Parthenogenetic activation: biology and applications in the ART laboratory.

Parthenogenesis is a reproductive strategy typical of lower species where a female gives birth to offsprings without a paternal contribution. On the contrary, parthenogenesis is not a form of natural reproduction in mammals even if mammalian oocytes, under appropriate stimuli, can undergo to parthenogenetic activation. This review describes the biological mechanisms regulating parthenogenetic activation in mammals and illustrates the fundamental differences between embryos and parthenotes. Ethical, legal and political concerns on the value of human embryos regulate and limit human embryological studies founded on the widespread belief that human embryos should not be created and studied for research purposes only. Based on the differences between parthenotes and embryos the use of parthenogenesis is proposed as an experimental tool to investigate embryo development which may solve many of the ethical concerns associated with the use of human embryos for experimental purposes. Examples of the possible uses of parthenotes in many field of research such as in vitro assays aimed to study some aspects of assisted reproductive technologies (ART), toxicology or stem cell are described and their validity is discussed.

Abnormal renal responses to calcium entry blockade in normotensive offspring of hypertensive parents.

In nine young normotensive subjects with no family history of hypertension and nine age-matched normotensive subjects with one parent with essential hypertension, effective renal plasma flow (p-aminohippuric acid clearance), glomerular filtration rate (inulin clearance), and excretion of sodium and exogenously administered lithium were measured for 90 minutes before and after administration of a single 20-mg oral dose of the calcium entry blocker nifedipine. Segmental tubular handling of fluid and sodium was estimated using lithium clearance as a marker of proximal tubular reabsorption. Nifedipine did not cause any change in subjects with no family history of hypertension, but in those with one hypertensive parent there was a marked increase in effective renal plasma flow (from 644 +/- 39 to 847 +/- 42 [SEM] ml/min x 1.73 m2; p less than 0.001) and a decrease in filtration fraction (from 17.6 +/- 1.0 to 12.6 +/- 0.4%; p less than 0.001), while the glomerular filtration rate was unchanged, thus suggesting a prevailing efferent vasodilation. Sodium excretion rate (p less than 0.02) and fractional sodium excretion (p less than 0.025) increased slightly but significantly in subjects with one hypertensive parent, but not in normotensive subjects with no family history of hypertension. Lithium clearance also rose (from 29.0 +/- 2.0 to 32.8 +/- 1.9 ml/min, p less than 0.001), and the derived value of fractional proximal reabsorption diminished (from 75.8 +/- 1.0 to 71.3 +/- 1.2%, p less than 0.001). Estimated distal delivery of sodium and absolute distal sodium reabsorption both increased significantly (p less than 0.005), while fractional distal sodium reabsorption was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Intercellular adhesion molecule-1 is expressed on human granulosa cells and mediates their binding to lymphoid cells.

Some immune cellular components have been recently demonstrated to play a critical role in ovarian physiology. Resident ovarian white blood cells are known to produce cytokines that modulate granulosa cell (GC) functions and differentiation. Moreover, it has been postulated that, during the formation of the corpus luteum and luteolysis, human luteal cells are able to interact with lymphocytes and macrophages through some adhesion molecules. This study was designed to examine, at messenger RNA and protein levels, whether intercellular adhesion molecule (ICAM)-1, known to be involved in leukocyte-cell binding, is expressed by human GCs. Furthermore, we also investigated whether this molecule could be involved in the complex events that allow the interaction between the ovary and the immune system. GCs, obtained from women undergoing in vitro fertilization procedures, were enzymatically dispersed with collagenase and cultured for different time periods. To assess the presence of ICAM-1 messenger RNA, total RNA obtained from freshly aspirated GCs and GCs luteinized in culture was reverse transcribed and then amplified using two oligonucleotide primers specific for the human ICAM-1 gene. A single major DNA band of the expected size (943 bp) was obtained. The identity of this material with the human ICAM-1 sequence was further confirmed by restriction enzyme analysis. Surface ICAM-1 protein was detected by flow cytometric analysis on luteinized GCs cultured for 7 and 15 days. Finally, to evaluate a possible functional activity of ICAM-1, a 51Cr-release-binding assay between peripheral blood lymphocytes and luteinized GCs was performed in the presence and absence of a monoclonal antibody against ICAM-1. As a result, lymphocyte adhesion to GC monolayers was significantly, but not completely, inhibited by the anti-ICAM-1 monoclonal antibody. These findings demonstrate that intercellular interactions between GCs and the immune system are, at least in part, mediated by the adhesion molecule ICAM-1. Based on this data, we might speculate that this molecule could participate in the remodeling processes of the ovarian endocrine compartment.

Testicular dysfunction in Hodgkin's disease before and after treatment.

Over a 7-year period, semen analysis was performed in 92 male patients with Hodgkin's disease prior to therapy. In 67% of patients semen revealed a decreased chance for fertility (i.e. oligozoospermia, asthenozoospermia and/or teratozoospermia). The mean basal levels of follicle-stimulating hormone (FSH), luteinising hormone, testosterone and prolactin were in the normal range. In 77 patients in complete remission after alternating MOPP/ABVD (mechlorethamine, vincristine, procarbazine, prednisone; doxorubicin, bleomycin, vinblastine, dacarbazine), testicular function was assessed. 87% of patients were azoospermic, 9% had semen abnormalities and only 4% were normospermic. Recovery of spermatogenesis was documented in only 17 of 42 (40%) reassessed patients after a median time of 27 months and was generally not affected by pretreatment sperm quality. After chemotherapy, the mean value of FSH [20.45 (S.E. 1.7) mUI/ml] was significantly superior compared with that of the mean pretreatment values. No difference was documented in the mean testosterone and prolactin values tested before and after treatment. Our findings indicate that, of patients with Hodgkin's disease, about half are affected by hypogonadism before starting chemotherapy. By utilising alternating MOPP/ABVD, persistent testicular dysfunction was documented in half of the patients.

Dopaminergic modulation of acute renal effects of nicardipine.

In order to investigate whether dopamine receptors are involved in the acute natriuretic effects of calcium channel blockade, experiments were carried out in humans with uncomplicated essential hypertension. In 8 hypertensives 5 mg intravenous nicardipine produced a large decrease in blood pressure and renal vascular resistance, a slight but significant increase in glomerular filtration rate (GFR) and marked natriuresis. When a second identical experiment was performed in the same subjects with the addition of 10 mg metoclopramide as dopamine blocker, changes in sodium excretion and vascular resistance were the same, but GFR did not increase during nicardipine. In a second set of experiments (n = 5) the same design was used, but nicardipine was infused at a dose of 0.01 mg/kg, ie, seven times less than in previous experiments. Under these conditions, blood pressure, renal vascular resistance and GFR did not change, but significant natriuresis occurred. Natriuresis was almost completely prevented by metoclopramide. In 5 additional hypertensives, metoclopramide alone was infused and 0.01 mg/kg nicardipine was added to the infusion. No change in any parameter was observed. Our findings suggest that the dopaminergic system participates in maintaining or increasing GFR during blood pressure reduction by calcium antagonists, although renal vasodilation and natriuresis are largely independent of dopamine receptor activity. In addition, when low doses of calcium antagonists produce natriuresis in the absence of renal or systemic hemodynamic changes (direct tubular effect), dopamine blockade is able to blunt this effect. This indicates a modulation by dopaminergic receptors of the direct tubular action of calcium antagonists.

Inhibitory effects of galanin on growth hormone (GH) release in cultured GH-secreting adenoma cells: comparative study with octreotide, GH-releasing hormone, and thyrotropin-releasing hormone.

The aim of the present study was to characterize in a large series (N = 12) of cultured somatotrope adenomas the in vitro effects of the neuropeptide galanin on growth hormone (GH) secretion. This was contrasted with two peptides known to be GH secretagogues (GH-releasing hormone [GHRH] and thyrotropin-releasing hormone [TRH]) and a peptide with a known GH-inhibitory effect (the somatostatin analog octreotide). Groups of three wells were incubated for 4 hours with growth medium alone (control incubation), galanin, GHRH(1-29)NH2, TRH, or octreotide. Galanin and octreotide were applied at concentrations of 0.1, 1, and 10 mumol/L, and GHRH and TRH at concentrations of 0.01, 0.1, and 1 mumol/L. Galanin was able to inhibit GH release in nine of 12 cultured somatotrope adenoma cells. This inhibitory effect was clearly dose-dependent in five adenomas. Overall, the mean GH nadir after galanin was -36.1% in nine responder adenoma cultures versus control wells. Octreotide inhibited GH release in five of eight cultured somatotrope adenoma cells. The mean GH nadir after octreotide was -32.7% in five responder adenoma cultures compared with control wells. GHRH and TRH were able to stimulate GH release, respectively, in seven of 11 and in six of seven cultured somatotrope adenoma cells. The mean GH peaks after either GHRH or TRH in responder adenoma cultures were, respectively, +71.5% and +143.7% compared with levels in the control wells. In conclusion, the consistency and potency of the in vitro GH-inhibitory effect of galanin in a large series of somatotrope adenomas are at least similar to those of the most effective available GH-lowering agent, the somatostatin analog octreotide.

Reliability of ovulation tests in infertile women.

OBJECTIVE: To assess the reliability of the most widely used clinical methods for predicting or confirming ovulation. METHODS: We monitored spontaneous cycles in 101 infertile women using basal body temperature (BBT), transvaginal ultrasound, a urinary stick system for LH surge, and three serum progesterone measurements in the midluteal phase. Transvaginal ultrasound monitoring was standard for ovulation detection and sensitivity. We calculated specificity and accuracy of each method compared with that standard. RESULTS: Follicular development and ultrasound evidence of ovulation were confirmed in 97 of 101 cycles (96%). Urinary LH surge preceded follicular rupture assessed by ultrasonography in all cycles and showed concordance with ultrasound-evidenced ovulation in 98 of 101 cases. The timing of BBT nadir had wide variability, and BBT and ultrasonography agreed in a similar percentage of cases (74%). Midluteal serum progesterone assessments showed ovulatory values in 93 subjects, and ovulation was concordant with ultrasonography in 90 subjects. CONCLUSION: Urinary LH was accurate in predicting ovulation with ultrasonography as the standard for detection, but time varied widely. The nadir of BBT predicted ovulation poorly. The BBT chart was less accurate for confirming ovulation, whereas a single serum progesterone assessment in midluteal phase seemed as effective as repeated serum progesterone measures.

Hexarelin, a novel GHRP-6 analog, stimulates growth hormone (GH) release in a GH-secreting rat cell line (GH1) insensitive to GH-releasing hormone.

Previous studies demonstrated that GHRP-6 has modest GH-releasing activity in primary pituitary cell monolayer cultures. However, the effects of this peptide have always been tested on cells very sensitive to GHRH. We have previously reported that GHRH is unable to stimulate GH secretion in the GH1 rat tumor cell line. The aim of the study was to assess for the first time the effect on GH secretion of the GHRP-6 analog, hexarelin, in the GH1 cells; moreover, we investigated the potential involvement of GHRH in the effects of hexarelin in the GH1 rat cell line. The GHRP-6 analog hexarelin (0.01-1 microM) significantly stimulated GH release in both normal and GH1 rat cells. The greatest GH-releasing effect of hexarelin was observed with the 1 microM dose both in GH1 (155+/-25% vs. control wells) and in normal rat pituitary cells (185+/-23% vs. control wells). GHRH significantly stimulated GH secretion in normal rat somatotrophs (3-fold increase). In this latter cell model, GHRH and hexarelin were demonstrated to have additive stimulatory effects on GH secretion. Conversely, GHRH did not affect hexarelin-stimulated GH release in GH1 cells at any of the doses used. Finally, 8Br-cAMP significantly stimulated GH secretion in both normal rat and GH1 cells. These results provide in vitro evidence that non-GHRH-mediated pathways for GHRP action exist. Moreover, the observation that cells not sensitive to GHRH can be significantly stimulated by hexarelin strongly suggests that GHRPs and GHRH have two distinct sites and modes of action at the pituitary level.

Computerized slow-staged freezing of semen from men with testicular tumors or Hodgkin's disease preserves sperm better than standard vapor freezing.

Two methods of freezing semen taken from patients with testicular tumors or Hodgkin's disease before treatment were compared. Many patients already have semen abnormalities, so an optimal method is extremely important. Ejaculates from 8 patients with testicular tumors and 20 with Hodgkin's disease were frozen by fast-freezing or by slow-staged freezing. Effects of motility, viability, and swelling after thawing were significantly impaired with both methods. However, cryosurvival was better after slow- than fast-freezing: motility 24% +/- 12.4% versus 15% +/- 11.2%; viability 24.1% +/- 11.4% versus 17.3% +/- 10.4%, swelling 33.3% +/- 11% versus 27.6% +/- 12.8%. The effects were equal for normal and abnormal sperm. Sperm from tumor patients should be frozen by slow-staged freezing method in spite of the higher cost and longer time.

Evaluation of semen and pituitary gonadotropin function in men with untreated Hodgkin's disease.

The reproductive capacities of 35 patients with Hodgkin's disease were assessed before treatment by semen evaluation and determination of basal hypothalamic-hypophyseal function and after stimulation with gonadotropin-releasing hormone (GnRH). Sixty-five percent of the patients had asthenozoospermia, 46% had teratozoospermia, and 28% had oligozoospermia. Normal semen was more frequently seen in asymptomatic patients (7 of 18) than in symptomatic patients (2 of 15). All the patients had normal basal follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels and significantly low testosterone (T) levels (P less than 0.01). The FSH response to 100 micrograms of GnRH was normal, but the LH responses were all significantly low, both as delta % (28.4% +/- 18.7% versus 52.4% +/- 25.3%, P less than 0.005) and as peak values (36.7 +/- 20.7 mIU/ml versus 59.4 +/- 26.4 mIU/ml, P less than 0.01). It is believed that abnormal semen is a specific symptom of Hodgkin's disease, secondary to functional insufficiency of the hypothalamic-hypophyseal axis, with a relative decrease in T production.

Intraperitoneal insemination in the treatment of male and unexplained infertility.

For couples with unexplained or male infertility, intraperitoneal (IP) insemination in induced cycles is a method that increases the chance of fertilization. Seventy-seven couples with male subfertility were subjected to 120 IP insemination cycles and 31 with unexplained sterility to 44 cycles. As a consequence of the treatment, 23 pregnancies were obtained, with pregnancy rates of 23% per cycle and 32% per patient for unexplained infertility and 11% and 17% for male subfertility. Pregnancy loss rate was quite elevated: 9 clinical abortions and 1 ectopic. Intraperitoneal insemination appears to be a relatively noninvasive way to deal with unexplained or male factor infertility, well worth trying before moving on to more invasive and costly approaches, such as gamete intrafallopian transfer or in vitro fertilization.

Efficacy of double intrauterine insemination in controlled ovarian hyperstimulation cycles.

OBJECTIVE: To investigate the effectiveness of double IUI and to determine the optimal timing of IUI in relation to hCG administration. DESIGN: Prospective randomized study. SETTING: Infertility Center, Department of Obstetrics and Gynecology, University of Milan. PATIENT(S): Patients with male factor and unexplained infertility undergoing controlled ovarian hyperstimulation (COH) and IUI. INTERVENTION(S): After COH with clomiphene citrate and gonadotropins, patients were randomly assigned to one of the following groups: group A received a single IUI 34 hours after hCG administration, group B received a double IUI 12 hours and 34 hours after hCG administration, and group C received a double IUI 34 hours and 60 hours after hCG administration. MAIN OUTCOME MEASURE(S): Number of follicles > 15 mm in diameter on the day of hCG administration, number of motile spermatozoa inseminated, clinical pregnancy rate. RESULT(S): Two hundred seventy-three patients underwent 449 treatment cycles: 90 patients were treated for 156 cycles in group A, 92 patients for 144 cycles in group B, and 91 patients for 149 cycles in group C. The overall pregnancies rates for groups A, B, and C were 13 (14.4% per patient and 8.3% per cycle), 28 (30.4% per patient and 19.4% per cycle), and 10 (10.9% per patient and 6.7% per cycle), respectively. There was a statistically significant difference between group B and groups A and C. CONCLUSION(S): Our data indicate that two IUIs performed 12 hours and 34 hours after hCG administration is the most cost-effective regimen for women undergoing COH cycles with clomiphene citrate and gonadotropins. Although the second insemination adds up to a slightly higher cost, it significantly increases the chance of pregnancy.

Effect of galanin on growth hormone (GH) response to thyrotropin releasing hormone of rat pituitary GH-secreting adenomatous cells (GH1) in culture.

The growth hormone (GH) releasing effect of thyrotropin-releasing hormone (TRH) and galanin, a 29-amino acid peptide widely distributed in mammalian CNS, alone or in combination was investigated in cultured rat pituitary tumor cells (GH1). TRH stimulated GH secretion in GH1 cells (maximal stimulation at the dose of 0.1 microM). Galanin alone had a significant GH inhibitory effect in GH1 cells at all the doses used. When the two peptides were administered in combination, no significant changes as compared to baseline levels were observed. The results of this study indicate that galanin has potent direct inhibitory effects on baseline and TRH-stimulated GH release from rat tumor cells.

Adenomyosis: a déjà vu?

Adenomyosis is a relatively frequent finding in series of hysterectomies performed for menorrhagia and dysmenorrhea. Evident selection biases of the available studies on adenomyosis have always limited the possibilities of defining the real clinical importance of the condition. Until now the only certain diagnoses have been made by histopathologists on uteri removed at surgery, but recently various sufficiently accurate techniques have been suggested which allow diagnosis on the uterus in situ. With the these methods it might be possible to obtain correct information on the epidemiologic characteristics of adenomyosis and to clarify whether it has a pathogenic role in unexplained ovulatory menorrhagia and juvenile dysmenorrhea. Furthermore, resectoscopic treatment has been proposed in some mild forms of adenomyosis to avoid hysterectomy, whereas it seems improbable that medical treatment can offer a definitive solution. The adoption of standard histologic criteria for adenomyosis seems important. Until this is done, it will be difficult to establish whether adenomyosis is really a disease or merely a paraphysiologic condition.

Long-term treatment with cimetidine does not essentially affect the hypothalamic-pituitary-gonadal axis in man.

The effect of cimetidine maintenance treatment on gonadal function has been assessed by seminal analysis plus prolactin and gonadotropin blood level concentrations. Nine patients, all with duodenal ulcer, 5 of whom had received 400 mg cimetidine at night for 18, 3 for 24 and 1 for 36 months, were studied. All patients were presumably fertile, having fathered a child born not more than 3 years before the start of cimetidine treatment. In all patients two seminal analyses were performed and blood specimens were obtained for radioimmunoassay estimation of FSH, LH and prolactin concentrations both in basal conditions and 20', 30' and 60' after the i.v. injection of Gn-RH (100 micrograms) and TRH (200 micrograms). All but one patient, in whom mild oligoasthenozoospermia was detected, had normal sperm counts and motility; moreover hormonal levels both in basal conditions and after releasing factor stimulation were found to be similar to those observed in a control group. Cimetidine maintenance treatment has no effect on sexual activity, the quality of seminal fluid or the pituitary secretion of gonadotropins or prolactin.

Abnormal uterine bleeding associated with iron-deficiency anemia. Etiology and role of hysteroscopy.

We reviewed the clinical and histologic records of 61 consecutive premenopausal women with abnormal uterine bleeding and moderate to severe iron-deficiency anemia investigated in a tertiary care and referral center. Excessive bleeding was caused by benign lesions in 67% of the cases and by anovulation in 25% and was unexplained in 8%. Hysteroscopy revealed an organic intrauterine lesion (submucous myomas in 38%, endometrial polyps in 13%, submucous adenomyomas in 3%) that could be treated endoscopically in more than half the patients. In populations without nutritional deficiencies, a woman of reproductive age with sideropenic anemia and no other evident cause of blood loss or systemic disease should be considered menorrhagic until proven otherwise. Hysteroscopy should be included in evaluations of abnormal uterine bleeding.

[Evolution and radiologic sequelae of pulmonary tuberculosis]. / Evoluzione e sequele radiologiche della tbc polmonare.

During the treatment of pulmonary tuberculosis, the radiologist is often asked the following questions: 1) is it really TB?; 2) is there any improvement?; 3) when is the X-ray check-up required?. In our opinion, the radiologist is in a position: 1) to confirm the diagnosis of TB; 2) to give a radiological diagnosis of "abnormality" but without a prognostic opinion; 3) to suggest, case by case, when the X-ray check-up is required. After some month or years, when radiological-clinical sequelae are present, the radiologist is often asked equally difficult questions: 1) is it still TB?; 2) is it still active?; 3) could it have caused the hemoptysis?. Again, in our opinion, the radiologist must bear in mind: 1) the not infrequent possibility of reinfections; 2) that the fine, smooth contour of the cavity, whose shape remains unchanged, does not necessarily signify absence of activity; 3) that, at the present time, other illnesses (bronchitis, bronchiectasis, lung cancer) are more frequently the cause of hemoptysis.

[Nuclear medicine in pneumonology in the light of 9 year's experience at the S. Luigi Gonzaga Hospital]. / Medicina nucleare in pneumologia alla luce di 9 anni di sperimentazioni all'Ospedale S. Luigi Gonzaga.

An attempt is made to draw a picture of the results obtained with nuclear medicine in the pneumological field in the light of 9 years experience. An attempt is also made to identify from among recently introduced techniques (T.A.C., echotomography) which ones might be substituted for radioisotopes and when, and on what occasion they can be used as complementary treatment. It is concluded that nuclear medicine is of absolute value for functional examinations of the respiratory apparatus (perfusors and ventilators) and for lung onconscannning with Ga67 citrate and Bleomycin-Co57 under present circumstances and that in other fields certain morphological scans (bone scans) are still useful by virtue of their cost-benefit ratio.

[Indications for radiotherapy of lung cancer in the light of its clinical reality]. / Indicazioni del trattamento radiante del carcinoma del polmone alla luce della sua realtá clinica.

Reference is made to a large series and the literature in an attempt to single out cases where radiation management is indicated in the curative, palliative and anatalogic treatment of lung cancer, an in association with other therapies. It is pointed out that each case much be examined on its merits in the light of the histological type, the extent of the process, and its possible metastasis. The conclusion is drawn that radiotherapy plays a useful part in the albeit slight increase in survival of lung cancer patients under acceptable general conditions.

[Echography in the study of thoracic pathology. II-- Guided biopsy]. / L'ecografia nello studio della patologia toracica. Nota II--Guida alla biopsia.

The usefulness of ultrasonic studies for guiding the needle during biopsy of solid lesions or evacuation of saccate effusions and empyemas. Is a explained possible diagnostic protocol is then proposed which includes the ultrasonic examination in the diagnostic approach to parietal, mediastinic or diaphragmatic lesions.