was performed 37-40 h thereafter. The monitoring of a urinary LH peak was carried out using a rapid urinary LH test. IUI took place approximately 22 h after detection of the LH surge. Overall, the pregnancy rates were 9.3% (4/43) after HCG induced ovulation and 20.5% (9/44) after spontaneous ovulation (P = 0.12). Analysis of mid-cycle events showed that following sonographic criteria, the HCG injection was performed significantly earlier in the cycle compared with the spontaneous LH surge. In addition, the mean diameter of the preovulatory follicles was significantly smaller and insemination was substantially earlier in the HCG induced cycles. These findings suggest that a beneficial effect arises from allowing the natural process of final follicular maturation to occur.

Forty-eight patients with male (n = 16) or idiopathic (n = 32) infertility were stimulated with human menopausal gonadotropin. Intrauterine insemination (IUI) or natural intercourse were performed after either human chorionic gonadotropin (hCG)-induced or spontaneous, urinary luteinizing hormone (LH) surge-monitored ovulation. A total of 148 cycles were analyzed. In 40 cycles treated with hCG-induced ovulation and IUI, 3 (7.5%) patients conceived, whereas 37 women accomplished natural intercourse after hCG-induced ovulation and 2 (5.5%) became pregnant. When inseminated after a spontaneous LH surge, 3 (8.8%) of 34 patients achieved a pregnancy; no conception occurred in 37 spontaneously ovulatory cycles combined with timed intercourse. Pregnancy rates did not substantially differ between the treatment modalities or between mono-ovulatory and polyovulatory cycles. The cycle characteristics between spontaneous ovulatory and hCG-induced cycles significantly did differ.

In the present prospective study we compared, in terms of pregnancy rates, the differences between intrauterine insemination (IUI) of in vitro capacitated husband's semen and timed natural intercourse in spontaneous or clomiphene citrate (CC) stimulated cycles. A rapid urinary luteinizing hormone peak detection test was used for timing of ovulation. Forty patients suffering from longstanding infertility of male (n = 17), cervical (n = 2), and idiopathic (n = 21) origin were randomly assigned into four distinct treatment modalities during 4 consecutive cycles. A total of 132 cycles were analyzed. In 35 cycles treated with CC plus IUI, five conceptions were achieved, whereas three pregnancies occurred in 32 inseminated spontaneous cycles. Only 1 patient conceived after timed intercourse in 31 CC stimulated cycles, and no pregnancy resulted from 34 spontaneous cycles combined with timed intercourse. There was a statistically significant higher conception rate in cycles in which IUI was performed, whereas the use of CC does not seem to improve the pregnancy rate. Analysis of results for other modifying factors did not substantially affect the relative risk (odds ratio) of pregnancy.

Osteoblastic and osteoclastic activity was studied in avian medullary bone in vivo. During the active period of eggshell calcification, medullary bone active resorption surface increased ninefold. This correlated with a sevenfold increase in the percentage of active osteoclasts. Osteoblast activity is also increased during the active period, as demonstrated by a twofold increase in the active osteoblastic surface. These findings and our observation that the medullary bone volume remains the same (+/- 13%) whether the eggshell is being formed (active period) or not (inactive period) led to the conclusion that the activities of osteoblasts and osteoclasts rapidly return to balance.

PTH bioactivity during the egg-laying cycle of the chicken has been measured by the cytochemical bioassay for PTH. During the period of eggshell calcification, PTH bioactivity is elevated. After completion of the shell the plasma PTH falls to a low level but is slightly raised again 2 h after ovulation. These results indicate that changes in bioactive PTH play an important role in the calcium metabolism of the chicken during this physiological calcium stress.

Glass wool filtration and two-layer, discontinuous Percoll (Pharmacia, Uppsala, Sweden) density gradient centrifugation resulted in an average recovery of 50% to 70% of the progressively motile and about 50% of the hypoosmotic swelling (HOS)-positive spermatozoa. Glass wool filtration tended to be more successful than Percoll centrifugation when the ejaculates were asthenozoospermic or produced a suspect/abnormal HOS test. After selection, the acrosin activity increased approximately two- to threefold, but no significant improvement in the percentage of normal sperm forms occurred. Experiments with mixtures of untreated and frozen-thawed ejaculates confirmed that glass wool filtration is more effective in removing nonmotile and HOS-negative spermatozoa than the two-layer Percoll centrifugation technique when the percentage of these types of spermatozoa in the ejaculate is high. The simplicity of these techniques and the good recovery of apparently viable spermatozoa makes these methods more desirable than other, more complicated techniques or procedures that yield a lower recovery of motile spermatozoa.

LDH is localized along various intracytoplasmatic membranes of osteoclasts. Macrophages show membrane-bound LDH only after phagocytosis of calcium hydroxylapatite, the main mineral constituent of bone. The localization of LDH in these macrophages is almost the same as in osteoclasts. The significance of this finding, and its possible implication in the process of bone resorption and the origin of osteoclasts are discussed.

Chick epiphyseal chondrocytes are isolated from 15 day old embryos and subsequently grown as small high density cultures (aggregates consisting of 2 X 10(5) cells), which can be handled individually. Histological evaluation showed a considerable increase in the dimensions of the nucleus and the cells 2-6 hours after initiating the growth of the chondrocytes by adding 10% serum to the culture medium. After 24 hours of culture the proportion of the matrix in the aggregate was increased from 0% to about 45%. In a series of experiments these aggregates were exposed to intermittent compressive force (ICF) with a peak value of 130 mbar above ambient and a frequency of 0.3 Hz. The nuclear and cell-dimensions of ICF-exposed chondrocytes were significantly larger than under control conditions when measured after 24 and 48 hours. Concomitantly, chondrocytes in ICF-exposed aggregates synthesized and deposited significantly more matrix in the aggregates as compared to controls. This model can be used to further study the direct effect of mechanical force on the synthesis and deposition of matrix components by cartilage cells in vitro.

In 29 domestic hens, four blood samples were collected during the period of egg formation, at time points representing different metabolic stages. Plasma concentrations of total calcium, total phosphorus, inorganic phosphorus and total protein were measured, and the effect of adding oyster shell particles to a standard laying diet was investigated. Analyses of covariance were made and correlations between the plasma characteristics were calculated. The following were significantly correlated: total and inorganic phosphorus, calcium and total phosphorus, and calcium and protein. Each of the four characteristics showed a significant decrease during egg formation. Inorganic phosphorus reached a peak 15 h after ovulation, probably related to medullary bone resorption. Plasma total phosphorus differed significantly between chickens. Addition of oyster shell to the diet did not affect the concentration or the pattern of the plasma characteristics during egg formation and did not reduce calcium mobilisation from medullary bone during shell formation.

In order to investigate whether the immune system is involved in the degradation of hydroxylapatite as implant material the adsorption behavior of several serum proteins to hydroxylapatite was studied and compared with the adsorption to other calcium phosphate compounds (Ca3(PO4)2, CaHPO4), calcium carbonate, aluminium oxide, and zinc oxide. It was found that complement factors, C4 and C3, and alpha 2-HS-glycoprotein adsorbed to the calcium phosphate compounds, but not to the other materials. IgG and albumin were adsorbed to all investigated compounds. These results suggest that the complement factors are directly adsorbed on the surface of the calcium phosphate compounds. Therefore, the activation of complement, if any, proceeds by the classical pathway. The binding of complement factors to the calcium phosphate salts investigated points to a more specific involvement of the immunosystem in the degradation process of calcium phosphate implants.

These investigations were intended to determine whether local and systemic skeletal effectors--3'5'-cyclic adenosine monophosphate (cAMP), prostaglandin E2 (PGE2), parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25(OH)2D), calcitonin, and NaF--could regulate 3[H]-thymidine incorporation (i.e., into DNA) in serum-free, monolayer cultures of embryonic chick calvarial cells, and/or modulate the activity of embryonic chick bone extracts to increase 3[H]-thymidine incorporation. In the absence of added bone extract, we found that calcitonin (0.1 U/ml), NaF (100 microM) and low-dose PTH (0.1 nM) stimulated 3[H]-thymidine incorporation, P less than .05 for each; isobutylmethylxanthine (IBMX--1 mM), 1,25OHD (10 nM), and high-dose PTH (10 nM) decreased 3[H]-thymidine incorporation; and PGE2 (1 microM) had no effect. The stimulatory actions of calcitonin, fluoride, and low-dose PTH were inductive, and the inhibitory actions of IBMX and 1,25(OH)2D were acute. PTH had complex time-dependent actions on 3[H]-thymidine incorporation, being inhibitory after 4-8 hours of exposure and stimulatory after 20-24 hours (P less than .001 for each). The effects of calcitonin, fluoride, and low-dose PTH to increase 3[H]-thymidine incorporation were greater in calvarial cell cultures enriched for undifferentiated osteoprogenitor cells than in cultures enriched for differentiated osteoblastlike cells. PTH inhibited 3[H]-thymidine incorporation in the latter (i.e., osteoblastlike) cultures (P less than .005). The inhibitory actions of IBMX and 1,25(OH)2D were independent of cell differentiation. Additional studies further revealed that these local and systemic skeletal effectors could also modulate the activity of embryonic chick bone extracts to increase 3[H]-thymidine incorporation in calvarial cell cultures. We found that calcitonin, fluoride, and low-dose PTH enhanced the effect of the extracts to increase 3[H]-thymidine incorporation (P less than .001 for each). These activations were noncompetitive, indicating (1) mechanistic differences between the stimulatory actions of the effectors and the chick bone extract (i.e., different rate-limiting steps for the effects of each on 3[H]-thymidine incorporation); and (2) that neither calcitonin, fluoride, nor 0.1 nM PTH altered the apparent affinity of the cells for stimulatory activity(s) in the extract. High-dose PTH was a noncompetitive inhibitor with respect to bone extract activity, indicating that the effect of 10 nM PTH to decrease 3[H]-thymidine incorporation was mechanistically distinct from the effect of the bone extract to increase 3[H]-thymidine incorporation.(ABSTRACT TRUNCATED AT 400 WORDS)