Study of aneuploidy in normal and abnormal germ cells from semen of fertile and infertile men.

This study was undertaken with the aim of investigating the cytogenetic constitution of normal as well as abnormal spermatozoa and immature germ cells found in semen of normal men and infertile patients. A specific protocol of double in-situ hybridization for chromosomes 1 and 17 based on colorimetric detection of the hybridization signals (ISH) and brightfield microscopy analysis of cellular morphology was applied. Also the influence of paternal age on sperm aneuploidy was investigated. We found that, at least in the age range analysed (28-54 years) and for semen of good quality (total normal motile counts above 10 x 10(6)) (n = 17), paternal age has no influence on baseline rates of sperm aneuploidy. However, with decreasing semen quality (total normal motile sperm counts below 5 x 10(6)) (n = 6) significantly higher rates of sperm aneuploidy for autosomes 1 and 17 were scored (0.8 versus 1.43%) (P < 0.001). Regardless of the type of semen analysed, a number of morphologically abnormal spermatozoa were found to be hyperhaploid or diploid in a high percentage of cases (20 and 10% respectively). The same was found for immature germ cells (aneuploidy rate of 18%). We conclude that in infertile men with poor quality semen a direct relationship may exist between the impairment of the spermatogenesis process (as reflected by an increased production of morphologically and cytogenetically abnormal germ cells) and rates of baseline aneuploidy occurring in normal spermatozoa. Infertile couples undergoing assisted reproduction treatment need to be counselled about the risk of using spermatozoa which may carry higher rates of non-disjunction for different chromosomes. While sperm hyper- or hypohaploidy for some chromosomes (X,Y) implies counselling couples about the risk of abnormal phenotype in their offspring, most autosomal sperm aneuploidies probably translate only into lower rates of embryo fertilization and survival.

Comparison of a blocking vs. a flare-up protocol in poor responders with a normal and abnormal clomiphene citrate challenge test.

This study aimed to standardize the clomiphene citrate test (CC-t) in our laboratory while comparing two different protocols of controlled ovarian stimulation in poor responders. One hundred and forty-four patients scheduled for assisted reproductive techniques were submitted to the CC-t within 3 months before starting stimulation; 133 underwent controlled ovarian stimulation with a blocking protocol. Poor responders in the first cycle (n = 30) were subsequently treated with a flare-up protocol. Although it was not statistically significant, more patients reached oocyte retrieval with the flare-up protocol. In the completed cycles, more gonadotropin ampules (55 +/- 15 vs. 34 +/- 13; p < 0.001) and more stimulation days (12.6 +/- 1 vs. 11.6 +/- 1.2; p < 0.005) were needed in the blocking than in the flare-up protocol. No difference was observed in peak 17 beta-estradiol levels, preovulatory follicles, oocytes retrieved or pregnancy rate between the two protocols. According to the threshold values, established on CC-t of patients who obtained a clinical pregnancy (n = 44), the incidence of abnormal results was 10%. All but one patient with abnormal CC-t were poor responders during the first stimulation cycle. The flare-up protocol did not improve the ovarian response in these patients.

Comparison of gonosomal aneuploidy in spermatozoa of normal fertile men and those with severe male factor detected by in-situ hybridization.

The purpose of the study was to analyse the frequency of sex-chromosome numerical abnormalities in human spermatozoa of infertile men by using a standardized experimental protocol of double target in-situ hybridization (ISH). The experiments were performed on decondensed sperm heads from 15 infertile patients (six cases of unexplained infertility and nine cases of severe oligoasthenoteratozoospermia). Three men of proven fertility were used as controls. The probes employed recognized the centromeric regions of human X chromosome and the long arm of the Y chromosome. In a smaller number of cases, additional experiments of double ISH were performed using centromeric probes for chromosomes 1 and 17. Signal detection was based on protocols of enzymatic cytochemical reactions. A total of 24,508, 24,679 and 42,285 cells were scored in the control, unexplained infertility and severe male factor groups of patients respectively. In all the patients in the ISH efficiency result was approximately 98%. In controls, unexplained infertility and severe male factor patients, the frequency of morphologically normal sperm cells carrying an abnormal chromosome constitution (XX or YY or XY or > 2 sex chromosomes signals) was 0.86, 0.75 and 1.35% respectively. The value of this last group of patients (severe male factor) was significantly higher than in the other two groups of patients (P < 0.008). The same findings were made using the autosomic probes. Our preliminary data support the possibility of an increased risk from paternal origin sex chromosome aneuploidies in children born after intracytoplasmic sperm injection (ICSI). Further investigations of the cytogenetic constitution of spermatozoa from severe male factor patients is warranted.

Constitution of semen samples from XYY and XXY males as analysed by in-situ hybridization.

A brightfield microscopical in-situ hybridization (ISH) technique was applied to semen samples of two 47,XYY males, one 46,XY/47,XXY/XXY male with fertility problems, and two normal 46,XY men, who served as controls. The use of a standardized nuclear DNA decondensation method, together with double-target ISH and morphological staining, allowed an accurate study of the sex chromosomal content and morphology of spermatozoa. In the males carrying an extra sex chromosome, we detected X- and Y-bearing spermatozoa in a ratio which did not differ significantly from the 1:1 ratio found in normal males. Aneuploidy for the sex chromosomes was found in approximately 15% of the spermatozoa of both XYY males and in 3% of the XXY male. The most striking finding was the relatively low percentage of spermatozoa in these patients, with an average of 65% in the XYY males and 84% in the XXY male. The other cells represented immature germ cells (IGC), including spermatogonia and spermatocytes arrested at various stages of spermatogenesis. Apparently, in XYY or XXY men, these IGC are shed into the semen to an increased extent as compared to normal, fertile men. The sex chromosome constitution of these IGC was heterogeneous. However, the finding that the majority of spermatozoa in semen of 47,XYY and 47,XXY males carried a single sex chromosome strengthens the hypothesis that a 46,XY germ cell line must be present, apparently with a proliferative advantage over the 47,XYY or 47,XXY cells.

Computerized colposcopy: results of a pilot study and analysis of its clinical relevance.

OBJECTIVE: To evaluate the clinical usefulness of a computer based system that stores and analyzes high-resolution digital colposcopic images. METHODS: Three hundred patients referred to our institutions for evaluation of abnormal cervical cytology underwent colposcopy with development of digital images. The first 70 patients were used to standardize the system. The images obtained from 188 evaluable patients were analyzed and scored by software according to internationally recognized colposcopic criteria. The results were compared with traditional colposcopic diagnoses and with corresponding histology results using kappa statistics for inter-observer agreement and McNemar test for significance. RESULTS: The exact concordance rate between computer-aided impressions and histology (85.1%, kappa = 0.77) was significantly higher (P < .001) than that observed between traditional colposcopic findings and histology (66%, kappa = 0.40). The computer-assisted colposcopy was much more accurate than traditional colposcopy in diagnosing high-grade lesions (91.2 versus 61.8% of exact concordance, P < .001) as well as normal histology (74.1 versus 34.5%, P < .001), but not significantly different when evaluating low-grade lesions (89.6 versus 86.5%). CONCLUSION: The integration of computer imaging and colposcopy can improve the colposcopic diagnostic accuracy. An inexperienced colposcopist may benefit from computerized support to obtain the most appropriate histologic specimen, and eventually access to distant consultation via modem or through on-line services. An additional advantage is the ability to develop a space-saving permanent record of digitized images readily available to review a patient's cervical history or perform effective programs of quality control in colposcopy.

Unexpected pregnancies after tubal recanalization failure with selective catheterization.

OBJECTIVE: To evaluate selective salpingography sensitivity. DESIGN: Retrospective study. SETTING: Obstetrics and Gynecology Department, University of Genoa, Italy. PATIENTS: One hundred seventeen patients previously submitted to selective salpingography because of unilateral or bilateral proximal tubal injection failure. RESULTS: Seven pregnancies, one of which was ectopic, were obtained in 17 patients who had only recanalized tubes available for conception; 15 pregnancies were obtained in 39 patients who had one tube recanalized and one already patent; 3 tubal pregnancies were obtained in 12 patients who had only one tube already patent; 4 pregnancies, one of which was ectopic, were obtained in 19 patients who had neither patent nor recanalized tubes. CONCLUSIONS: Selective salpingography can give false-positive results; therefore, it is possible to obtain a pregnancy even after selective salpingography failure.

Luteal phase defects in assisted reproduction: the role of luteal phase supplementation.

Data from literature show that whereas in non-GnRH-a controlled ovarian hyperstimulation cycles luteal phase supplementation is not crucial, its use in GnRH-a/gonadotropins protocols seems to lead to a definite reduction of the negative effects of such drugs. The most important side effect of the hCG use as luteal support is the increased rate of the ovarian hyperstimulation syndrome (OHSS). Therefore its use should be reserved for very selected patients, for those subjects with preovulatory levels of E2 < 2500 pg/ml. As regard the use of progesterone, most authors agree that it has less efficacy than hCG but this depends on the administration route.

Endometrial morphology after 12 months of vaginal oestriol therapy in post-menopausal women.

The endometrial effect of long-term vaginal oestriol (E3) therapy for urogenital atrophy was assessed in 23 post-menopausal women. Hysteroscopic and histological examinations were performed in each patient to assess endometrial atrophy before treatment and after 6 and 12 months of therapy (0.5 mg vaginal E3 for 21 days, then 0.5 mg twice weekly). The primary atrophic picture was confirmed at the end of the 6th month in all but one of the patients. In one case, the histology showed an abnormal stromal reaction with no epithelial alterations. Treatment was continued and after the 12th month complete atrophy was confirmed both hysteroscopically and histologically in all patients. Efficacy as regards vaginal and urogenital complaints was good. Our results demonstrate that in women with endometrial atrophy effective and well-tolerated treatment with vaginal E3 can be safely continued for up to 12 months.

Transcervical selective salpingography: a diagnostic and therapeutic approach to cases of proximal tubal injection failure.

OBJECTIVE: Evaluation of selective salpingography for diagnosis and treatment of tubal injection failure during hysterosalpingography (HSG). DESIGN: Prospective study. SETTING: Obstetrics and Gynecology Department, University of Genoa (Italy)--tertiary care. PATIENTS: One hundred eighty infertile women with unilateral or bilateral proximal tubal injection failure during HSG were submitted to the procedure. INTERVENTION: Under fluoroscopy, a 4.5-F nylon catheter (3-F tip) was inserted into the ostium with or without the aid of a J-shaped, coaxial, angiographic guide wire, and 2 to 3 mL of contrast medium were injected. The procedure lasts 20 to 30 sec/tube. MAIN OUTCOME MEASURES: Of 155 tubal ostia, 145 (94.2%) were catheterized. RESULTS: Of the 146 catheterized tubes, 110 (75%) were rendered patent. Of the others, 21 (14.3%) presented hydrosalpinx or distal obstructions, and isthmic obstruction was present in 5 (3.4%). Patency of at least one tube was achieved in 82 (81.2%) of the 101 catheterized women; 8 conceived spontaneously and 11 after gamete intrafallopian transfer to the recanalized tube. CONCLUSIONS: During HSG, selective salpinography can be performed when proximal injection failure is observed to determine its cause or to restore patency.

'Blind' transcervical transfer of gametes in the fallopian tube: a preliminary study.

The aim of the study was to assess, in a small number of volunteers, the feasibility of transferring gametes by blind transcervical tubal catheterization. Forty patients underwent ovarian stimulation with a combination of a luteinizing hormone-releasing hormone analogue and exogenous gonadotrophin. Twenty-six patients were submitted to echographical oocyte retrieval and subsequent blind transcervical tubal transfer of the gametes. Successful cannulation of the tube was achieved in 18 subjects, at the first attempt and in four subjects at the second attempt; in the remaining four subjects, three or more attempts were needed. No complications were recorded during the tubal transfer. Seven clinical pregnancies (27% of all transfers) were obtained: three ended in abortion (11%), one was ectopic (4%) and three were ongoing (11%). These preliminary results encourage larger studies using this approach.

Comparison of different ovarian stimulation protocols for gamete intrafallopian transfer in patients with minimal and mild endometriosis.

Three different stimulation protocols were tested in patients affected by stage I and II endometriosis with no other causes of infertility, and scheduled for the gamete intrafallopian transfer technique. In two protocols a gonadotropin hormone-releasing hormone analog was used. The analog was started 6 months before stimulation in the former and along with the exogenous gonadotropin in the latter. Patients receiving only gonadotropin served as controls. Sixty patients were selected for this study; 55 reached laparoscopy. Whereas patients receiving either gonadotropin alone or simultaneous analog and gonadotropin had similar pregnancy rates, this was much higher in the patients undergoing a prolonged, medically induced hypoestrogenism. Prolonged analog pretreatment before ovarian stimulation may give better chances of success in endometriosis patients undergoing assisted reproduction techniques.

The duration of pituitary suppression by means of intranasal gonadotropin hormone-releasing hormone analogue administration does not influence the ovarian response to gonadotropin stimulation and success rate in a gamete intrafallopian transfer (GIFT) program.

The aim of the study was to compare the ability to prevent endogenous luteinizing hormone interferences, ovarian response, and success rate between two groups of patients undergoing GIFT procedures and treated with the same stimulatory protocol but with a different timing in the administration of the gonadotropin hormone-releasing hormone analogue (GnRH-a). The former underwent a concomitant administration of gonadotropin and analogue; the latter started stimulation only after the achievement of complete hypogonadotropinism. The analogue was always given intranasally and stimulation was identical in the two groups. Our results showed that (1) prevention of premature luteinization is obtained with both approaches and (2) no significant difference in terms of length of stimulation, gonadotropin doses, ovarian response, and success rate was recorded between the two groups.

Gamete intrafallopian transfer and plasma chorionic gonadotropin dynamics: evidence of a delayed appearance.

The purpose of this study was to compare early human chorionic gonadotropin secretion in two groups of pregnancies. These pregnancies were obtained, in one group, during a natural cycle, while, in the other group, they resulted from gamete intrafallopian transfers performed in hyperstimulated cycles. A logarithmic regression analysis was chosen to allow statistical comparison of serial plasmatic evaluation as hormone determinations were obtained, among patients, at different postovulatory days. The regression lines of the two groups of patients presented similar slopes. The unpaired t test performed on calculated data for 3 different days constantly revealed significantly higher levels (P less than 0.005) of natural vs stimulated cycles; thus a different origin on the x axis (days of the luteal phase) is deduced. These findings support the possibility of a delay in the appearance of human chorionic hormone in the patients undergoing gamete intrafallopian transfer technique.

Dibromochloropropane (DBCP) effects on the reproductive function of the adult male rat.

In order to evaluate the effect of dipromochloropropane (DBCP) on reproductive function, groups of male adult Wistar rats were injected subcutaneously with different doses of DBCP. A strictly dose-dependent effect resulting in histological alteration principally toward the seminiferous tubules was observed. Sperm count and sperm motility showed azoospermia or oligoasthenospermia with a significative recovery in the lower dose of DBCP treated rats. Among the enzymatic activities evaluated as biochemical markers of testicular function: LDH was not appreciably affected by the DBCP treatment while GGT and NADPH-cyt P450-reductase significantly enhanced suggesting an induction of the detoxification processes.

Biology of nidation and ectopic implantation.

The incidence of ectopic pregnancy has been steadily growing during the past decade; this fact can only partially be related to known factors (PID, use of IUDs, minipill, inductors of ovulation, sterilization reversal). Ectopic pregnancy is still a frequently misdiagnosed pathology; nevertheless, the recent advances in early diagnosis have allowed a decrease in the mortality rate and an improvement in subsequent fertility. In absence of obvious tubal abnormalities it is difficult to assess the cause of an ectopic implantation. Yet the factors regulating and nidation of the embryo in the human are far from clear. The transport of the embryo in the oviductal fluid depends on ciliary beat (generally regarded as the leading factor) and on muscle contractions mainly in the ampullary-isthmic and in the utero-tubal junction, where a sphincter action is stimulated by estrogens and relaxed by progesterone. Other substances (catecholamines, prostaglandins, oxytocin) are thought to be involved in ovum transport, although their role is unclear. Even less is known about local influence of the embryo on the oviduct. During preimplantation and implantation both the embryo and the mother engage in an elaborate interaction, elsewhere referred to as a 'conversation', involving long-and short-range signals. An essential role is played by the endocrine luminal milieu. A local action of estrogens on a progesterone-primed endometrium may be required for either the release of crucial signals for the blastocyst activation, or to make epithelial cells sensitive to the presence of the embryo, thus inducing the decidualization. In some animals, these estrogens are not ovarian, but synthesized by the blastocyst itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Variations in some of the body fluid hormone levels currently used for endocrine monitoring during the third trimester of pregnancy.

We have investigated the variability of four hormonal parameters used for the endocrine monitoring of the third trimester of pregnancy (E3, E4, HPL, SP1). We have observed no circadian rhythm of Estriol, Estetrol, HPL, and SP1. The within-a-day and the day-to-day variations are much greater for E4 than for E3 and for HPL than for SP1. The standard deviations were great for the steroids, especially for E4 and much less for the protein hormones, especially SP1. This implies that it is necessary to have serial assays of the steroids to have any confidence in the levels, while good monitoring could be guaranteed with a smaller number of assays for HPL, and SP1.

Estradiol and estetrol plasma levels before and after intravenous administration of dehydroepiandrosterone sulfate in normal and pathologic pregnancies.

Intravenous injections of 50 mg dehydroepiandrosterone sulfate (DHEAS) were given to 7 women with normal pregnancies and 5 with pathologic pregnancies and the serum levels of 17 beta-estradiol and estetrol were assayed before and at 15 or 30-minute intervals for three hours after the injection. All tests were carried out during the 25th to 36th week of amenorrhea. Serum estradiol rose rapidly in normal subjects and remained high to the end of the test. In patients with gestational pathology the estradiol pattern was not significantly different from that of the controls. Esterol plasma levels showed a biphasic pattern with an initial rise at 30 min. and a second rise at 90 min. in normal pregnancies, whereas in pathologic pregnancies this response was either lacking completely or was markedly reduced compared to the controls.