Veselko Grizelj

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.

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.

Detection and typing of human papillomaviruses by polymerase chain reaction in cervical scrapes of Croatian women with abnormal cytology

The association between certain human papillomaviruses (HPV) and cervical intraepithelial neoplasia (CIN) is well documented, but still unknown among Croatian women. In 1995, women between the age of 17 and 64 with cytomorphologically abnormal smears (CIN I–IV) were tested for the presence of HPV. Consensus and specific primers were used in the polymerase chain reaction (PCR) to detect the most common types: 6, 11, 16, 18, 31 and 33, as well as the unknown-risk HPV types (HPV X). Out of 379 specimens, 163 (43%) contained one or more HPV types. Coinfection with different HPV types in the same sample was observed in 16 cases. Beside low-risk HPV 6/11 (25.8%) the most frequently observed types were high-risk HPV types 16 (20.2%) and 31 (17.8%). Globally, the HPV positivity rate declines with age. The presence of HPV DNA significantly increased from 35.5 to 61.1% along with the severity of the cervical intraepithelial neoplasia (CIN I– IV). HPV type 6/11 was strongly associated with CIN I (33.8%), HPV type 31 with CIN II (22.9%), and HPV type 16 with CIN III (50%).

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.

Pregnancy following the laparoscopic bipolar electrocoagulation of polycystic ovaries resistant to medicamentous ovulation induction – a case report

The case of a primarily infertile patient with polycystic ovaries (PCOS) resistant to medicamentous ovulation induction is presented. The preoperative condition, laparoscopic ovarian drilling using an original technique of bipolar electrocoagulation and consecutive spontaneous pregnancy and delivery are described. This case suggests that bipolar forceps with jaws 1 mm wide could be a useful instrument for laparoscopic ovarian drilling.

Histochemical demonstration of a Δ5,3β-hydroxysteroid dehydrogenase activity of cumulus cells related to the maturity and developmental potential of recovered oocytes**Supported by Federal Scientific Council grant NG9 and Research grant No 2.04.01.03.10 from the Scientific Board of Croatia, Zagreb, Yugoslavia.††Presented in part at the XIII World Congress of Fertility and Sterility, Marrakesh, Morocco, October 2 to 6, 1989.

A simple and rapid histochemical technique is described for demonstration of Δ 5 ,3 β -hydroxysteroid dehydrogenase activity in cumulus cells from preovulatory follicles aspirated for in vitro fertilization (IVF) of corresponding oocytes. Histochemical activity of Δ 5 ,3 β -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 Δ 5 ,3 β -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 Δ 5 ,3 β -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.