Human gamete fusion test: an experimental model for assessing the fusion potential of sperm from nonfertilizing normospermic patients.

OBJECTIVE: To develop an experimental model for assessing the ability of sperm to bind and subsequently fuse with the oolema and to use this test for evaluating the fusion potential of nonfertilizing human sperm. DESIGN: Aged human oocytes (n = 108) were denuded of their zonae and loaded with DNA-specific bisbenzamidazole fluorochrome. Sibling oocytes were inseminated by semen samples from either normospermic patients (study group; n = 12) who have repeatedly failed to achieve fertilization in vitro or patients demonstrating high fertilization rates (control; n = 12). Gamete fusion was ascertained by fluorescent microscopy and validated by scanning electron microscopy. Test results were analyzed in relation to current partial zona dissection treatment outcome. SETTING: Infertility and IVF Unit of an academic tertiary referral medical center and university-based basic research laboratory. RESULTS: In samples achieving fertilization by partial zona dissection (7/12; 9% to 23% fertilization rate), the mean numbers of fused spermatozoa were similar to those of the control (2.1 compared with 2.6) and significantly higher than in the partial zona dissection-failed fertilization subgroup. In six of seven cases in which partial zona dissection yielded embryos, fluorescent spermatozoa were detected on the oolema, whereas in all but one partial zona dissection-failed cases gamete fusion was not observed. CONCLUSIONS: The suggested human gamete fusion test may help focus on specific sperm dysfunction in nonfertilizing patients. It correlates well with the outcome of partial zona dissection-assisted IVF trials and may provide useful information before this treatment is attempted.

Assisted hatching by partial zona dissection of human pre-embryos in patients with recurrent implantation failure after in vitro fertilization.

OBJECTIVE: To examine the potential of the partial zona dissection technique to promote successful implantation by assisting embryo hatching after IVF. DESIGN: The study and the control group included 72 and 82 patients, respectively, each had undergone at least three failed IVF-ET attempts. Assisted hatching was performed on four- to six-cell stage embryos by creating a slit in the zona pellucida using the partial zona dissection technique. After 90 minutes incubation (5% CO2, 37 degrees C), the embryos were transferred to the uterus. SETTING: Infertility and IVF Unit of an academic tertiary referral medical center. RESULTS: In the assisted hatching group, 230 micromanipulated embryos were replaced (3 or 4 treated embryos per patient) compared with 295 nonmanipulated embryos in the control group. Clinical pregnancy rates (PRs) were similar in the assisted hatching and control groups (n = 15; 20.8% and n = 12; 14.6%, respectively). However, the contribution of assisted hatching by partial zona dissection to successful implantation was related to the patients's age: patients older than 38 years showed a markedly higher PR after assisted hatching: 23.9% in the study group compared with only 7% of the controls. CONCLUSIONS: These results demonstrate that assisted hatching by partial zona dissection is a quick and efficient method that does not induce any visible damage to the embryos replaced. In a selected group of patients (aged over 38 years, who have failed to conceive in at least three previous IVF attempts) it significantly increases the chances for pregnancy after ET.

Maturation-associated changes in the rat zona pellucida.

Rat follicular oocytes, arrested at prophase I, cannot be fertilized in vitro. This capacity is acquired following resumption of meiosis and a series of changes involving both the oocyte and the cumulus cells surrounding it. Oocytes exposed to sperm at different hours before ovulation show a gradual increase in the permeability of their zona pellucida (ZP). Our study examined whether the ZP, in response to the physiological stimulus for maturation and concomitant with the other oocyte--cumulus components, undergoes maturational changes. Two ZP characteristics were assessed, sensitivity to proteolysis and sperm binding. ZP surrounding oocytes and eggs were collected from five sources: 1) germinal vesicle (GV)-intact oocytes, 2) preovulatory eggs, 3) ovulated eggs isolated from oviducts of immature females, 4) fertilized eggs, 5) ovulated eggs isolated from oviducts of mature females. All ZP surrounding oocytes/eggs from groups 1-5 were dissolved by trypsin. When solubility by pronase and alpha-chymotrypsin was examined, a large variation between groups was found. All ZP from group 2 were dissolved by 0.001% pronase, compared to 0% solubility in group 4. Only 10% of the ZP surrounding GV-intact oocytes (group 1) were dissolved by this enzyme, compared to 82% in group 3. Solubility in 0.01% alpha-chymotrypsin showed a similar pattern. Capacitated sperm were incubated with eggs from groups 1 and 3. The number of sperm binding to ZP in group 3 was repeatedly higher than that in group 1. In both tests it was found that the ZP surrounding the mature eggs differ in their characteristics from ZP of GV-intact oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous and follicular fluid-induced acrosome reaction in sperm samples from in vitro fertilizing and nonfertilizing normozoospermic patients.

PURPOSE: One of the most challenging and intriguing groups of infertile patients is that of normozoospermic men who have repeatedly not achieved fertilization in vitro. These cases probably present a wide range of gamete disorders manifested at different stages of the fertilization process. The occurrence of spontaneous and follicular fluid (FF)-induced acrosome reactions (ARs) in in vitro fertilizing and nonfertilizing specimens from normozoospermic IVF patients was assessed, and the effect of Percoll and cryopreservation on the incidence of ARs was evaluated. METHODS: Semen samples from 62 normozoospermic (15 in vitro nonfertilizing and 47 fertilizing) patients were analyzed. Spermatozoa were double-stained with FITC-conjugated Pissum sativum lectin, to assess their acrosomal status, and propidium iodide, to evaluate their vitality, using flow cytometry analysis. RESULTS: A lower average of AR incidence was observed in the nonfertilizing than in the fertilizing group with all treatments. Both groups exhibited an increase in the proportion of ARs following incubation with FF. This rise was most prominent when Percoll-separated fractions were used (15.8 and 25.6% AR in the nonfertilizing and fertilizing groups, respectively). Thawed cryopreserved and fresh fertilizing samples exhibited similar AR rates. CONCLUSIONS: That normozoospermic recurrently nonfertilizing compared to fertilizing semen samples have a lower capacity to undergo ARs is suggested.

Expression of cadherin adhesion molecules on human gametes.

The presence of cadherins, Ca(2+)-dependent cell-cell adhesion molecules which may be involved in gamete interaction, was investigated in human gametes. Expression of cadherin molecules was demonstrated using an anti-pan-cadherin antibody and specific antibodies against the three classical cadherins: E- (epithelial), P- (placental) and N- (neural) cadherins. Samples of 48 h old unfertilized oocytes and spermatozoa from in-vitro fertilizing semen samples were lysed and separated by electrophoresis. Localization of cadherins was determined on intact, fixed, permeabilized spermatozoa and oocytes by immunocytochemisty assessed by confocal microscopy. Immunoblotting with the pan-cadherin antibody revealed a single band of approximately 120 kDa in spermatozoa (whether 'fresh', capacitated, or frozen-thawed) and oocyte extracts. Oocytes presented all three classical cadherins with the appropriate molecular weights of 120-130 kDa. In sperm lysate we demonstrated the presence of E-cadherin but not N-cadherin. The anti-P antibody detected a 90 kDa peptide as the only immunoreactive antigen. Following immunocytochemistry of human oocytes all cadherin molecules were allocated predominantly to the plasma membrane with only traces in the cytoplasm. In spermatozoa, several staining patterns were observed with each of the pan-cadherin, N-cadherin and E-cadherin antibodies mostly confined to different head regions. We conclude that cadherin molecules are present on plasma membranes of both human spermatozoa and oocytes and may play a role in the intricate recognition process preceding gamete fusion.

Thiol status in human sperm.

The passage of spermatozoa along the epididymis is characterized by a gradual stabilization of intracellular organelles mainly through the oxidation of thiol groups. In this study, we examined the relationship between the thiol-disulfide status of human spermatozoa (using a specific fluorescent probe, monobromobimane) and routine semen analysis parameters. Fluorescence intensity was measured by spectrofluorimeter and its frequency distribution within samples, using a fluorescence-activated cell sorter. The mean proportion of reactive thiols SH/(SS + SH) in 29 semen samples was 29.8% +/- 2.5%. When comparing thiol labeling patterns, oligozoospermic samples differed from normozoospermic ones (P less than 0.05). However, within the normozoospermic group, no correlation was found between thiol-labeling patterns and routine sperm parameters or fertilizing capacity in vitro. No difference in thiol labeling patterns was found between "swim-up" and "whole semen" preparations.

Superovulation before IVF by recombinant versus urinary human FSH (combined with a long GnRH analog protocol): a comparative study.

PURPOSE: Gonal-F (Serono, Aubonne, Switzerland) is a recombinant human follicle stimulating hormone (FSH) synthesized in vitro by cells into which genes encoding for FSH subunits have been inserted. This preparation exhibits physiochemical, immunological, and pharmacological properties that bear great similarity to those of native human FSH. It has a high specific activity and can be administered subcutaneously. To compare the efficacy and safety of Gonal-F with those of urinary human FSH (Metrodin; Serono) in achieving superovulation for IVF purposes in a prospective, randomized study. METHODS: Twenty infertile patients (normo-ovulatory healthy women) were recruited for the study and allocated at random to the Gonal-F or Metrodin groups. The treatment protocol consisted of pituitary down regulation by GnRH analog (Buserelin; Hoechst, Frankfurt, Germany) employing the "long" protocol initiated at the mid-luteal phase (900 micrograms/day, intranasal administration). Gonal-F (SC) or Metrodin (IM) was injected daily (225 IU/day) starting on cycle day 3. Dose adjustment was performed, when necessary, from cycle day 7. RESULTS: Of the 20 cycles analyzed, none was canceled due to poor response. No cases of adverse effects (including local intolerance) or ovarian hyperstimulation syndrome were recorded in either group. They did not differ significantly in the following treatment-dependent variables: hormone profile, duration of FSH treatment, total FSH dose required to achieve follicular maturation, and the number of oocytes retrieved, fertilized, and replaced. CONCLUSIONS: These preliminary data concur with previous studies in demonstrating that Gonal-F is as effective and safe as Metrodin (when given in combination with a "long" protocol of GnRH analog) in inducing controlled ovarian hyperstimulation for IVF purposes. Its mode of administration (SC instead of IM) offers an additional advantage over the urinary human FSH.

Treatment variables in relation to oocyte maturation: lessons from a clinical micromanipulation-assisted in vitro fertilization program.

OBJECTIVE: In an effort to understand the mechanism underlying the improved pregnancy rate observed in IVF cycles when gonadotropin-releasing hormone analogues (GnRH-a) are applied, we investigated a possible relationship between treatment variables and oocyte nuclear maturity. DESIGN: Nuclear maturity was retrospectively assessed in cumulus-free, denuded oocytes, obtained from women undergoing micromanipulation-assisted IVF treatment following controlled ovarian hyperstimulation with GnRH-a and menotropins. SETTING: The setting was the infertility and IVF unit of a tertiary academic medical center. PARTICIPANTS: Two hundred twenty-one patients underwent 435 treatment cycles. MAIN OUTCOME MEASURE: This was the proportion of germinal vesicle-intact immature (GVII) oocytes. RESULTS: One hundred fifty-four of the 3520 oocytes studied (4.4%) were in the GVII stage. These oocytes were found in 66 of the treatment cycles (15.2%) and in 54 of the patients (24.4%). Cycles in which GVII oocytes were detected did not differ from those in which all the aspirated oocytes were mature in the following respects: patient age, type and duration of infertility, controlled ovarian hyperstimulation protocol and time of ovum pickup. However, the GVII group was characterized by a significantly higher peak estradiol level, as well as a higher number of mature follicles visualized sonographically (diameter, > 14 mm) and oocytes retrieved. CONCLUSIONS: Comparing the present findings with previously published data, it appears that the inclusion of GnRH-a in the stimulation regimen is associated with a lower proportion of immature oocytes. A higher occurrence of oocyte-nuclear immaturity is apparently associated with a significantly better ovarian response to stimulation. The high incidence of immature oocytes observed in patients with normospermic partners and low fertilization rates in previous cycles may suggest that the fertilization failure in some of these cases is due to oocyte, rather than sperm, dysfunction.

Gonadotropin stimulation following GnRH-a priming for poor responders in in vitro fertilization-embryo transfer programs.

The effect of gonadotropin-releasing hormone agonist (GnRH-a) administration before gonadotropin superovulation on the stimulation characteristics of poor responder patients was assessed in an in vitro fertilization (IVF) program. Thirty consecutive patients who had exhibited low ovarian response (fewer than four retrieved oocytes) in at least two previous IVF cycles (control cycles, n = 60), were eligible for the study. GnRH-a (nafarelin) was administered daily for 7-10 days from the mid-luteal phase of the previous cycle until the first day of menstruation. Menotropin treatment was commenced on cycle day 3 (with no additional GnRH-a) (study cycles, n = 39). A significantly higher number of oocytes was retrieved (p < 0.0002) and a higher number of embryos transferred (p < 0.003) in the study cycles than in the control cycles. No cases of premature luteinizing hormone surge were recorded. Pregnancy rates per embryo transfer and per cycle were 10.4% and 7.7% for the study cycles and 2.8% and 1.6% for the control cycles, respectively. GnRH-a, administered prior to gonadotropin treatment, should be an additional option of ovulation induction protocol for poor responders in IVF programs.

Doppler parameters of uterine and ovarian stromal blood flow in women with polycystic ovary syndrome and normally ovulating women undergoing controlled ovarian stimulation.

OBJECTIVE: To test the hypothesis that the increased ovarian sensitivity to gonadotropins observed in women with polycystic ovary syndrome (PCOS) may be due to changes in ovarian stromal blood flow in these patients. DESIGN: Uterine and ovarian stromal arterial blood flow (with transvaginal color Doppler ultrasound) were measured in ten women with PCOS and 12 normo-ovulatory women (control group), undergoing gonadotropin stimulation before in vitro fertilization. METHODS: A careful ovarian stimulation strategy was adopted for the study group in order to avoid ovarian hyperstimulation syndrome and achieve an ovarian response which was comparable to that of the control group. Resistance and pulsatility indices (RI and PI) of the uterine and ovarian stromal arteries were calculated before the onset of gonadotropin treatment, on cycle day 5 (after commencing treatment), day of human chorionic gonadotropin injection, day of ovum pick-up as well as on the day of embryo transfer, and 7 and 12 days later. RESULTS: No significant differences were found in RI and PI between the study and control groups throughout the treatment cycle. CONCLUSIONS: It seems that polycystic ovaries do not bear an inherent disturbance in blood flow dynamics of the uterine and ovarian arteries, as measured by color Doppler, which would explain the increased sensitivity of polycystic ovaries to stimulation with gonadotropins.

Inclusion of standard and low-dose gonadotropin releasing hormone-analog (short protocol) in controlled ovarian hyperstimulation regimens in normogonadotropic patients aged 40-48 years who are undergoing in vitro fertilization.

We aimed to compare the efficiency of three controlled ovarian hyperstimulation protocols in achieving superovulation in normogonadotropic patients aged 40 years or more, who were undergoing in vitro fertilization (IVF) treatment. This was a prospective randomized clinical study, carried out in the Infertility and IVF Unit of an academic tertiary hospital. A total of 219 normogonadotropic patients (serum follicular stimulating hormone level < 15 mIU/ml) aged 40-48 years, with regular menstrual cycles, were randomly allocated to one of three short follicular protocols: menotropins only (group A), menotropins plus a mini-dose of gonadotropin releasing hormone (GnRH)-analog (600 microg/ day) (group B), or menotropins plus a standard dose (900 microg/day) of a GnRH-analog (group C). Those cycles that reached the stage of oocyte retrieval (67, 70 and 71 cycles, respectively) were analyzed. The mean daily dose of menotropins needed for ovarian stimulation was higher when GnRH-analog was used (groups B and C) (p < 0.02; ANOVA), although there was no significant difference in the time of human chorionic gonadotropin injection (average: cycle day 11). Peak estradiol levels (p < 0.02), number of oocytes retrieved (3.9, 5.4 and 5.5 oocytes/cycle, respectively, p < 0.02) and number of embryos transferred (1.6, 1.8 and 2.1 embryos/cycle, respectively, p < 0.05) were higher when GnRH-analog was included in the controlled ovarian hyperstimulation protocol. The IVF treatment resulted in 19 pregnancies (9.1% implantation rate), with a similar distribution among all three groups (11.9%, 8.6% and 7.0%). However, a higher miscarriage rate was noted in the menotropins-only group (67.5% vs. 33.3% and 40.0% of pregnancies). No differences were observed in any of the aforementioned variables between the mini-dose and standard dose GnRH-analog groups (groups B and C). In conclusion, controlled ovarian hyperstimulation before IVF treatment in normogonadotropic patients aged 40 years or more is more effective when a GnRH-analog (short protocol) is included in the treatment regimen. In this selected group of patients, reducing the daily dose of GnRH-analog does not improve the treatment results.

Is diagnostic testicular fine needle aspiration necessary in azoospermic men before sperm aspiration/extraction for intracytoplasmic sperm injection cycles?

PURPOSE: To determine whether diagnostic testicular fine needle aspiration (TEFNA) sampling needs to be performed in azoospermic men prior to obtaining testicular sperm cells for IVF-ICSI procedures. METHODS: Ten azoospermic patients underwent TEFNA in 1993-1996. During 1997, all patients underwent testicular sperm aspiration (TESA) and/or testicular sperm extraction (TESE) to retrieve spermatozoa for IVF-ICSI cycles. The results of the two procedures performed in two separate hospitals were compared. RESULTS: Diagnostic TEFNA revealed spermatozoa in five patients; identical results in four were found during IVF-ICSI cycles. In three patients, only Sertoli cells were found on TEFNA, in two of them TESA/TESE showed identical results, and in one, two spermatozoa were detected by Cyto-SEM. In the remaining two patients, spermatids or spermatocytes were found on both procedures. CONCLUSIONS: There was a very good correlation between the diagnostic and therapeutic procedures. We suggest that in azoospermic patients, diagnostic TEFNA is valuable in order to avoid unnecessary controlled ovarian hyperstimulation in the female partner for IVF. In patients in whom spermatozoa are detected, cryopreservation may be performed for later IVF-ICSI cycles.