Sperm chromosomal abnormalities in patients with unexplained recurrent abortions.

Cytogenetic studies showed that about half of concepti were chromosomally abnormal in first trimester abortions. Sperm chromosomal abnormalities in men with normal karyotype could occur during spermatogenesis. The objective of this study was to evaluate sperm chromosomal abnormalities in patients with unexplained recurrent abortions. A total of 14 couples with normal karyotype, and negative workup for endocrine, immune and anatomical causes of recurrent abortion was investigated. Semen analysis was performed and chromosomal abnormalities were assessed by fluorescent in situ hybridization for chromosomes 13, 18, 21, X and Y. The average number of abortions was 5.8. The incidence of chromosomal abnormalities was 16.5% that was higher when compared to baseline (4.6%). In conclusion, a high rate of sperm chromosomal abnormalities was observed in recurrent abortion patients. These abnormalities might form during spermatogenesis since all patients had normal karyotype. Sperm chromosomal abnormality analysis can be included into recurrent abortion workup when no other cause is detected.

Isolation, purification and partial characterization of early pregnancy factor (EPF) from sera of pregnant women.

Early pregnancy factor (EPF) is a pregnancy protein, which is secreted into the maternal serum 12-16 hours after fertilization. It is thought to be an immunosuppressive molecule. EPF is detected in pregnant woman's serum by the rosette inhibition assay (RIA). In this study, EPF was purified from the pregnant woman's sera by using ion exchange chromatography and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins which showed a positive result with the RIA, were found to be 35 kDa and 17 kDa molecular weights. The biological activities of these proteins were stable upon heat treatment at 56 degrees C for 30 min. Proteins isolated and purified in this study might be of great significance to the field of human reproduction with particular reference to pregnancy and recurrent abortion.

Effects of the hypo-osmotic swelling test on the outcome of intracytoplasmic sperm injection for patients with only nonmotile spermatozoa available for injection: a prospective randomized trial.

OBJECTIVE: Hypo-osmotic swelling test (HOST) has been shown to be an effective method for the selection of live sperm. On-going pregnancies were obtained by using HOST-selected sperm. The aim of this study was to evaluate the effect of using HOST-selected "live" sperm versus nonselected sperm on the outcome of intracytoplasmic sperm injection cycles when only nonmotile sperm were available for injection. DESIGN: Prospective randomized study. SETTING: Governmental tertiary care hospital. PATIENT(S): Thirty ICSI cycles with no motile sperm were included in this study. INTERVENTION(S): For the HOST group, potentially live spermatozoa detected by hypo-osmotic reaction of the tail were injected into oocytes. For the No-HOST group, the sperm were randomly injected into the oocytes without checking the viability. MAIN OUTCOME MEASURE(S): The fertilization, cleavage, embryo quality, pregnancy, and implantation rates were assessed for the two groups. RESULT(S): Among 30 cycles, 15 fall into each group. Fertilization, cleavage rates, and the number of good quality embryos were similar between two groups. CONCLUSION(S): HOST-selected live spermatozoa can be safely used for intracytoplasmic sperm injection to establish pregnancies. There is a tendency for higher pregnancy and implantation rates to result, but it does not reach statistical significance.

Localization of connective tissue growth factor in human uterine tissues.

Connective tissue growth factor (CTGF) is a recently described heparin-binding mitogen for fibroblasts and smooth muscle cells. The aim of this study was to investigate the production of CTGF by human uterine tissues using immunohistochemical and Northern blotting analyses. For immunohistochemistry, formalin-fixed human proliferative (n = 5), early secretory (n = 5; days 15-19), mid-secretory (n = 5; days 20-23), late secretory (n = 5; days 24-28) endometrial, and decidual (n = 5) tissues were stained using a highly specific affinity-purified polyclonal antibody raised against residues 81-94 of human CTGF. Myometrial (n = 5) and leiomyoma (n = 5) tissues were also used for CTGF immunochemistry. In proliferative endometrium, epithelial and vascular endothelial cells showed strong CTGF immunoreactivity, whereas stromal cells were negative or only weakly positive for the CTGF protein. Throughout the entire secretory stage, CTGF was detected in epithelial and endothelial cells of endometrium. Stromal cells showed strong immunoreactivity to CTGF only in oedematous areas for early and mid-secretory endometrium, and in decidualized regions of late secretory endometrium. During pregnancy, the decidual, epithelial and endothelial cells of the endometrium were all immunoreactive to CTGF. In myometrial and leiomyoma samples, CTGF immunoreactivity was found only in the endothelial cells. Northern blotting of mRNA from normal uterus (n = 2) or leiomyoma (n = 6) using a 320 bp human CTGF cDNA probe revealed a single 2.4 kb transcript. This study is the first to demonstrate CTGF gene expression and localization of its encoded protein in human uterine tissues. The cell- and cycle-specific localization of CTGF support a role for this molecule in regulating aspects of uterine cell growth, migration, and/or matrix production during the menstrual cycle and pregnancy.

Day 5 versus day 3 embryo transfer: a controlled randomized trial.

Blastocyst transfer has been suggested to improve implantation rate without affecting pregnancy rate. The aim of this study was to compare the pregnancy and implantation rates of day 3 and 5 transfers in a prospective randomized manner. Patients with four or more zygotes were randomly allocated on day 1 to either day 3 or 5 transfers. Fertilization was achieved through regular IVF or intracytoplasmic sperm injection. Zygotes were kept in Medicult IVF medium for day 3 transfers and transferred into G1.2 and G2.2 on day 1 and 3 respectively for day 5 transfers. The morphologically best two or three embryos or blastocysts were chosen for transfer in both groups. Overall pregnancy rates per embryo transfer were the same (39%) in day 3 and 5 transfers. Implantation rates were 21 and 24% for day 3 and 5 transfers respectively. The pregnancy and implantation rates for day 5 transfers were significantly affected by the availability of at least one blastocyst to transfer and the number of zygotes. The number of good quality embryos on day 3 also significantly affected pregnancy and implantation rates on day 5 transfers. Multiple gestation rate, number of abortions and ongoing pregnancies were similar in both groups. In conclusion, day 3 and 5 transfer had similar pregnancy, implantation and twinning rates. Currently, day 5 transfers have no advantages over day 3 transfers.

Advanced surgical sperm recovery is a viable option for intracytoplasmic sperm injection in patients with obstructive or nonobstructive azoospermia.

OBJECTIVE: To determine whether advanced sperm retrieval is appropriate in cases of obstructive and nonobstructive azoospermia. DESIGN: Prospective controlled study. SETTING: Tertiary care center. PATIENT(S): Men with obstructive and nonobstructive azoospermia, and their partners. INTERVENTION(S): Surgical sperm retrieval followed by intracytoplasmic sperm injection (ICSI) after 4 or 48 hours. MAIN OUTCOME MEASURE(S): Fertilization and pregnancy rates. RESULT(S): Advanced and fresh surgical sperm recoveries for ICSI were performed in 54 and 230 cycles, respectively. Patient demographics and cycle parameters were comparable. Two hundred forty-one (56.3%) of 428 injected eggs in the advanced retrieval group were fertilized, compared with 955 (56.6%) of 1,686 eggs in the fresh retrieved group (P=.94). There was no statistically significant difference in the pregnancy rates per ET between groups: 38.2% (18 of 47) in the advanced retrieval group and 39.9% (73 of 183) in the fresh sperm recovery group (P=.97). CONCLUSION(S): Testicular and epididymal sperm recovery can be safely performed 48 hours before ICSI. This facilitates planning, and, in cases of failure to retrieve sperm, hCG administration and ovum pick-up can be canceled, thereby reducing costs and eliminating the risk of ovarian hyperstimulation.

Embryo development and pregnancies from in-vitro matured and fertilized human oocytes.

There is an increasing interest in retrieving immature oocytes in the absence of or with limited gonadotrophin exposure, with the aim of maturing them in vitro for embryo transfer purposes. The aim of this report is to present our experience of fertilization, embryonic development and pregnancies from in-vitro maturation cycles. A total of 18 patients underwent 21 cycles in which an average of 8.1 immature oocytes was retrieved after limited exposure to human menopausal gonadotrophin (HMG) and no exposure to human chorionic gonadotrophin (HCG). In one cycle, no oocytes were recovered. The oocytes were cultured for 44 h and 121 oocytes which reached MII were injected with a single spermatozoon. A total of 71 oocytes showed two pronuclei and 53 zygotes cleaved. Forty-four embryos were transferred in 17 cycles. Five weeks after embryo transfer, ultrasound examination indicated the presence of one gestational sac and one fetal heart beat in two patients. The results suggest that in-vitro matured oocytes can undergo fertilization and the resulting embryos may result in pregnancies. However, the success rate was not sufficient to recommend widespread use of the technique without further research.

Effects of reducing insemination time in human in vitro fertilization and embryo development by using sibling oocytes.

PURPOSE: Recent studies showed a beneficial effect of reducing the time of sperm-oocyte interaction on fertilization, division, and implantation rates of the oocytes obtained from randomized patients. In the present study, the effects of reduced insemination time on fertilization and embryo development were evaluated by using sibling oocytes from the same patient. METHODS: A total of 464 oocytes from 36 patients was randomly allocated to be inseminated for either 1 hr (reduced) or 18 hr (regular). RESULTS: Fertilization rates were not significantly different between reduced (135/229; 59%) and regular (150/235; 64%) groups. Cleavage rates and embryo quality were similar in both groups. A total of 135 embryos (73 from the reduced and 62 from the regular group) was transferred to 36 patients. Thirty-four embryos implanted in 18 patients (25.2% implantation and 50.0% pregnancy rates). CONCLUSIONS: Fertilization, cleavage, and embryo development from 1-hr insemination is comparable, not superior, to those from an 18-hr insemination time, which is commonly used in in vitro fertilization programs. These data suggest that reduced insemination time can be used during in vitro fertilization to avoid unnecessarily longer exposure to spermatozoa.

Effect of human chorionic gonadotropin on cytokine production from human endometrial cells in vitro.

PROBLEM: To examine whether human chorionic gonadotropin (hCG) is involved in the regulation of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and leukemia inhibitory factor (LIF) secretion from cultured human endometrial cells. METHOD OF STUDY: A mixed population of endometrial cells from six in vitro fertilization/embryo transfer patients was cultured and incubated with various doses of hCG (0, 1, 10, 50, 100, and 500 IU/ml) for 24 hr. IL-6, TNF-alpha, and LIF levels in the culture medium were measured with enzyme-linked immunosorbent assay. RESULTS: IL-6 and TNF-alpha levels were stimulated by hCG in a dose-dependent manner. Stimulation of IL-6 and TNF-alpha levels by 500 IU/ml of hCG increased their production by 3.7- and 2.8-fold, respectively (P < 0.05). Stimulation of IL-6 by 100 IU/ml of hCG was also significant (P < 0.05). However, there was no significant effect of hCG on LIF secretion by endometrial cells (P = 0.31). CONCLUSIONS: hCG is involved in the regulation of endometrial cytokine production from human endometrial cells in vitro. This finding supports the recently emerging notion that hCG could have important local roles within the uterus besides its well-known luteotrophic role on the corpus luteum for maintenance of pregnancy.

Presence of leukemia inhibitory factor and interleukin-12 in human follicular fluid during follicular growth.

PROBLEM: Cytokines have been shown to be present in human follicular fluid and have regulatory functions on follicular maturation. The presence of leukemia inhibitory factor (LIF) and interleukin (IL)-12 in human follicular fluid obtained at different stages of maturation was investigated. METHOD OF STUDY: Follicular fluids and granulosa cells were obtained from preovulatory and immature follicles. Follicular fluids from both groups were assayed for IL-12 and LIF by enzyme-linked immunosorbent assay. Granulosa cells from preovulatory and immature follicles were treated with human chorionic gonadotropin (hCG) in vitro and subsequent LIF and IL-12 production were measured. RESULTS: The average concentration of LIF was significantly higher in preovulatory follicles (7.6 +/- 1.3 pg/ml, n = 24) than in immature follicles (2.0 +/- 1.3 pg/ml, n = 6). The concentration of IL-12 was significantly higher in follicular fluid obtained from immature follicles (10.9 +/- 5.0 pg/ml) than in preovulatory follicles (1.3 +/- 0.4 pg/ml). hCG only stimulated LIF production from mature granulosa cells; it had no effect on IL-12 production. CONCLUSIONS: IL-12 and LIF are present in follicular fluid and their levels are regulated differently during follicular maturation. hCG stimulates LIF production from granulosa cells in vitro.

Recovery and maturation of immature oocytes in patients at risk for ovarian hyperstimulation syndrome.

PURPOSE: Our purpose was to examine the rate of immature oocyte recovery and their potential for in vitro maturation from canceled human menopausal gonadotropin cycles due to the risk of having ovarian hyperstimulation syndrome develop. METHODS: Patients underwent ultrasound-guided immature oocyte pickup. The number of oocytes recovered from these patients was recorded, and then cultured in vitro. Cumulus expansion and the stage of nuclear maturation were observed after 24 and 48 hr, respectively. RESULTS: Seventeen patients underwent 20 immature oocyte recoveries. A total of 162 oocytes (8.1 oocytes/patient) was obtained. All of the oocytes were enclosed in dense layers of cumulus cells. Among them, 78.4% showed cumulus expansion after 24 hr and 66% completed meiotic maturation to metaphase II after 48 hr in culture. There was only one immature oocyte pickup in which no oocytes were recovered (95% recovery rate). None of the patients had ovarian hyperstimulation syndrome develop. CONCLUSIONS: Immature oocytes can be recovered from canceled human menopausal gonadotropin cycles in patients who are at potential risk for severe hyperstimulation syndrome. These oocytes can be matured in vitro and can be used for clinical and research purposes as well.

Pregnancy after transfer of embryos which were generated from in-vitro matured oocytes.

In-vitro maturation of human oocytes is an important technique in assisted reproduction due to its potential for reducing the use of fertility drugs. We offered this technique as an alternative to cancelling the cycle to a patient who was at risk of ovarian hyperstimulation syndrome (OHSS) after treatment with gonadotrophin-releasing hormone analogue (GnRHa) and human menopausal gonadotrophin (HMG). The patient had 40 visible antral follicles with a maximum diameter of 13 mm and an oestradiol concentration of 14,000 pmol/l on cycle day 12. Immature oocytes were aspirated transvaginally under ultrasound guidance. Ten cumulus-enclosed oocytes were harvested and nine of them completed nuclear maturation to metaphase II after 48 h in culture. By 18 h after an intracytoplasmic sperm injection (ICSI) procedure, seven of these metaphase II stage oocytes displayed two distinct pronuclei and two polar bodies. All fertilized oocytes but one underwent cleaveage; four of these were transferred 2 days later. Endometrial priming was initiated with 8 mg oestradiol valerate daily from the day of oocyte retrieval and 50 mg progesterone was injected i.m. daily starting 2 days after that. A single intrauterine sac was seen containing one fetus with positive fetal heart beat on ultrasound at 7 weeks of gestation. Unfortunately, the pregnancy ended at 24 weeks shortly after premature rupture of membranes; a live healthy-looking girl was delivered who died 18 days later.

High fertilization prediction by flow cytometric analysis of the CD46 antigen on the inner acrosomal membrane of spermatozoa.

The study was set up to determine the relationship between the human sperm acrosome reaction and fertilization in couples undergoing routine in-vitro fertilization (IVF) treatment. Prospective data analysis was carried out on all IVF patients during a 6 month period. Exceptions were those patients having insufficient sperm concentration to allow both acrosome reaction determination and insemination. The main outcome measures were the prediction of fertilization in IVF patients using flow cytometric analysis of the spontaneous and ionophore-induced acrosome reaction [giving the acrosomal response to ionophore challenge (ARIC) score] in the male partner's spermatozoa versus standard analytical methods of sperm motion parameters and morphology. Stepwise logistic regression indicated only two independent factors predictive of fertilization: ARIC score (chi 2 = 109.6, P < 0.0001) and post-Percoll % motility (chi 2 = 8.8, P < 0.003). Of patients with an ARIC score of > 10, 92% had > 30% of oocytes fertilized; 100% of patients with an ARIC score of < 10 had < 30% fertilization of oocytes. The sensitivity and specificity of the assay system were 1.00 and 0.82 respectively. The results would indicate that the ARIC test as measured by flow cytometric analysis of CD46 binding is a sensitive and specific assay for use in the prediction of fertilization in IVF patients, thus enabling direct channelling of those patients with ARIC scores of < 10 into the more invasive micro-assisted fertilization schemes.

Immunoreactive endothelin-1, endothelin-2 and big endothelin-1 in follicular fluids of women undergoing ovulation induction for in-vitro fertilization.

Endothelin-like immunoreactivity specific for endothelin-1 (ET-1), endothelin-2 (ET-2) or big endothelin-1 (big ET-1) was measured, using commercially available radioimmunoassay kits, in follicular fluid collected at the time of oocyte aspiration from 36 women undergoing ovulation induction by human menopausal gonadotrophin (HMG). The relationship of ET concentrations to HMG dose, peak serum oestradiol concentration, the number and size of follicles (by ultrasound), the number of retrieved oocytes and the fertilization rate per retrieved oocyte were studied. Overall, 94% of follicular fluid samples were positive for ET-1, 92% were positive for ET-2, and 100% were positive for big ET-1. Mean ET-1, ET-2 and big ET-1 concentrations were 17.23 +/- 12.20, 32.42 +/- 14.32 and 34.55 +/- 16.34 pg/ml respectively. Endothelin-like immunoreactivity in follicular fluid samples was found in an order of ET-1 < ET-2 < big ET-1. There was a highly significant positive correlation (r = 0.8711,P = 0.001, n = 32) between follicular ET-1 and ET-2 concentrations. No significant correlation of follicular big ET-1 was established either with ET-1 or ET-2. However, big ET-1 was found to be negatively correlated with number of oocytes (P = 0.03) and number of follicles (P = 0.04). Control plasma ET-1 and follicular ET-1 were not significantly different. There was no significant correlation between ET concentrations and any of the other studied parameters. The results demonstrated that immunoreactive ET-1, ET-2 and big ET-1 exist in human follicular fluid collected at the time of oocytes retrieval for in-vitro fertilization and may be involved in the regulation of reproductive function. The clinical significance and physiological role of follicular fluid ET deserve further studies.

Progesterone does not potentiate the acrosome reaction in human spermatozoa: flow cytometric analysis using CD46 antibody.

The study was designed in order to investigate the action of progesterone on the spontaneous and ionophore-induced human spermatozoa acrosome reaction in vitro. The principle of the assay system is flow cytometric analysis of CD46 antibody binding to the inner acrosomal membrane. The technique is a simple and objective method of analysis, allowing fluorescent analysis of a large segment (5000 spermatozoa) of the spermatozoa population under investigation, with concomitant isolation of the live fraction of the spermatozoa population. Four concentrations of progesterone (1, 25, 50, and 100 microg/ml) were examined for their effects on spermatozoa capacitated for 4 and 24 h. In addition, motility parameters were examined by the CellSoft 2000 automated semen analyser system. Analysis of variance revealed that progesterone had no effect on either the spontaneous acrosome reaction or the ionophore-induced acrosome reaction at both 4 h and 24 h of spermatozoa capacitation times. Further, no effects on sperm motility parameters or on spermatozoa viability could be attributed to progesterone. We therefore conclude that progesterone has no objectively measurable effects on either the sperm acrosome reaction or sperm motility parameters, as measured in normal sperm populations.

High prevalence of obesity in a Saudi infertility population.

A menstrual history was taken from the female partners of all new infertility couples seen in our clinic between 1988 and 1990. The body mass index (Kg/M2) was measured in all females. The ovulatory status was studied using a combination of serial transvaginal ultrasound investigations and progesterone measurements in the second half of the cycle in females with regular menstrual cycles or progesterone measurements one week before the expected onset of menstruation in females with oligomenorrhea. Amenorrheic patients were considered anovulatory if no anatomical abnormality was found. Out of the 1755 patients, only 17% were in the normal weight category (BMI 19-24), 42% were overweight (BMI25-29) and 38% were obese (BMI 30 or more), while the remaining 3% were underweight. With increasing BMI, the percentage of oligomenorrhea increased from 18% to 32%, the percentage of amenorrhea increased from 2% to 13%. The overall percentage of anovulation increased from 32% to 55%.

Results of 2426 in vitro fertilization cycles from King Faisal Specialist Hospital and Research Centre, 1986-1992.

The report consists of summary in vitro fertilization (IVF) results for the period of 1986 to 1992, concerning 2426 IVF cycles on 954 patients. Tubal factor accounted for 48.7% of cases; male factor, 15.2%; unexplained factors, 15.8%; tubal plus male factor, 12.2%; with 7.7% miscellaneous pathologies. Stimulation regimes were of seven varieties over the period described. All cycles were monitored by transvaginal ultrasound and serum estradiol/LH. All oocyte retrievals were carried out at 34 hours post-HCG. Standard laboratory procedures were utilized for oocyte and pre-embryo culture and a maximum of four embryos were replaced approximately 48 hours after ovum pickup (OPU). Luteal support was by either progesterone suppository or intramuscular injection for 14 days following embryo transfer. Average number of oocytes per retrieval was 7.8, with a fertilization rate of 54% over all groups. Two hundred and sixty-eight pregnancies were initiated (16.1% per embryo transfer [ET]) with 53 of these being biochemical. Pregnancy rates followed a learning curve in the early years with a plateau at 17.8% per ET. Thus, the IVF program has evolved to yield acceptable results after refinement of stimulation regimes, laboratory techniques (especially with regard to sperm preparation), ET techniques and luteal support, thereby tailoring its protocols to suit the local population.