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Insulin resistance is one of the key factors in the pathogenesis of polycystic ovary syndrome (PCOS). The peroxisome proliferator-activated receptor gamma (PPARG) plays a role in the regulation of insulin sensitivity. The aim of the present study was to establish a possible association of the PPARG Pro12Ala polymorphism with PCOS and its effect on family and personal history, as well as on the metabolic and endocrine parameters in PCOS patients. A total of 151 PCOS patients and 179 healthy women of reproductive age were enrolled. History, body mass index (BMI), waist-to-hip ratio and the presence of phenotypic hyperandrogenism were recorded. Hormonal, metabolic and biochemical profiles were assessed. A molecular analysis for the genetic polymorphism was performed. One third (29.8%) of the PCOS patients were found to be carriers of at least one variant of the Ala allele (X/Ala), while 70.2% carried two wild-type Pro alleles (Pro/Pro), with an equal distribution observed in the control group. The PCOS patients carrying the X/Ala alleles exhibited lower serum fasting insulin levels, homeostatic model assessment of insulin resistance (HOMA-IR) and BMI compared to Pro/Pro carriers. This finding was significant only in the lean PCOS group. The polymorphic genotype exerted no effect on history, hormonal and clinical hyperandrogenism, lipid status or C-reactive protein, leptin, adiponectin, resistin and ghrelin serum levels in women with PCOS. In conclusion, although the PPARG Pro12Ala polymorphism is not a major determinant of PCOS in the Croatian population, it may exert a positive effect on insulin sensitivity and BMI. As these associations were recorded exclusively in the lean group of patients with PCOS, this polymorphism potentially contributes to a protective role against hyperinsulinemia and obesity.

The aim of this review is to analyze the relationship between the preovulatory progesterone (P) rise and the in vitro fertilization (IVF) pregnancy outcome. It also investigates the sources and effects of P level increase, including the underlying mechanisms and potential strategies in preventing its elevation during ovarian stimulation. The origin of production of P in the early follicular phase is adrenal which shifts toward the ovaries prior to the ovulation. Several factors contribute to the etiology of P level increase including the number of multiple follicles, the overdose of gonadotropins and poor ovarian response. Nowadays, the influence of the preovulatory P rise on IVF outcome remains controversial. Several authors have failed to demonstrate any negative impact, while others reported a detrimental effect associated with the rise of P. It seems that P rise (≤ 1.5 ng/ml or 4.77 nmol/l) may have deleterious effects on endometrial receptivity, namely, accelerating the endometrial maturation process that subsequently narrows the time-frame for implantation and thus decreases pregnancy rates. To prevent a P rise, it might be preferable to use milder stimulation protocols, earlier trigger of ovulation, cryopreservation of all embryos and transfer in the natural cycle.

Objective: To compare the efficacy and tolerability of two different types of vaginal progesterone (P), Crinone 8% gel (Fleet Laboratories Ltd., Watford, United Kingdom) and Utrogestan capsules (Laboratories Besins International, Paris, France), used for luteal support after in vitro fertilization (IVF) cycles., Design: Cohort study., Setting: In Vitro Fertilization Polyclinic, Zagreb, Croatia., Patients: A total of 285 women aged < or =37 years undergoing IVF-embryo transfer treatment., Interventions: Patients were treated with either Crinone 8% vaginal P gel (90 mg) administered daily, or Utrogestan vaginal capsules (2 x 100 mg) administered three times daily. Progesterone was administered from the day of oocyte retrieval (day 0) to menses or, in a case of pregnancy, until week 12., Main Outcome Measure: Clinical pregnancy rate. The tolerability and acceptability of both preparations were determined by a questionnaire given to patients., Results: The similar rates of clinical pregnancies (33.1% vs. 30.9%) [corrected] were obtained by using either Crinone 8% vaginal P gel or Utrogestan vaginal capsules. Overall tolerability and acceptability were significantly better in the Crinone group than in the Utrogestan group., Conclusions: The efficacy of the two vaginal P formulations was nearly the same, but the tolerability and acceptability of Crinone 8% gel were superior, in the opinion of patients.

Thermal dimerization of nitroso compounds in the solid state was investigated by using para-substituted nitrosobenzenes as model compounds. A mechanism that includes the interplay of topochemical reaction trajectories and phase transfer was proposed on the basis of FT-IR spectroscopic kinetics, time-resolved powder diffraction, and low-temperature X-ray structure determination. From shapes of the kinetic curves analyzed on the basis of the Avrami model, it was found that phase transfer could be triggered by a dimerization reaction of para-substituted nitrosobenzene to azodioxide, which, in turn, can be caused by different packing factors such as disorder in the starting nitroso monomer crystals. Since the represented model can be extended to a broad series of compounds, we propose it as a general method for investigations of solid-state reaction mechanisms.

Objective: To analyze the role of B-mode ultrasound, color and power Doppler examination and three-dimensional ultrasound with and without power Doppler facilities in evaluation of the peri- and postmenopausal ovaries., Methods: Review of the literature on ultrasonic assessment of the ovaries in postmenopausal patients., Results: Ultrasound has assumed an increasing role in the evaluation of the peri- and postmenopausal ovaries. Ovarian lesions are a cause of great concern because of their malignant potential and the limited ability to distinguish accurately between benign and malignant lesions using morphological sonographic criteria alone. Superimposed color Doppler imaging enables the visualization of vascularization and blood flow characteristics of the ovaries and/or ovarian lesions. It becomes possible to detect normal, suspicious and pathologic blood flow characteristics even in small blood vessels. Owing to superb quality of the images, three-dimensional sonography enables precise assessment of the ovarian lesions. Three-dimensional power Doppler ultrasound permits effective targeting and rendering of ovarian tumor vasculature, thus providing new diagnostic possibilities for early detection of ovarian malignancy and differentiation of benign from malignant ovarian pathology., Conclusions: Further technological development will contribute to more objective evaluation of ovarian tumor morphology and vasculature which will give us tremendous support in clinical decision making and planning the management.

Background: We present a 12-year-old girl with a 5-year history of progressive virilization., Results: Regarding elevated plasma levels of 17-hydroxyprogesterone (17-OHP) and androgens, normal ultrasound and CT scan of ovaries and adrenal glands, the nonclassic form of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency was presumed the cause of virilization. As the glucocorticoid therapy did not normalize high levels of 17-OHP and androgens, and the DNA analysis did not demonstrate a mutation causing CAH, a laparotomy was performed. Near the right ovary a tumor was found and extirpated. Pathohistological studies determined it to be a rare steroid cell tumor, 'not otherwise specified'. Within the next months the signs of virilization resolved and menarche occurred., Conclusions: Steroid cell tumor should be considered in differential diagnosis of virilization in childhood. Regarding the age of our patient and pathohistological findings of the tumor, her prognosis is favorable., (Copyright 2001 S. Karger AG, Basel)

Objective: To evaluate the influence of cryopreservation medium and freezing-thawing techniques on human sperm motility and morphology., Study Design: 63 semen samples were obtained from 39 donors to the artificial insemination programme. Possible effects of the sperm dilution with cryomedium on the motility were examined 10 min after exposure of 24 high initial quality semen samples to TEST-yolk ¿zwitterion-citrate-egg yolk extender containing TES [N-Tris (hydroxymethyl) methylaminoethane sulfonic acid] and Tris [(hydroxymethyl) aminomethane]¿ and human sperm preservation medium (HSPM). Post-thaw sperm motility from 24 frozen semen samples was examined comparing the cryoprotective efficacy of TEST-yolk and HSPM following different freezing techniques (vapour freezing, fast programmable freezing and slow programmable freezing). The relationship of sperm morphology to the effects of freezing was investigated on 39 semen samples following different freezing techniques. Post-thaw sperm motility from 39 frozen semen samples was compared among three groups divided according to the percentage of morphologically normal cells (<40, 40-50 and >50%) in fresh semen., Results: Exposure of spermatozoa to cryomedia for 10 min at room temperature significantly reduced motility in TEST-yolk treatment group for 9% and in HSPM treatment group for 18% (P<0.01). The recovery of motile sperms (mean+/-standard deviation) was 49+/-15.7, 43+/-15.2 and 52+/-16.8% when TEST-yolk was used and 34+/-17.8, 32+/-18.2 and 50+/-13.6% when HSPM was used as a cryopreservative following vapour freezing, and fast and slow programmable freezing, respectively. Following vapour freezing and also following fast programmable freezing, the recovery of motile sperm was significantly higher (P<0.05) after addition of TEST-yolk medium than after addition of HSPM. Post-thaw motility of the sperm cryopreserved in HSPM showed significant differences (P<0.05) after three different freezing techniques. The recovery of motile sperms was 57+/-26.4, 38+/-8.6 and 38+/-17.3% in groups with >50, 40-50 and <40% morphologically normal cells, respectively. The percentage of morphologically normal spermatozoa was reduced 8% after vapour freezing and 6 and 3% after fast and slow programmable freezing, respectively. The results were statistically analysed using SAS/STAT software., Conclusions: Slow programmable freezing was superior to vapour freezing and fast programmable freezing as a method for sperm cryopreservation. However, none of these methods of freezing had discernible effects on sperm morphology. Motility of spermatozoa decreased due to the exposure of semen to cryomedium. TEST-yolk was a superior cryomedium to HSPM. Fresh semen with more than 50% of morphologically normal cells showed the best recovery of motile cells after freezing and thawing.

A case of combined interstitial and intrauterine pregnancies after bilateral salpingectomy and in-vitro fertilization with embryo transfer is reported. The case was incorrectly diagnosed ultrasonographically as intrauterine triplets at 7 weeks gestation. The patient suffered from intra-abdominal bleeding at 14 weeks gestation. At laparotomy, a ruptured left interstitial pregnancy with a non-viable fetus was found in the left abdominal quadrant. The uterine defect was successfully repaired and gestational contents and blood were evacuated from the abdominal cavity. The intrauterine twin pregnancy progressed without incident, and a Caesarean section was performed at 36 weeks gestation, resulting in healthy male and female infants.

A case of simultaneous bilateral tubal pregnancy resulting from in-vitro fertilization and embryo transfer is presented. Repeated transvaginal ultrasound examinations confirmed an intrauterine sac but no fetus. A diagnosis of early missed abortion was incorrectly made and a curettage was performed. The pathological examination showed the presence of decidua and Arias-Stella phenomenon but no chorionic villi. Diagnostic laparoscopy and laparotomy performed 40 days after embryo transfer (eighth week of gestation), revealed bilateral tubal pregnancy. Bilateral salpingectomy was performed.

Objectives: To determine the concentrations of hyaluronic acid, FSH, P, and E2 in the follicular fluid (FF) obtained from IVF-ET patients and to assess the value of these measurements in predicting the outcome of fertilization., Design: One hundred eleven samples were retrospectively analyzed for the hyaluronic acid and hormone contents., Setting: University-based tertiary care center., Patients: Preovulatory FF samples were collected from 67 women undergoing IVF-ET treatment because of tubal absence or obstruction., Main Outcome Measures: The FF hyaluronic acid and hormone concentrations were compared according to the type of ovulation induction, follicular development, and IVF outcome., Results: According to the type of ovulation induction, a significantly lower hyaluronic acid concentration was found in FF harvested from the patients treated with GnRH agonist-hMG. No significant correlation was found between FF hyaluronic acid and either morphological maturity of the oocyte-cumulus complex or fertilizability of oocytes. The level of FSH was significantly higher in FF, yielding a mature oocyte-cumulus complex and from which the oocyte obtained successfully fertilized and cleaved. A significant increase in the E2 concentration was found in FF in which mature cumuli oophori were present. The levels of hyaluronic acid significantly correlated with FSH in FF., Conclusions: Expansion of the human oocyte-cumulus cell complex is an FSH-dependent phenomenon. The data are in agreement with the hypothesis that intrafollicular FSH plays an important role in the secretion of hyaluronic acid by granulosa cells and may act synergistically with E2 to enhance cytoplasmic maturation, resulting in successful fertilization.

Preovulatory serum estradiol and progesterone levels as well as their ratio were compared in different types of ovulation induction in order to determine whether these findings could be used to predict the number of preovulatory follicles, number of oocytes aspirated and embryos obtained. Significantly more oocytes were retrieved by follicular aspiration and significantly more embryos developed in patients receiving gonadotropin-releasing hormone agonist and human menopausal gonadotropins than in those given other ovulation inductors. On days -2 and -1 of the cycle, serum estradiol levels were significantly lower in pure follicle-stimulating hormone induction. Serum progesterone was significantly higher in pure follicle-stimulating hormone cycles on days -4 and -3. In clomiphene citrate and human menopausal gonadotropin induction, progesterone levels were significantly lower on days -2 and -1, and on the day of follicular aspiration. Ratios of estradiol/progesterone were lower in pure follicle-stimulating hormone group from day -3 to day -1 of the cycle. A significant correlation was found between estradiol and progesterone serum levels and the numbers of preovulatory follicles, oocytes and embryos. The study revealed the usefulness of serum estradiol and progesterone determinations in assisted reproduction.

A simple and rapid histochemical technique is described for demonstration of delta 5,3 beta-hydroxysteroid dehydrogenase activity in cumulus cells from preovulatory follicles aspirated for in vitro fertilization (IVF) of corresponding oocytes. Histochemical activity of delta 5,3 beta-hydroxysteroid dehydrogenase was demonstrated in samples of cumulus obtained from 62 oocytes recovered from 24 women. Patients were treated with clomiphene citrate in combination with human menopausal gonadotropins and human chorionic gonadotropin injections. The cumulus was found to contain small and large cell types. Small cells possessed more delta 5,3 beta-hydroxysteroid dehydrogenase activity predominantly in the area near the oocyte. Cytoplasmic vacuolation has been noted in large, pale cells with moderate or low enzyme activity. The most active cells were predominant in cumulus from which oocytes were fertilized. Significant differences have been found between high and low delta 5,3 beta-hydroxysteroid dehydrogenase activity of cumulus cells from mature oocyte-corona-cumulus complexes leading to a successful fertilization and cleavage of oocytes and between groups with different histochemical activity when aspirated complexes were scored immature and the IVF of oocytes has failed.

Sixty follicular fluids obtained from 26 women with either clomiphene citrate and human menopausal gonadotropins (hMG) or hMG-induced ovulation were analyzed for the contents of total proteins, fibrinogen, plasminogen, antithrombin III, ceruloplasmin, alpha-2 macroglobulin, alpha-1 antitrypsin and immunoglobulins (IgG, IgA, IgM). Concentrations of these proteins was correlated to the type of ovarian follicle growth induction. Follicular fluids from patients stimulated with clomiphene citrate-hMG contained significantly higher concentrations of ceruloplasmin than those treated with hMG alone. No significant differences in the concentrations of other proteins were noted between the two types of ovarian induction. A multivariate data analysis resulted in three Varimax factors (VRX I) suggesting that proteins with antiprotease activity in the follicular fluid may play a role in human follicle maturation. Follicular fluid Ig may reflect the degree of follicular wall permeability under hMG treatment. Accordingly, it may be assumed that a combination of different proteins described by VRX factors could be used for evaluation of ovarian stimulation.

Prostaglandin F2 alpha (PGF2 alpha), progesterone (P4) and estradiol-17 beta (E2) in follicular fluid were measured by radioimmunoassay in patients undergoing in vitro fertilization and embryo transfer. Follicular growth was induced using clomiphene citrate-hMG-hCG (15 patients) and FSH-hMG-hCG (4 patients). There was no significant difference in follicular fluid PGF2 alpha and P4 concentrations relative to oocyte maturity as assessed morphologically. The highest PGF2 alpha concentration was found in fluid from FSH-hMG-hCG cycles where fertilization occurred. The value is significantly higher (p less than 0.002) than in fluid from clomiphene-hMG-hCG cycles whether fertilization took place or not. There was no significant difference in P4 and E2 levels in relation to the type of ovarian induction or success in fertilization. Positive correlation between P4 and E2 in follicular fluid was found (r = 0.404). The positive correlation between total dose of hMG given to the patients and PGF2 alpha concentration in their preovulatory follicular fluid (r = 0.434) suggests that PGF2 alpha is secreted locally as the result of hMG and hCG stimulation. It is proposed that PGF2 alpha could be a biochemical marker for assessing the success of in vitro fertilization.

Forty-eight zygotes with more than two pronuclei were identified after in vitro fertilization, representing 6.1% of all fertilized oocytes. The chromosome preparations from pronuclear stage to the cleaved human embryos were examined. Prophase was found in eight out of ten zygotes. The spreading of chromosomes allowed an adequate counting in only two cases. Six of the eight preparations displayed a late prophase. In this stage each haploid group of chromosomes can be analysed separately. Kariogamy usually occurred 4 to 5 h after the pronuclei had disappeared, and polyploid number of chromosomes were found in well-spread metaphases. The chromosomal preparations were made for eleven human embryos arising from zygotes with three pronuclei. Out of ten preparations, where the chromosomes could be counted, seven embryos (70%) contained hypodiploidic groups of chromosomes. In two of the cases, however, triploid metaphases were found, and in the last one a triploid/diploid mosaicism.