Suppression of LH during ovarian stimulation: effects differ in cycles stimulated with purified urinary FSH and recombinant FSH

There has been much debate about the role of luteinizing hormone (LH) during follicle stimulating hormone (FSH)-treated ovarian stimulation for assisted reproduction, where the endogenous LH is suppressed using a gonadotrophin-releasing hormone analogue. The requirement for LH in oestradiol biosynthesis is established, but other effects of 'insufficiency' are less clear, and little attention has been paid to the specific origin of the FSH used. The aim of this study was to examine the roles of profoundly suppressed circulating LH concentrations in cycles of ovarian stimulation for IVF, which were affected in two large separate cohorts of patients undergoing assisted reproduction. They were stimulated by either purified urinary FSH (MHP) or recombinant human FSH (rFSH). Within each dataset, outcomes were examined with respect to the circulating concentrations of LH in the mid-follicular phase, as plasma samples were stored prospectively, and assayed retrospectively. Patients with profoundly suppressed LH showed much reduced oestradiol concentrations at mid-follicular phase and at human chorionic gonadotrophin administration in cycles treated with either MHP or rFSH. However, gross ovarian response, as became evident by FSH dose demands, duration of stimulation, and also oocyte and embryo yields and embryo cryopreservation were influenced only in cycles treated with MHP. Furthermore, no effect upon pregnancy survival was observed. Thus, it is concluded that there is a demand for additional exogenous LH treatment only in cycles treated with purified urinary FSH where the LH is profoundly suppressed.     

The expression patterns of mRNA-encoding stem cell factor, internal stem cell factor and c-kit in the prepubertal and adult porcine ovary

The receptor, c-kit, and its ligand, stem cell factor (SCF), are important regulators of ovarian follicle growth and development. The aim of this study was to identify the sites of expression of mRNA for c-kit and SCF in prepubertal and mature (pregnant and non-pregnant) animals. Ovaries were recovered from prepubertal animals, non-pregnant sows and five sows at approximately 3 months of gestation. Ovine SCF and c-kit DNA were cloned into plasmid vectors to produce RNA probes. Expression of mRNA encoding SCF and c-kit were detected via in situ hybridization. Both mRNA were detected throughout ovaries from all animals. This study provides evidence that the growth-factor complex is required throughout follicle development, and also for continued maintenance of the corpus luteum (CL) in the mature animal. SCF mRNA was localized to the granulosa cell layer and was also extensively expressed in endothelial tissue and throughout the CL. c-kit mRNA was detected in the theca layer, oocytes and also in CL. In conclusion, expression of SCF and c-kit mRNA in granulosa and theca cells, respectively, indicate an important interaction between somatic cells throughout follicle development and that in the mature animal, SCF and c-kit potentially have a role in maintaining progesterone secretion by the CL. The observations of continued expression of SCF and c-kit throughout development suggest that there may be differences in the role of this receptor-ligand complex between large mono- vs. poly ovulatory species, such as the pig.     

Hemoglobin levels in Tibet: different effect of age and gender for Tibetans vs Han

Tibetans form a population which has resided on the Qinghai–Tibetan plateau for thousands of years, and are reported to have less hemoglobin than Han Chinese lowlanders who have migrated to the plateau. However, for various altitudes, detailed comparisons of hemoglobin in the two ethnic groups has not been reported. We investigated the hypothesis that the effects of altitude, age, and gender on hemoglobin concentration would differ between Han and Tibetan residents of the plateau. Hematological parameters for both genders were determined in healthy Tibetan adults (n=3,000) and children (n=332), and healthy Han Chinese adults (n=2,612) and children (n=275), aged 5–60 years living at four different altitudes (mean altitude of 2,664, 3,813, 4,525, and 5,200 m). Hemoglobin values increased with altitude for all ages in both ethnic groups and in both genders. The gain in hemoglobin with altitude had the rank order: Han males > Han females > Tibetan males and females. Even before puberty, Han children had more hemoglobin than Tibetan children. An effect of age on hemoglobin was seen at the time of puberty in men, but not in women. A positive correlation was found between hemoglobin concentration and age in adult Han males and females, but not in Tibetan males, and only at the higher altitudes, in Tibetan females. In both Tibetans and Hans, males had higher hemoglobin values than females at each altitude, but the gender differences increased with altitude in Han, whereas it either decreased or did not change in Tibetans. Examination of hemoglobin levels by histogram showed non-Gaussian distributions: Tibetan men and women had skewing to higher values, whereas Han men and women had skewing in the opposite direction. We conclude that increasing age and the effect of gender in Tibetans are associated with different hemoglobin responses to altitude than in Han, and we speculate that genetic influences may be involved.     

Controlled ovulation of the dominant follicle: a critical role for LH in the late follicular phase of the menstrual cycle

BACKGROUND: A method was sought to control ovulation of the dominant follicle and to test the importance of LH during the late follicular phase of the menstrual cycle. Menstrual cycles of rhesus monkeys were monitored, and treatment initiated at the late follicular phase (after dominant follicle selection, before ovulation). METHODS: The 2-day treatment consisted of GnRH antagonist plus either r-hFSH and r-hLH (1:1 or 2:1 dose ratio) or r-hFSH alone. In addition, half of the females received an ovulatory bolus of hCG. RESULTS: When treatment was initiated at estradiol levels >120 pg/ml, neither the endogenous LH surge, ovulation nor luteal function were controlled. However, when treatment was initiated at estradiol levels 80-120 pg/ml using either 1:1 or 2:1 dose ratios of FSH:LH, the LH surge was prevented, and ovulation occurred following hCG treatment. FSH-only treatment also prevented the LH surge, but follicle development appeared abnormal, and hCG failed to stimulate ovulation. CONCLUSIONS: Control over the naturally dominant follicle is possible during the late follicular phase using an abbreviated GnRH antagonist, FSH+LH protocol. This method offers a model to investigate periovulatory events and their regulation by gonadotrophins/local factors during the natural menstrual cycle in primates.     

Total lymphocyte count of 1200 is not a sensitive predictor of CD4 lymphocyte count among patients with HIV disease in Kampala, Uganda

Introduction

Total Lymphocyte Count (TLC) has been found to be an inexpensive and useful marker for staging disease, predicting progression to AIDS and death and monitoring response to ART. However, the correlation between TLC and CD4 has not been consistent. Access to HAART is expanding in Kampala, Uganda, yet there are no published data evaluating the utility of TLC as inexpensive surrogate marker of CD4 cell count to help guide therapeutic decisions.

Objective

To evaluate clinical illnesses and total lymphocyte count (TLC) as surrogate markers of the CD4 cell count in HIV infected persons being considered for ART.

Methods

A total of 131 patients were enrolled and evaluated by clinical assessment, TLC and CD4 count. Clinical illnesses and TLC dichotomized at various cut-point values were used to determine the sensitivity, specificity, and positive and negative predictive values (PPV and NPV) for the diagnosis of CD4 count <200 cells/mm³ among 100 participants fulfilling criteria for WHO clinical stage 2 and 3.

Results

A strong correlation was observed between TLC and CD4 (r = 0.73, p<0.0001). For all clinical syndromes, except pulmonary tuberculosis, the positive predictive values (PPV) for a CD4 count <200 cells/mm³ were high (>80%) but the negative predictive values (NPV) were low. Using the WHO recommended TLC cut-off of 1200 cells/mm³ to diagnose a CD4 less than 200 cells/mm³, the PPV was 100%, and the NPV was 32%.

Conclusion

Our data showed a good correlation between TLC and CD4 cell count. However, the WHO recommended TLC cutoff of 1200 did not identify the majority of WHO stage 2 and 3 patients with CD4 counts less than 200 cells/mm³. A more rational use of TLC counts is to treat all patients with WHO stage 2 and 3 who have a TLC <1200 and to limit CD4 counts to patients who are symptomatic but have TLC of >1200.     

Binding proteins for insulin-like growth factors in the human ovary: identification, follicular fluid levels and immunohistological localization of the 29-32 kd type 1 binding protein, IGF-bp1.

The occurrence of pregnancy-associated endometrial alpha 1-globulin (alpha 1-PEG), a 29-32 kd insulin-like growth factor binding protein, now termed type 1 or IGF-bp1, has been examined in the human ovary by monoclonal and polyclonal antibody based radioimmunoassay and immunohistological techniques. Follicular fluids aspirated from 51 follicles of 32 women undergoing hyperstimulation involving buserelin or clomiphene-based protocols contained 35.5-276.0 ng/ml (mean 101.0 mg/ml) of immunoreactive IGF-bp1. Mean fluid concentrations were three times the level of IGF-bp1 detected in paired serum samples, available for 21 women. Immunoreactive IGF-bp1 in follicular fluid exhibited similar dose-response curves to purified protein and amniotic fluid and immunoreactive IGF-bp1 coeluted in gel filtration with a peak of [125I]-IGF-1 binding corresponding to the elution profile of purified IGF-bp1. Gel filtration also revealed the presence in follicular fluid of a greater than 100 kd binding protein with a binding capacity equal to IGF-bp1 under the conditions employed. A highly significant correlation (P less than 0.001) was found between follicular fluid progesterone and IGF-bp1 and a correlation of lower significance was found between oestradiol and IGF-bp1 (P less than 0.05). However, only low levels of immunoreactive IGF-bp1 were detected in supernatant media of granulosa cells in culture (range undetectable to 2.3 ng/ml). Employing monoclonal antibody-based immunohistology, immunoreactive IGF-bp1 was consistently associated with luteinized granulosa cells of corpora lutea rather than paraluteal cells and its intensity of reactivity appeared to reflect luteal phase steroid hormone profiles. No consistent reactivity was detected in preovulatory follicles and granulosa cells in culture, although reactivity was associated with primordial oocytes. Immunoreactive IGF-bp1 was detected in six of nine supernatant media of explants of luteal tissue obtained from five corpora lutea, with levels ranging from undetectable to greater than 200 ng/ml. These observations suggest that IGF-bp1 is primarily related to luteinization of the granulosa and the resultant luteal cells, and if produced by the luteal cells, additional exogenous factors are required to induce production by granulosa cells in vitro.     

Complete and partial luteinized unruptured follicle syndrome after ovarian stimulation with clomiphene citrate/human menopausal gonadotrophin/human chorionic gonadotrophin

A total of 31 clomiphene citrate/human menopausal gonadotrophin(HMG)/human chorionic gonadotrophin (HCG)-stimulated cyclesin 28 patients were investigated to determine the fate of eachof the matured follicles. A standard stimulation regimen wasadhered to, and ultrasound as well as hormonal monitoring wasperformed. All follicles were measured by vaginal ultrasoundat –12, +35 and +45 h relative to HCG administration andat 7 days after HCG administration. Of the 220 follicles, 107(48.6%) ruptured. The number of ruptured follicles per cyclewas correlated with the mid-luteal progesterone concentration(r = 0.63, P = 0.0005). The probability of follicular rupturewas related to follicular diameter at 12 h before HCG administration;6% of follicles <12 mm in diameter ruptured compared with87% of follicles 18–19 mm. A complete luteinized unrupturedfollicle (LUF) syndrome was observed in six cycles (20%). Inthese cycles, follicular growth and oestradiol, progesterone,luteinizing hormone (LH) and follicle stimulating hormone (FSH)concentrations at 12 h before HCG administration were similarto those in cycles with follicular rupture. However, mid-lutealprogesterone concentrations were lower in complete LUF cycles(46.97 ± 8.95 nmol/1 versus 108.74 ± 12.27 nmol/1;P = 0.02). These data demonstrate that in stimulated cyclesmany follicles, usually the smaller ones, fail to rupture, evenafter HCG administration. Complete LUF syndrome, despite a strongexogenous ovulatory signal, and the absence of any differencein peri-ovulatory hormonal parameters, indicates that the defectcausing LUF resides in the follicle itself and/or hormonal changesduring the follicular phase.     

Thrombocytosis in preterm infants: a possible involvement of thrombopoietin receptor gene expression

Transient thrombocytosis is commonly observed in preterm infants after birth, but its physiological mechanism is still unknown. To understand the mechanism of the transient thrombocytosis in preterm infants we firstly evaluated a correlation between platelet counts and thrombopoietin (TPO) levels in preterm infants and next c-mpl mRNA levels on platelets in healthy preterm infants longitudinally during a half-year of life. The mean platelet counts in 45 very low birth weight infants (mean gestational age 27.4±1.8 weeks, mean birth weight 1047±249 g) was 230±71×10⁹/l just after birth and thereafter gradually increased to 579±178×10⁹/l by 5 weeks of age. The platelet counts continued this level for about next 8 weeks. Serum TPO levels soon after birth and at 1 month of age were significantly higher than those at the age of 2–6 months. There was a significant negative correlation between platelet counts and serum TPO values. The c-mpl expression levels on platelets at birth and at 1 month of age tended to be lower than those on platelets from adults, and the c-mpl levels gradually increased through 6 months of age, although they were still lower than those of adults. Our results suggest that low expression of TPO receptor on platelets until 1 month after birth cause a decreased TPO clearance and keep a high level of free TPO in blood, thereby promoting platelet production from megakaryocytes or their progenitors in bone marrow, resulting in the subsequent thrombocytosis in preterm infants.     

Apoptosis of Merkel cells in neurotrophin-3 null mice

Postnatal mice lacking neurotrophin-3 (NT3) are deficient in Merkel cells of touch domes and whisker follicles. We examined the mechanism of Merkel cell loss by immunocytochemistry and electron microscopy. Merkel cell of whisker follicles of NT3 null newborns exhibited decreased immunoreactivity for cytokeratin 8 and contained apoptotic bodies that were positive for cleaved caspase-3, a marker of active apoptosis. By electron microscopy, the Merkel cells displayed aggregation of chromatin along the nuclear membrane, with the marginated chromatin forming caps at the periphery of the nucleus. Ribosomes aggregated in the cytoplasm, while dense core granules characteristic of Merkel cells were still discernible. Finally, the Merkel cells and their nuclei fragmented into apoptotic bodies. None of the apoptotic Merkel cells were contacted by nerve fibers, and their desmosomal contacts with surrounding keratinocytes disappeared. After postnatal day 6 apoptotic Merkel cells were no longer observed, and the number of surviving Merkel cells was severely reduced. They were flat and contained few osmiophilic granules. We conclude that perinatal apoptosis is responsible for the loss of Merkel cells lacking innervation in NT3 null mice.     

Rapid accurate absolute granulocyte count determination during severe leukopenia using the flow cytometer

The absolute granulocyte count (AGC) in 125 blood samples from patients with total white blood cell counts of less than 1,000/microliter was estimated using three different methods, which were then compared for efficiency and accuracy. The three methods were 25 cell differential counts using Wright's-stained blood smears, granulocyte percentage estimates from WBC counting chambers, and combined narrow- and wide-angle light-scatter characteristics determined on a flow cytometer. A survey of clinical laboratories at University Hospital Cancer Centers revealed that the smear differential was the most-often-used method in those laboratories even when less than 25 cells could be counted. Consequently the data obtained from the counting chamber and flow cytometer methods were compared to the smear differential "standard" using linear regression, and outliers were identified. There was good correlation between AGC determined by smear differential and WBC counting chamber (correlation coefficient .911) and excellent correlation between the AGC determined by smear differential and the flow cytometer method (correlation coefficient .970). The flow cytometer method used in this investigation required minimal specimen preparation, and test results were available at a rate of 60 seconds/sample. The ease of sample preparation, speed, and statistical reliability of test results makes the flow cytometer an attractive alternate method of determining granulocyte counts on leukopenic patients as compared to the stained blood smear differential.