Velimir Šimunić

Local estrogen treatment in patients with urogenital symptoms.

Objectives: Determination of the efficacy and safety of vaginally administered low dose (25 μg) micronized 17β‐estradiol in the management of patients with urogenital symptoms. Methods: A total of 1612 patients with urogenital complaints were randomized to receive 25 μg of micronized 17β‐estradiol (n=828) or placebo (n=784) in a multicenter double‐blind placebo‐controlled study running for 12 months. Female patients were treated once a day over a period of 2 weeks, and then twice a week for the remaining of the 12 months with an active or placebo tablet. The assessment included full history‐questionnaire, micturition diary, gynecologic and cystometric examination, transvaginal ultrasound, and serum 17β‐estradiol level determination. It was carried out at the beginning, and after 4 and 12 months of treatment. Results: The overall success rate of micronized 17β‐estradiol and placebo on subjective and objective symptoms of postmenopausal women with vaginal atrophy was 85.5%, and 41.4%, respectively. A significant improvement of urinary atrophy symptoms was determined in vaginal ERT group as compared with the beginning of the study (51.9% vs. 15.5%, P=0.001). The maximal cystometric capacity (290 ml vs. 200 ml, P=0.023), the volume of the urinary bladder at which patients first felt urgency (180 vs. 140, P=0.048), and strong desire to void (170 ml vs. 130 ml, P=0.045) were significantly increased subsequent to the micronized 17β‐estradiol treatment. The number of patients with uninhibited bladder contractions significantly decreased following micronized 17β‐estradiol as compared with pretreatment values (17/30, P=0.013). Side effects were observed in 61 (7.8%) patients treated with low dose micronized 17β‐estradiol. Therapy with 25 μg of micronized 17β‐estradiol did not raise serum estrogen level nor stimulated endometrial growth. Conclusions: Local administration of 25 μg of micronized 17β‐estradiol is an effective and a safe treatment option in the management of women with urogenital complaints.

Follicular fluid contents of hyaluronic acid, follicle-stimulating hormone and steroids relative to the success of in vitro fertilization of human oocytes * †

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.

Comparison of protective media and freezing techniques for cryopreservation of human semen

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.

Prostaglandin F2α, progesterone and estradiol concentrations in human follicular fluid and their relation to success of in vitro fertilization

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.

Ultrasonic assessment of the peri- and postmenopausal ovary

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.

Protein concentration in pre‐ovulatory follicular fluid related to ovarian stimulation

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, α‐2 macroglobulin, α‐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.

Number of follicles, oocytes and embryos in human in vitro fertilization is relative to serum estradiol and progesterone patterns during different types of ovarian hyperstimulation

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.

Lipid and Lipoprotein Contents of Human Follicular Fluid

The concentrations of total cholesterol, phospholipids, triacylglycerols and lipoproteins were measured in 87 follicular fluids obtained from 35 women undergoing in vitro fertilization and embryo transfer. The results were correlated with the levels of progesterone in follicular fluid. Two different types of ovarian stimulation were used. High density lipoproteins were the dominant lipoproteins found in the preovulatory follicular fluid. Low density lipoproteins were absent or appeared in trace amounts. Significantly higher triacylglycerol and high density lipoprotein levels were found when stimulation with human menopausal gonadotropins and chorionic gonadotropin was applied, as compared to the clomiphene citrate-menopausal gonadotropin-chorionic gonadotropin menstrual cycle. In both groups, extracorporal fertilization resulted in cleavage of oocytes and embryo transfer. No significant correlation between any follicular fluid lipid and progesterone concentration was found. The lipids estimated in the follicular fluid appeared to have no influence on the oocyte fertilizability. The presence of triacylglycerols and high density lipoproteins in the follicular fluid may indicate follicular wall permeability under the treatment with menopausal gonadotropins.

Chromosomal preparations of human triploid zygotes and embryos fertilized in vitro

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.

Fertility preservation with ovarian stimulation protocols prior to cancer treatment

Abstract: An increasing trend towards later childbearing has been reported recently in many developed countries. Although the incidence of reproductive age in women who have delayed pregnancy with cancer is 10%, they may be concerned regarding the preservation of ovarian function due to advanced fertile age and with the impact of cancer treatment on later fertility. Among multiple strategies controlled, ovarian stimulation for embryo or oocyte cryopreservation is currently the most established method for fertility preservation. It is important to choose the appropriate ovulation induction protocol prior to oncologic treatment, because most of these patients have only the chance of a single cycle to conceive. Current treatment protocols offer a minimal time delay until oncologic treatment is commenced. In urgent settings, random-start ovarian stimulation represents a new technique which provides a significant advantage by decreasing the total time of the treatment, because it may be started irrespective of the phase of the cycle without compromising oocyte yield and maturity before cancer treatment. However, in patients with oestrogen-sensitive cancers stimulation, protocols using letrozole are currently preferred over tamoxifen regimens, and therefore, it may be highly advisable to use letrozole with gonadotrophins routinely as a safe, effective and novel protocol of ovulation induction.