Induction of the human sperm acrosome reaction with mannose-containing neoglycoprotein ligands

In the interest of classifying cases of male factor infertility, we have paid particular attention to the sugar ligand binding properties of the human sperm surface and the functional capacity of the acrosome for exocytosis--key parameters for assessing sperm fertilizing ability. Zona recognition and binding involve the interactions of sperm surface mannose receptors (lectins) with mannose ligands on the zona pellucida. Sperm surface mannose lectins can be visualized by their ability to bind a synthetic model zona ligand, fluorescein isothiocyanate (FITC)-conjugated mannosylated bovine serum albumin (BSA) (Man-FITC-BSA). We now report that Man-FITC-BSA biologically also mimics the effects of solubilized authentic human zonae, in that binding of Man-FITC-BSA results in a time-dependent receptor aggregation and the induction of acrosome exocytosis in capacitated sperm populations from fertile donors. In our assay, the addition of mM amounts of mannose monosaccharide to Man-FITC-BSA increases the number of polyvalent mannose ligands bound by individual spermatozoa and increases the rate of the acrosome reactions induced by Man-FITC-BSA, thereby increasing specimen processing efficiency. We conclude that exposure of human spermatozoa to polyvalent mannose ligands + D-mannose monosaccharide offers a new, convenient and readily available system to study sperm capacity for induced acrosome loss.     

Studies of lysophospholipids related to the hamster sperm acrosome reaction in vitro

1. One of the metabolic features of acquired immunodeficiency syndrome is increased tissue glucose uptake documented by euglycaemic-hyperinsulinaemic clamp studies, suggesting increased insulin sensitivity. However, these results may also be related to the confounding effect of increased non-insulin-mediated glucose uptake in acquired immunodeficiency syndrome, which will result in an erroneously presumed increased insulin sensitivity. To study the contribution of non-insulin-mediated glucose uptake to total tissue glucose uptake in acquired immunodeficiency syndrome, we conducted a hypoinsulinaemic clamp study in clinically stable human immunodeficiency virus-infected (Centers for Disease Control class IV) men (n = 7) and healthy subjects (n = 5). Glucose uptake was measured by a primed, continuous infusion of [3-3H]glucose in the postabsorptive state and during somatostatin-induced insulinopenia at euglycaemic (approximately 5.3 mmol/l) and hyperglycaemic (approximately 11 mmol/l) glucose concentrations. 2. Basal glucose concentration (patients, 5.2 +/- 0.1 mmol/l; control subjects, 5.3 +/- 0.1 mmol/l) and basal glucose tissue uptake (patients, 15.9 +/- 0.5 mumol min-1 kg-1 fat-free mass; control subjects, 15.2 +/- 0.4 mumol min-1 kg-1 fat-free mass) were not different between the two groups. 3. Euglycaemic glucose uptake during somatostatin infusion, reflecting non-insulin-mediated glucose uptake, decreased to 82 +/- 3% in patients and 78 +/- 2% in control subjects (not significant). Under hyperglycaemic (approximately 11 mmol/l) conditions with sustained insulinopenia, no differences in glucose uptake existed between the two groups (patients, 16.8 +/- 0.6 mumol min-1 kg-1 fat-free mass; control subjects, 16.1+/- 0.3 mumol min-1 kg-1 fat-free mass).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of gamma-aminobutyric acid, progesterone and ionophore A23187 on acrosome reaction of tree shrew sperm in vitro: examination of acrosome reaction with an improved fluorescence microscopy

A number of acrosome reaction (AR) initiators have been found to be effective in inducing AR of human, laboratory and domestic animal sperm. Using an improved simple fluorescence microscopy, effects of gamma-aminobutyric acid (GABA), progesterone and ionophore A23187 on sperm AR of tree shrew, a useful animal model in biomedical research, have been investigated. Spontaneous AR in 4.92-7.53% of viable sperm was observed. Complete AR in 10.31-18.25% of viable tree shrew sperm was obviously induced by 5 microM and 10 microM calcium ionophore A23187, 1 mM GABA, and 5 microM progesterone, and there were no significant differences between their abilities to initiate complete AR. No significant differences of AR percentages between 1- and 2-h treatments with A23187, progesterone and/or GABA were observed. These results suggested that the responses of tree shrew sperm to these AR initiators are similar to that of human and other mammalian sperm.     

Morphology and kinetics of the hamster sperm acrosome reaction

The morphology and kinetics of the normal acrosome reaction were examined in vitro using hamster sperm incubated in detoxified sera. The reaction involved either swelling and elevation or crenulation and fragmentation of the acrosomal cap. Swelling and elevation occurred during both normal and degenerative reactions, as reported by others. Crenulation with subsequent fragmentation of the cap was observed during normal reactions. Early crenulation of the acrosome could be induced by cold shock (5 degrees C, 25 minutes), but this did not decrease the incubation time required (at 37 degrees C) for completion of the normal reaction. In appropriate sera, the occurrence of normal and degenerative acrosome reactions in motile sperm was significantly separated in time to study the reactions independently. The duration of the normal reaction, i.e., the time between the first morphological change in the acrosome (initiation)until the actual detachment of the cap (termination) was estimated to be 20 minutes. Saline dilution of these sera delayed initiation of the reaction and increased the duration of the reaction once it had started. Data from cold-shock and serum dilution experiments indicate that the mechanisms which govern the initiation and termination of the normal reaction are independently variable, and further suggest that initiation involves a change in membrane permeability and that termination includes membrane vesiculation.     

Usefulness of the acrobead test in evaluating human acrosome function in fresh and cryopreserved sperm

PURPOSE: Assays for the acrosome reaction are usually cumbersome and lack reproducibility. Accurate determination of acrosomal status is important in patients diagnosed with male infertility before proceeding with intrauterine insemination or in vitro fertilization. We determined the optimum capacitation time and acrosomal status of fresh semen specimens in normal fertile men with the Acrobead test, and whether the assay could be used to evaluate cryopreserved semen specimens. MATERIALS AND METHODS: Semen samples from 13 normal donors were divided, with half of the fresh ejaculate used for the Acrobead test and half cryopreserved for a minimum of 24 hours in liquid nitrogen before testing. Fresh and frozen specimens were prepared with the swim-up technique. Sperm concentration was adjusted to 4, 2, 1 and 0.5 x 10(6)/ml. in 4 wells of a 96-well tissue culture plate. Ten microl. polyacrylamide beads (1.5 x 10(6)/ml.) coated with anti-CD46 monoclonal antibodies (MH61 beads) were added to each well. The attachment of beads with acrosome reacted spermatozoa was scored after 0, 1, 3, 6 and 24 hours of incubation. Results were graded on a scale of 0 (no bead binding to the sperm) to 4 (complete attachment to the beads). Specimens with scores of at least 2 were considered normal. RESULTS: A score of at least 2 was noted in 3 of 13 fresh specimens (15.3%) at 1, 9 (69.2%) at 3, 11 (84.6%) at 6 and 13 (100%) after 24 hours. However, a significantly greater number of frozen specimens (8 of 13, or 62%) had a score of 2 or more at 1 hour of incubation and 100% bead attachment to sperm occurred at 3 hours. CONCLUSIONS: Our results indicate that in fresh semen specimens an incubation period of 6 to 24 hours can be used to screen individuals who present with normal sperm characteristics but have slow acrosome reactions. Early acrosome reaction observed in cryopreserved specimens indicates that these spermatozoa may have membrane damage and leakage of acrosome contents as a result of the freeze-thaw process. The Acrobead assay is a simple and objective test that can be used at a clinical andrology laboratory to evaluate the acrosomal status of fresh but not frozen human spermatozoa.     

Characterization of two second messenger pathways and their interactions in eliciting the human sperm acrosome reaction

Actinobacillus actinomycetemcomitans, a gram-negative, capnophilic bacterium, is associated with several human diseases and is the suspected etiologic agent in certain forms of periodontal disease. We have previously shown that this organism produces an immunosuppressive factor (ISF) which is capable of inhibiting both T- and B-cell activation. Furthermore, these effects appear to be associated with the activation of a population of suppressor cells. We now report that the ISF induces a unique population of CD4+CD8+ dual-positive T-cells. By utilizing multiparameter flow cytometric analysis, we were able to detect the presence of dual-positive cells in cultures of human T-cells treated with PHA and ISF. The cells appeared within 48 hr and their induction was dependent upon the presence of both CD4 and CD8 cells in the culture. Dual expression of CD4 and CD8 was stable in that the cells continued to express both surface proteins after being sorted and cultured for an additional 24 hr. Phenotypic analysis indicates that these cells are also CD3+, CD2+, CD5+, TCR alpha beta+, CD45RA+ (and RO+), and CD29+. The dual-positive cells express surface markers associated with T-cell activation: CD25+, CD69+, CD71+, and HLA-DR+. In contrast, the cells were negative for CD34, CD57, CD56, and CD16. Cell cycle analysis indicates that > 80% of the dual-positive cells were in the S phase. Finally, functional analysis of these cells indicates that they are capable of suppressing the proliferative response of autologous T-cells to PHA.     

Progesterone initiation of the human sperm acrosome reaction: the obligatory increase in intracellular calcium is independent of the chloride requirement

The progesterone-initiated human sperm acrosome reaction (AR) requires a rise in intracellular Ca2+ ([Ca2+]i), extracellular Cl- and apparently increased Cl- flux through a unique steroid receptor/Cl- channel resembling but not identical to a GABA(A)/Cl- channel complex. The present study uses fura-2 loaded human sperm, GABA(A)/Cl- channel blockers (picrotoxin and pregnenolone sulfate) and Cl(-)-containing and Cl(-)-deficient media to determine whether the progesterone-mediated increase in [Ca2+]i is dependent on the Cl- requirement. There was no significant difference between the progesterone-mediated increases of [Ca2+]i obtained in Cl(-)-containing and Cl(-)-deficient media. Picrotoxin did not significantly inhibit the progesterone-mediated increase in [Ca2+]i, and pregnenolone sulfate increased [Ca2+]i to the same extent as progesterone. These results strongly suggest that the increase in [Ca2+]i essential to the AR is independent of the AR Cl- requirement and could be explained by the existence of two different sperm plasma membrane progesterone receptors.     

The characteristics of monoclonal antibodies and their antigens associated with human sperm acrosome reaction. I. The induction of acrosome reaction and monoclonal antibody production

A low blood glucose level is associated with impairment of higher cerebral function and an increase in cerebral blood flow. This study examined whether there are differences in the physiological responses to hypoglycaemia between the cerebral hemispheres. Eight healthy men participated in two hyperinsulinaemic glucose clamp studies: after 60 min at 4.5 mmol/l, blood glucose was either lowered to 2.0 mmol/l and "clamped" there for 60 min (hypoglycaemia) or continuously maintained at 4.5 mmol/l (euglycaemia). Cardiac output, middle cerebral artery velocity (transcranial Doppler) and cerebral blood flow (133-xenon inhalation) were measured during the studies. Neuropsychological tests were used to determine whether hypoglycaemia caused differential impairment of hemispheric cognitive function. Hypoglycaemia was associated with symmetrical impairment of cognitive function in both cerebral hemispheres and a rise in cardiac output (from 5.5 [0.2] to 8.7 [0.2] l.min-1, p < 0.0001, mean [standard error]), middle cerebral artery velocity (from 55 [2.6] to 64 [2.8] cm.s-1, p < 0.002), and global cerebral blood flow (from 56 [2.6] to 69 [2.9] ml.100 g-1.min-1, p < 0.005 compared to pre-insulin values). There were no differences in the blood flow response during hypoglycaemia between hemispheres and the increase in blood flow did not correlate with either the change in cardiac output or rise in plasma catecholamine levels. After 120 min of hyperinsulinaemic, euglycaemia, global cerebral blood flow rose significantly above baseline (from 58 [2.4] to 63 [2.2] ml.100 g-1.min-1, p < 0.05). In conclusion, using the techniques described, the physiological and cognitive responses of each cerebral hemisphere to hypoglycaemia were symmetrical.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of acrosome reaction in human spermatozoa accelerates the time of pronucleus formation of hamster oocytes after intracytoplasmic sperm injection

OBJECTIVE: To assess the relationship between the incidence of acrosome reaction (AR) and the timing of pronucleus (PN) formation after intracytoplasmic sperm injection (ICSI). DESIGN: Prospective study. SETTING: Infertility Research Center, Jeil Women's Hospital. MAIN OUTCOME MEASURE(S): Human semen obtained from fertile donors was prepared by one of the following methods: washing only (washed control); Percoll gradient; pentoxifylline; human follicular fluid (FF); pentoxifylline + FF; or platelet-activating factor (PAF) treatment. The AR of each group was assessed by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin or Arachis hypogea agglutinin. Spermatozoa of washed control, pentoxifylline + FF, and PAF treated groups, with significantly higher AR rate than others, were injected into mature hamster oocytes. Spermatozoon-injected oocytes were cultured for 6, 9, 12, or 15 hours. Then they were stained with Toluidine blue for PN formation examination under a light microscope. RESULT(S): Acrosome reaction rates of washed control, Percoll gradient, pentoxifylline, FF, pentoxifylline + FF, and PAF treated groups were 10.5% +/- 2.6%, 10.3% +/- 1.7%, 16.4% +/- 1.8%, 24.8% +/- 5.6%, 28.4% +/- 3.8%, and 33.3% +/- 5.2%, respectively. Pronuclear formation rate in washed control, pentoxifylline + FF, and PAF treated groups were 5.6% (3/54), 19.0% (11/58), and 18.9% (10/53) at 6 hours; 32.7% (18/55), 51.8% (29/56), and 57.4% (31/54) at 9 hours; 36.1% (22/61), 53.6% (30/56), and 50.0% (27/54) at 12 hours; and 47.2% (25/53), 64.8% (35/54), 53.6% (30/56) at 15 hours after ICSI. Pronuclear formation rate was significantly higher in pentoxifylline + FF, and PAF treated groups than that in the washed control group at 6 and 9 hours after ICSI. CONCLUSION(S): Pronuclear formation of oocytes takes place faster on those that were injected with acrosome-reacted spermatozoon than those injected with acrosome-intact spermatozoon. It could be concluded that induction of the AR of spermatozoa accelerates the time of PN formation and early development of the embryo in ICSI.     

The duration of estradiol and progesterone exposure prior to progesterone withdrawal regulates oxytocin mRNA levels in the paraventricular nucleus of the rat

The nonapeptide oxytocin (OT) is important for milk ejection during lactation, uterine contractility at parturition, and the onset of maternal behavior. Sequential exposure to estradiol (E2) and progesterone (P) followed by P withdrawal increases OT mRNA in the paraventricular nucleus (PVN), and to a lesser degree the supraoptic nucleus (SON), of the rat 48 hours after the P is removed. Although increases in PVN OT mRNA are not accompanied by changes in posterior pituitary OT peptide content, the PVN contains OT neurons that project to both the posterior pituitary (magnocellular group) and extra pituitary sites (parvocellular groups). Steroid-induced increases in OT mRNA occur in both the magnocellular and the parvocellular regions of the PVN. The latter are believed to contribute to CNS release of OT which may be important for certain behaviors including the onset of maternal behavior. The same steroid sequence that increases PVN OT mRNA also induces maternal behavior in virgin ovariectomized rats. Exposure of animals to E2 and P for 2 weeks resulted in the shortest latency to the onset of maternal behavior in ovariectomized rats, whereas exposure for 6 days was associated with a longer latency. In this study we questioned if the duration of E2 and P exposure prior to P withdrawal is an important regulator of PVN OT mRNA levels. We compared OT mRNA levels in the PVN of virgin ovariectomized rats administered no steroid or sequential E2 and P for 2 weeks versus 6 days. On day 1 animals received steroid-filled or empty capsules followed by P-filled or empty capsules on day 3. In one steroid-treated group, E2 and P were continued for 6 days and in the other group for 14 days prior to P removal. Animals were sacrificed 48 hours after P removal. Levels of OT mRNA were compared among 6 day and 2 week steroid-treated animals and sham-treated animals. The relative abundance of OT mRNA was significantly increased, P < 0.05, in animals receiving the 2-week, but not the 6-day, steroid treatment compared to sham-treated animals. Pituitary OT peptide content was not significantly different among the three groups. We conclude that the duration of steroid exposure may be an important regulator of the level of OT mRNA in the PVN of the rat.     

Effects of cholesterol reduction on BP response to mental stress in patients with high cholesterol

Rotating instruments are becoming increasingly significant in the scaling and planing of the root surface. The objective of this in vitro study was to test various root-planing instruments on extracted teeth and then to compare the treated surfaces using scanning electron microscopy. Two manual instruments (scaler and curette) and five mechanically rotating instruments (Desmo-Clean; Perio-Set; Viking-Set; and 40-microns and 15-microns diamond finishers) were investigated. From a total of 42 teeth, six root surfaces were treated with each instrument. The results confirm the clear superiority of the manual instruments (especially the curette). The manual instruments permit good root planing with minimum ablation from the root surface and only a thin smear layer (a compound of grinding dust, dentinal fluid, and water). The best planing results by rotating instruments were achieved with the Desmo-Clean and the 15-microns diamond finisher, whose performance was almost equal to that of the manual instruments. The rotating instruments, however, were associated with higher ablation and a marked smear layer. Manual instruments remain the media of choice on easily accessible root surfaces; however, rotating instruments are of advantage in inaccessible areas (eg, furcations) because of their handling properties.     

Motivational state regulates the content of learned flavor preferences.

Rats acquired a preference for an aqueous odor (almond) presented in simultaneous compound with sucrose. Separate presentations of saccharin reduced this preference in rats with ad-lib access to food during training or at test, but not in rats that were hungry during both training and test. In contrast, separate presentations of sucrose reduced the preference for the almond irrespective of deprivation state during training and test. We interpret the results to mean that a hungry rat forms odor–taste and odor–calorie associations, and its motivational state on test determines which of these associations controls the preference. In contrast, a rat that is not hungry during training only forms an odor-taste association, and its performance on test is independent of its level of hunger. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Differential agonist regulation of the human kappa-opioid receptor

Opiates are potent analgesics used clinically in the treatment of pain. A significant drawback to the chronic use and clinical effectiveness of opiates is the development of tolerance. To investigate the cellular mechanisms of tolerance, the cloned human kappa-opioid receptor was stably expressed in human embryonic kidney (HEK 293) cells, and the effects of opioid agonist treatment were examined. The receptor-expressing cells showed specific high-affinity membrane binding for a kappa-selective opioid, 3H-labeled (+)-(5alpha,7alpha,8beta)-N-methyl-N-[7-(1-pyrrolidiny l)-1-oxaspiro [4,5] dec-8-yl] benzeneacetamide ([3H]U69,593), and a nonselective opioid antagonist, [3H]diprenorphine. Pretreatment with pertussis toxin or guanosine 5'-O-(3-thiotriphosphate) reduced [3H]69,593 binding, indicating that the human K receptor coupled to G proteins of the Gi or Go families in HEK 293 cells. The receptor-mediated inhibition of adenylyl cyclase was abolished by pertussis toxin pretreatment and was blocked by a kappa-selective antagonist, norbinaltorphimine. A 3-h pretreatment with a kappa-selective agonist, (+/-)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide (U50,488), caused receptor down-regulation, whereas no receptor down-regulation was found after levorphanol pretreatment. U50,488 or dynorphin A(1-17) pretreatments (3 h) desensitized the ability of U50,488 or dynorphin A(1-17) to inhibit cyclic AMP accumulation, as evidenced by a decrease in functional potency. Also, U50,488 pretreatment desensitized the ability of levorphanol to inhibit forskolin-stimulated cyclic AMP accumulation. In contrast, pretreatment of cells with either levorphanol or a potent nonselective opioid, etorphine, resulted in no apparent receptor desensitization. Taken together, these results demonstrate that the human kappa receptor is differentially regulated by selective and nonselective opioid agonists, with selective agonists able to desensitize the receptor.     

An agonist murine monoclonal antibody to the human c-Mpl receptor stimulates megakaryocytopoiesis

Thrombopoietin (TPO) is a hematopoietic growth factor that stimulates megakaryocytopoiesis and platelet production in vivo and promotes the development of identifiable megakaryocytes in vitro. We have developed a murine monoclonal antibody, BAH-1, raised against human megakaryocytic cells, which specifically recognizes the c-Mpl receptor and shows agonist activity by stimulating megakaryocytopoiesis in vitro. BAH-1 antibody specifically binds to platelets and to recombinant c-Mpl with high affinity. Similar to TPO, BAH-1 alone supported the formation of colony-forming unit-megakaryocyte (CFU-MK) colonies. The combination of BAH-1 plus interleukin-3 or of BAH-1 plus human TPO significantly increased the number of human CFU-MK colonies. In addition, BAH-1 monoclonal antibody stimulated the proliferation and maturation of primary bone marrow megakaryocytes in a dynamic heterogeneous liquid culture system. Individual large megakaryocytes as well as small megakaryocytic cells were observed in cultures of CD34(+) CD41(+) cells in the presence of BAH-1 antibodies. Similar to TPO, BAH-1 antibody induced a significant response of murine immature megakaryocytes as observed by an increase in the detectable numbers of acetylcholinesterase-positive megakaryocytes. No effects of BAH-1 antibody were observed on colony-forming unit-granulocyte-macrophage, burst-forming unit-erythroid, or colony-forming unit-erythroid colonies. In vivo studies showed that BAH-1, alone or in combination with TPO, expands the numbers of megakaryocytic progenitor cells in myelosuppressed mice. This antibody should prove useful in understanding the structure-function aspects of the c-Mpl receptor as well as in evaluating the effects of the sustained activation of this receptor in preclinical models of severe thrombocytopenia.     

Triiodothyronine (T3) modulates hCG-regulated progesterone secretion, cAMP accumulation and DNA content in cultured human luteinized granulosa cells

Ample clinical evidence indicates that women with thyroid disorders frequently exhibit menstrual disturbances and impaired fertility. In order to characterize the nature of thyroid hormone action in the ovary, the direct effects of triiodothyronine (T3) were investigated in vitro using a culture system of human luteinized granulosa cells. The presence of T3 receptors was also searched in such cells. The cell cultures were maintained in serum-free Ham's F-10 medium in the absence or presence of hCG, with or without graded doses of T3 (10(-11)-10(-7) M), and cell proliferation (assessed by DNA content) as well as cell function (cAMP accumulation and progesterone secretion) determined. T3 alone stimulated cell proliferation. hCG, on the other hand, was anti-mitogenic and T3 combined with hCG inhibited cell growth even further, reaching levels below those reached by either control or hCG alone. Exposure of cells to T3 markedly enhanced hCG-induced cAMP accumulation. Addition of 1-methyl-3-isobutylxanthine (MIX) abolished the cAMP-stimulatory effect elicited by T3, suggesting that the thyroid hormone may act, as MIX, by inhibiting phosphodiesterase. T3 was devoid of any influence on basal progesterone secretion, but inhibited hCG-induced secretion of the steroid. The effects of T3 are not accounted for by changes in cell number since the influence of thyroid hormone on cAMP and steroid secretion were expressed per microgram DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of progesterone biosynthesis in the human placenta by estradiol 17 beta and progesterone

The information available concerning the effects of chemotherapy administered during pregnancy is limited and consists of case reports and small series. A registry has been established at the National Cancer Institute, but there are currently only several hundred cases of neonates exposed to chemotherapy registered. All clinicians who care for women receiving chemotherapy during pregnancy should report those experiences to the National Cancer Institute to increase the data base. When chemotherapy is used during the embryogenesis period in the first trimester there is an increased rate of spontaneous abortion and major birth defects. The most toxic chemotherapeutic agents administered during pregnancy are methotrexate and aminopterin and should be avoided when possible, particularly during the first trimester. Pregnancy-related physiologic changes should be kept in mind when dosing and administering cytotoxic chemotherapy. The risk of fetal malformation when chemotherapy is administered during the second and third trimesters is probably not greater than background rate, but there may be a greater risk of stillbirth, fetal growth restriction, premature birth, and maternal and fetal myelosuppression. Breastfeeding should be avoided in women receiving chemotherapy.     

The significance of serum oestradiol and progesterone levels in evaluating the response to clomiphene citrate

In practice, the prediction of ovulation after the administration of clomiphene citrate (Clomid) has been based on measurement of biphasic basal body temperature and menstrual changes. In this article a protocol is presented to gauge adequately the extent of the response to the drug and to monitor reliably the occurrence of ovulation. The measurement of oestradiol levels at two different times, coupled with a single progesterone estimation, has proved the method of choice in monitoring Clomid response. These assays may provide reliable criteria for establishing suitable doses for individual patients.