Effect of docosahexaenoic acid-enriched fish oil supplementation in pregnant women with Type 2 diabetes on membrane fatty acids and fetal body composition--double-blinded randomized placebo-controlled trial.

AIMS: To test if docosahexaenoic acid-enriched fish oil supplementation rectifies red cell membrane lipid anomaly in pregnant women with Type 2 diabetes and their neonates, and alters fetal body composition. METHODS: Women with Type 2 diabetes (n = 88; 41 fish oil, 47 placebo) and healthy women (n = 85; 45 fish oil, 40 placebo) were supplemented from the first trimester until delivery. Blood fatty acid composition, fetal biometric and neonatal anthropometric measurements were assessed. RESULTS: A total of 117 women completed the trial. The women with Type 2 diabetes who took fish oil compared with those who received placebo had higher percentage of docosahexaenoic acid in red cell phosphatidylethanolamine in the third trimester (12.0% vs. 8.9%, P = 0.000) and at delivery (10.7% vs. 7.4%, P = 0.001). Similarly, the neonates of the women with Type 2 diabetes supplemented with the fish oil had increased docosahexaenoic acid in the red cell phosphatidylethanolamine (9.2% vs. 7.7%, P = 0.027) and plasma phosphatidylcholine (6.1% vs. 4.7%, P = 0.020). Docosahexaenoic acid-rich fish oil had no effect on the body composition of the fetus and neonates of the women with Type 2 diabetes. CONCLUSIONS: A daily dose of 600 mg of docosahexaenoic acid was effective in ameliorating red cell membrane docosahexaenoic acid anomaly in pregnant women with Type 2 diabetes and neonates, and in preventing the decline of maternal docosahexaenoic acid during pregnancy. We suggest that the provision of docosahexaenoic acid supplement should be integrated in the antenatal care of pregnant women with Type 2 diabetes.

Anti-Mullerian hormone is increased in follicular fluid from unstimulated ovaries in women with polycystic ovary syndrome.

BACKGROUND: Anti-Mullerian hormone (AMH) may have a role in disordered folliculogenesis in polycystic ovary syndrome (PCOS). Though there have been several investigations into circulating AMH levels in patients with PCOS, no previous studies have compared AMH concentrations in the follicular fluid of unstimulated ovaries in women with PCOS with that of normally ovulating women. METHODS: Follicular fluid was aspirated from 4-8-mm follicles of unstimulated ovaries during routine laparoscopy or laparotomy from women with anovulatory PCOS (n = 11) and those with regular ovulatory cycles (n = 8). Follicular AMH was compared in the two groups. Serum samples were analysed for AMH and endocrine profile. RESULTS: Follicular fluid AMH levels were significantly higher (P < 0.0001) in women with anovulatory PCOS (median: 466.2 ng/ml) compared with normal-ovulatory controls (median: 78.0 ng/ml). Mean follicular fluid AMH levels in PCOS patients were 60 times higher than in the serum. Moreover, there was a significant correlation between the follicular fluid and serum concentrations of AMH in the PCOS group (r = 0.86; P = 0.007) but not in controls. CONCLUSIONS: Highly elevated AMH in follicular fluid from PCOS patients in contrast to age-matched normal controls suggests that increased circulating concentrations of AMH are partly due to the increased production of AMH by individual follicles and not simply attributable to the increased number of small antral follicles. This suggests an intrinsic abnormality in the ovarian follicles themselves in PCOS, which could contribute to disordered folliculogenesis.

Hormones and the auditory system: a review of physiology and pathophysiology.

This review explores the potential role of hormones in modulating the auditory function. The review describes four groups of hormones (the hormones of the circadian cycle, reproduction, stress response and the fluid and electrolyte balance), their physiological variations, interactions, as well as the physiological basis for their effect on the auditory system. Possible contribution of hormones to pathophysiology of auditory dysfunctions, including hyperacusis, tinnitus, Menière's disease and pre-menstrual auditory dysfunction, has also been discussed.

Granulosa cell survival and proliferation are altered in polycystic ovary syndrome.

CONTEXT: Polycystic ovary syndrome (PCOS) represents the most common endocrine abnormality in women of reproductive age. The cause of PCOS remains largely unknown, but studies suggest an intrinsic ovarian abnormality. OBJECTIVE: The objective of the study was to test our hypothesis that differences in granulosa cell proliferation and apoptosis may underlie abnormalities that affect follicular development. DESIGN: Granulosa cells were prepared from follicular fluid aspirated from 4- to 8-mm follicles of unstimulated ovaries during routine laparoscopy or laparotomy from women with anovulatory PCOS and those with regular ovulatory cycles. SETTING: The study was conducted at a university hospital. PATIENTS: Fourteen women with anovulatory PCOS and nine women with regular ovulatory cycles participated in the study. MAIN OUTCOME MEASURES: Immunocytochemistry on granulosa cells to investigate apoptotic and proliferation rates, together with real-time RT-PCR to analyze gene expression profiles of apoptotic regulators, was measured. RESULTS: Significantly lower apoptotic rates were found in granulosa cells from patients with PCOS, compared with women with regular ovulatory cycles (P=0.004). Lower apoptotic rates were associated with decreased levels of the apoptotic effector caspase-3 (P=0.001) and increased levels of the anti-apoptotic survival factor cellular inhibitor of apoptosis proteins-2 in the PCOS group that were coupled to higher proliferation rates (P=0.032). Gene expression profiling confirmed the immunocytochemical findings. CONCLUSIONS: Our findings indicate that there are significant differences in the rate of cell death and proliferation in granulosa cell populations in PCOS patients. These are associated with decreased expression of apoptotic effectors and increased expression of a cell survival factor. These results provide new insights that may be useful in developing specific therapeutic intervention strategies in PCOS.

The effect of interleukin-6 on ciliary beat frequency in the human fallopian tube.

OBJECTIVE: To investigate the effect of interleukin-6 (IL-6) on ciliary activity within the human fallopian tube in vitro. DESIGN: An experimental laboratory-based study. SETTING: University teaching hospital. PATIENT(S): Fallopian tube specimens were obtained from eight women undergoing hysterectomy for fibroid uterus. MAIN OUTCOME MEASURE(S): Ciliary beat frequency (CBF) in the human fallopian tube. INTERVENTION(S): After baseline CBF measurements, increasing concentrations of IL-6 (10 pg/mL, 100 pg/mL, and 1000 pg/mL) were applied to fallopian tube mucosa explants and CBF measurements repeated. An anti-IL-6 monoclonal antibody was added and CBF measured once more. Negative and antibody-only control samples were used. RESULT(S): The CBF remained unchanged with the addition of IL-6 at concentrations of 10 pg/mL and 100 pg/mL but IL-6 at 1000 pg/mL significantly decreased CBF, an effect abolished by the addition of the specific antibody. CONCLUSION(S): These data suggest that IL-6 has an inhibitory effect on ciliary activity, suggesting a possible role for this cytokine in the subfertility process. This finding may be important in conditions where there are increased IL-6 levels in the peritoneal fluid, such as endometriosis and pelvic inflammatory disease.

Reproductive ageing in women.

The traditional view in respect to female reproduction is that the number of oocytes at birth is fixed and continuously declines towards the point when no more oocytes are available after menopause. In this review we briefly discuss the embryonic development of female germ cells and ovarian follicles. The ontogeny of the hypothalamic-pituitary-gonadal axis is then discussed, with a focus on pubertal transition and normal ovulatory menstrual cycles during female adult life. Biochemical markers of menopausal transition are briefly examined. We also examine the effects of age on female fertility, the contribution of chromosomal abnormalities of the oocyte to the observed decline in female fertility with age and the possible biological basis for the occurrence of such abnormalities. Finally, we consider the effects of maternal age on obstetric complications and perinatal outcome. New data that have the potential to revolutionize our understanding of mammalian oogenesis and follicular formation, and of the female reproductive ageing process, are also briefly considered.

The reproductive significance of human Fallopian tube cilia.

Effective tubal transport of ova, sperm and embryos is a prerequisite for successful spontaneous pregnancy. Although there is much yet to be discovered about the mechanisms involved, it is evident that tubal transit is a far more complicated process than initially thought. Propulsion of gametes and embryos is achieved by complex interaction between muscle contractions, ciliary activity and the flow of tubal secretions. Evidence is accumulating of the important and possibly pre-eminent role of ciliary motion in this process; and this review describes current knowledge about ciliary activity and its physiological regulation. There is also a description of the effects on ciliary function of cigarette smoking and various pathological states, including endometriosis and microbial infection, with consideration given as to how altered ciliary activity may impact upon fertility.

The effect of ovarian follicular fluid and peritoneal fluid on Fallopian tube ciliary beat frequency.

BACKGROUND: The Fallopian tube undergoes well-recognized changes during the ovarian cycle. Ciliary beat frequency (CBF) increases during the secretory phase of the cycle. The stimulus is unknown, although CBF is known to be hormone responsive. At ovulation, follicular fluid is released into the peritoneal cavity and enters the Fallopian tube. We hypothesized that this fluid may provide the stimulus for the increase in CBF detected after ovulation. METHODS: Using a technique which records changes in light intensity, we have studied the effect of pre-ovulatory follicular fluid on CBF of Fallopian tube epithelial cells, and compared this with the effect of either peritoneal fluid or culture medium alone. Follicular fluid samples from 13 women undergoing IVF were collected by selective aspiration of individual follicles. Peritoneal fluid was collected from six women undergoing laparoscopic sterilization. Fallopian tubes were collected from 10 women who underwent hysterectomy for benign conditions. RESULTS: After 24 h incubation, there was a highly significant difference in CBF between the Fallopian tube samples bathed in follicular fluid (mean CBF +/- SEM: 6.34 +/- 0.02 Hz) compared with explants bathed in either medium (4.20 +/- 0.06 Hz) or peritoneal fluid (5.24 +/- 0.03 Hz) (P < 0.005). There was also a significant difference in CBF between tissues bathed in secretory (5.47 +/- 0.03 Hz) compared with proliferative phase peritoneal fluid (4.75 +/- 0.02 Hz) (P < 0.005). CONCLUSIONS: The increase in CBF detected after ovulation may aid ovum pick-up and transport along the Fallopian tube. Factor(s) within human follicular fluid and secretory phase peritoneal fluid may be responsible for this increase in CBF.

Effect of angiotensin II on ion transport across human Fallopian tube epithelial cells in vitro.

Angiotensin II type 1 receptors have been identified in Fallopian tube epithelia. Polarized confluent human Fallopian tube epithelial cell cultures were used under short-circuit conditions to study the actions of angiotensin II on electrogenic ion transport. The results demonstrate that angiotensin II increases baseline short-circuit current, implying a net transport of negatively charged ions from a basal to apical direction. This effect was inhibited by the selective angiotensin II type 1 receptor antagonist losartan. The effects of angiotensin II on short-circuit current were rapid in onset, brief in duration, and although less than those achieved with ATP, similar in amplitude to those described for other epithelia with angiotensin II. These findings reflect a significant retention of function for these cells in monolayer culture. Immunohistochemistry using the antibody 6313/G2, which is directed against a specific sequence in the extracellular domain of the angiotensin II type 1 receptor, confirmed that the receptor was retained in cultured cells. The results indicate that angiotensin II plays a role in regulating the composition of Fallopian tube secretions.

Fallopian tube ciliary beat frequency in relation to the stage of menstrual cycle and anatomical site.

BACKGROUND: The cyclical changes in ciliary structure and motion within the human Fallopian tube are well documented. Previous investigators have studied ciliary beat frequency (CBF) in relation to menstrual cycle and anatomical site, but with conflicting results. METHODS: Using a technique that records variations in light intensity, we have studied the changes in CBF in relation to the menstrual cycle and anatomical site. Fallopian tubes were collected from 26 women who underwent hysterectomy for benign conditions. Menstrual history, hormone profile and endometrial biopsy results were used to determine the stage of the cycle. Fourteen women were in the proliferative phase, and 12 women in the secretory phase. RESULTS: Mean CBF for all subjects was 5.3 plus minus 0.2 Hz. There was no significant difference in CBF in relation to anatomical site. In the fimbrial region the ciliary beat was faster in the secretory (5.8 plus minus 0.3 Hz) as compared with the proliferative phase (4.9 plus minus 0.2 Hz), P < 0.02. CONCLUSIONS: It is possible that this increase in fimbrial CBF may contribute to ovum retrieval and transport after ovulation. However, the reproductive significance of the changes in CBF in relation to the menstrual cycle needs further investigation.

The effect of cAMP on ion transport in Fallopian tube epithelial cells in vitro.

The coupled movement of ions and water across epithelia determines the composition and volume of fluid present in the lumen of organs. The second messenger cAMP is important in effecting electrolyte and water transport in many transporting epithelia; however, its role in Fallopian tube transport is uncertain. We have conducted electrophysiological studies on Fallopian tube epithelial cell monolayers in Ussing chambers and have demonstrated that exogenously added cAMP and agents that generate its intracellular production results in an increase in short-circuit current consistent with the transport of net electrical charge from a basal to mucosal direction. In contrast to the known effects of ATP in this tissue, the increase in short-circuit current was not explicable in terms of electrogenic chloride secretion as it was not affected by the chloride channel inhibitors, 4-acetamido-4'-isothiocyanostilbene-2,2-disulphonic acid 1 mmol/l (SITS) and frusemide. Instead the current was reduced by the sodium channel inhibitor, amiloride, and was therefore, in part, explicable in terms of electrogenic Na+ absorption. These findings will enhance our understanding of the physiological mechanisms responsible for human Fallopian tubal fluid formation and composition.

Importance of sperm-to-epithelial cell contact for the capacitation of human spermatozoa in fallopian tube epithelial cell cocultures.

OBJECTIVE: To investigate the mechanisms involved in the stimulatory effect of fallopian tube epithelial cell coculture on sperm movement characteristics. DESIGN: Human spermatozoa were cultured with human fallopian tube epithelial cell monolayers. A microporous membrane was used to prevent sperm-to-epithelial cell contact. Sperm movement characteristics were measured at 4 and 24 hours. SETTING: University hospital and fertility center. PATIENT(S): Voluntary donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Movement characteristics of human spermatozoa. RESULT(S): Fallopian tube epithelial cell coculture increased sperm motility, curvilinear velocity, amplitude of lateral head displacement, and hyperactivated motility, mainly at 24 hours, compared with controls. These stimulatory effects were inhibited when a microporous membrane prevented cell-to-cell contact between sperm and fallopian tube epithelial cells. CONCLUSION(S): Physical contact between sperm and epithelial cells in coculture systems seems to be the main factor in stimulating sperm movement characteristics, and this could be the main mechanism of in vivo sperm capacitation.

Comparison of the growth patterns and morphological characteristics of mechanically and enzymatically isolated fallopian tube epithelial cells.

This study set out to compare the growth patterns and morphological characteristics of human fallopian tube epithelial cells isolated: (1) mechanically; and (2) enzymatically. Cells were cultured in medium supplemented with fetal bovine serum and antibiotics and their epithelial nature was established by immunocytochemistry for cytokeratins. Primary cultures were polygonal in shape with centrally located nuclei, irrespective of the isolation method. Cells isolated enzymatically exhibited a higher growth rate, but the survival rate was poor after more than 2-3 passages. Mechanical isolation gave a lower yield of cells, but had a higher survival rate when sub-cultured, even beyond 8 passages. Thus, mechanically isolated cells might be useful for longer term cultures, whereas enzymatically isolated cells are best only for short-term work.

The pineal and extra-pineal origins of 5-sulphatoxy N-acetyl-serotonin in humans.

In humans 6-sulphatoxy melatonin (SaMT) is the principal metabolite of endogenous and exogenous melatonin. 5-sulphatoxy N-acetyl-serotonin (SNAS) is a minor metabolite of exogenous melatonin, but it has not been established whether the levels of endogenous SNAS in plasma derives principally from endogenous melatonin. We have developed the first radioimmunoassay (RIA) for SNAS and used it (together with RIAs for melatonin and SaMT) to determine whether endogenous SNAS derives from endogenous melatonin or from platelet serotonin. Our results show a) the values of endogenous SNAS, unlike endogenous SaMT, increased with blood collection procedures that increased the values of serotonin, b) the values of endogenous SNAS in urine or in platelet-poor plasma were approximately the same as those of endogenous SaMT, but, unlike SaMT, did not show a diurnal rhythm, and c) we confirmed that SNAS was a minor metabolite of orally ingested melatonin. Thus, our conclusion is that SNAS is a minor metabolite of exogenous melatonin, but is not a significant metabolite of endogenous melatonin. In all probability, endogenous SNAS is principally the metabolite of platelet serotonin.

The effect of ovarian steroids on epithelial ciliary beat frequency in the human Fallopian tube.

Using a method that detects variations in light intensity we have studied the effect of ovarian steroids on human Fallopian tube epithelial ciliary beat frequency in vitro. We have found that baseline ciliary beat frequency averages between 5-6 Hz. Cilia from ampullary segments of the Fallopian tube beat significantly faster (5.4 Hz+/-0.2) than those from fimbrial segments (4.8 Hz+/-0.2). There was no significant difference in baseline ciliary beat frequency at any other anatomical site in the Fallopian tube. Incubation with progesterone (10 micromol/l) suppresses human Fallopian tube epithelial ciliary beat frequency by 40-50%. This inhibition was observed at similar magnitudes in all Fallopian tubes studied irrespective of anatomical site. Progesterone-induced reductions in ciliary beat frequency were concentration dependent and prevented by the progesterone receptor antagonist mifepristone (RU486). Oestradiol alone (10 micromol/l) had no effect on ciliary beat frequency at any anatomical site in the Fallopian tube but did prevent the reduction in ciliary beat frequency seen with progesterone when tissues were incubated with these two steroids together.

Elevated nocturnal melatonin is a consequence of gonadotropin-releasing hormone deficiency in women with hypothalamic amenorrhea.

Elevated nocturnal melatonin is found in women with idiopathic hypogonadotropic hypogonadism (IHH), but it is not known whether this is implicated in the etiology of their GnRH deficiency. It is unlikely that nocturnal melatonin can be implicated in the etiology of the GnRH deficiency of Kallmann's syndrome (KS), because this condition is caused by defective neuronal migration in embryonic life. We therefore measured nocturnal melatonin in women with IHH and KS to determine whether it was elevated in one or both conditions and thereby to determine whether it was implicated as cause or consequence of GnRH deficiency. Four women with IHH, 3 women with KS, and 7 individually matched (age and body size) controls were recruited. Frequent day- and nighttime samples were taken for LH pulsatility studies. All patients showed absent or diminished LH pulsatility, compared with their respective controls. Samples were also taken over 24 h for melatonin and 6-sulphatoxymelatonin (the principle metabolite of melatonin and an independent marker of its secretion). Melatonin and 6-sulphatoxymelatonin levels were elevated in 6 of 7 patients (compared with their matched controls) and were significantly elevated in the KS group (compared with their controls). The finding of elevated nocturnal melatonin (and its metabolite) in GnRH-deficient women with KS (as well as IHH) suggests that nocturnal melatonin is elevated as a consequence of GnRH deficiency, irrespective of its etiology.

Radioimmunoassay of bound and free melatonin in plasma.

We describe a nonextraction procedure, and two extraction procedures, for RIA of melatonin in human plasma. All procedures showed a diurnal rhythm of melatonin in human subjects, with interindividual differences greater than interprocedure differences. However, further investigations demonstrated considerable variability of recovery in the nonextraction procedure, suggesting a variability of binding proteins between samples. Combining recovery and dialysis experiments in the extraction procedures, we demonstrated that chloroform was unable to extract albumin-bound melatonin from a human serum albumin solution but, paradoxically, was able to extract bound and free melatonin from a plasma sample. The methanol extraction procedure extracted free and bound melatonin from all sources. These results indicate that albumin binding can substantially affect the RIA procedures. We conclude that assays should be validated against free and bound melatonin and that the two forms should be independently investigated when assessing bioactivity.

Placental protein 14 production by human Fallopian tube epithelial cells in vitro.

We studied the in-vitro secretory function of non-polarized and polarized cultured Fallopian tube epithelial cells by measurement of the placental protein 14 (PP14) secretion in primary cultures and subcultures from Fallopian tubes obtained from eight premenopausal women in different phases of the ovarian cycle. Primary cultures were established in minimal essential medium in Earle's salts supplemented with fetal bovine serum and the cells were subcultured for six passages, in the polarized cell cultures, the cells being seeded on an extracellular matrix system. Cell freezing was carried out using 10% dimethyl sulphoxide. PP14 secretion into the culture media was measured by a radioimmunoassay using 125I-PP14 as label and rabbit anti-human PP14 serum. There was a large amount of PP14 secretion into the culture media in primary cultures, the secretion decreasing considerably after subculture 1. PP14 secretion after subculture 2 was not different from the control values. Polarized and non-polarized cells secreted similar amounts of PP14 and frozen-thawed cells did not appear to secrete PP14. Epithelial cells from Fallopian tubes obtained at different phases of the ovarian cycle did not appear to show any difference in PP14 secretion rates. Our data suggest that the in-vitro secretion of PP14 by human Fallopian tube epithelial cells is adversely affected by cell ageing and freezing.