Effect of transferring a morphologically impaired embryo with a good quality embryo on the pregnancy and implantation rates.

OBJECTIVE: This study aimed to investigate the effect of transferring embryos with different qualities on pregnancy and implantation rates. PATIENTS AND METHODS: In a retrospective multi-center study we analyzed 761 patients aged ≤ 35 years who had an elective transfer of one or two embryos. Embryos were scored morphologically by their developmental stage into good "A" and impaired "B" embryos. Pregnancy and implantation rates were compared between patients who had a transfer of: one grade "A" embryo; two grade "A" embryos, two embryos one grade "A" plus one grade "B" embryos; one grade "B" embryo and two grade "B" embryos. RESULTS: Higher pregnancy and implantation rates were observed in patients who had received one embryo of grade "A" (34.6%) and two grade "A" embryos (45.2%, 25.85% respectively), compared to patients who received two embryos, one of grade "A" plus one of grade "B" (25%, 13.77% respectively). CONCLUSIONS: Transferring a morphologically and developmentally impaired embryo, significantly lower the implantation chance of the good quality embryo.

Simultaneous administration of human acidic and recombinant less acidic follicle-stimulating hormone for ovarian stimulation improves oocyte and embryo quality, and clinical outcome in patients with repeated IVF failures.

BACKGROUND: Ovarian stimulation is an integral procedure in assisted reproduction treatment. It is achieved by the administration of exogenous gonadotropins to increase follicular recruitment and oocyte yield. Optimization of ovarian stimulation is an essential prerequisite for the success of IVF treatment. AIM: This study aimed to evaluate the effect of a combined stimulation protocol of human FSH and recombinant FSH, simultaneously administered, on oocyte and embryo quality and clinical outcome. PATIENTS AND METHODS: In a prospective randomized study 197 infertile patients with a history of previous IVF failures for at least 3-5 attempts, were enrolled for an in vitro fertilization treatment. All patients had a standard down-regulation with GnRH analog and were then stimulated with FSH. The patients were matched into three groups: group A (no = 66) received human FSH combined with recombinant FSH in equal doses, simultaneously administered; group B (no = 67) received human FSH alone and group C (no = 64) received recombinant FSH alone. RESULTS: There were significantly higher pregnancy (p < 0.04) and implantation rates (p < 0.03) in favor of group A (hFSH/rFSH) compared to groups B (hFSH) and C (rFSH). A significant increase in the proportion of mature metaphase II oocytes (p < 0.002) and grade 1 embryos (p < 0.03) was observed in group A with respect to group B and C. Significantly higher delivery rate (p < 0.01) was achieved in group A compared to groups B and C. No significant differences were observed between groups regarding miscarriage rate and risk of ovarian hyperstimulation syndrome. CONCLUSIONS: The results show that the combination of human and recombinant FSH for ovarian stimulation may produce a positive effect on follicular development as it improve oocyte quality, embryo development, and ultimately clinical outcome.

Successful pregnancies and deliveries after a simple vitrification protocol for day 3 human embryos.

OBJECTIVE: To evaluate the efficacy and efficiency of freezing cleaved human embryos through vitrification. DESIGN: Clinical study of vitrification of human embryos. SETTING: Assisted reproductive technology centers. PATIENT(S): Thirty-six patients undergoing IVF-ICSI treatment whose surplus embryos were frozen. INTERVENTION(S): Two hundred fifteen surplus embryos vitrified, subsequently thawed, and transferred in natural or controlled cycles. MAIN OUTCOME MEASURE(S): Embryo survival rate after thawing and resultant patient pregnancy rate. RESULT(S): From the 215 vitrified and thawed embryos, 106 survived, with an overall embryo survival rate of 49.3%. The survival rate was higher when embryos were vitrified at the eight-cell stage compared with at the six to seven-cell and six-cell stages (79.2%, 39.7%, and 21.1%, respectively). On average, 2.9 +/- 1.2 embryos per patient were transferred, resulting in 11 pregnancies (30.5%), with an implantation rate of 10.4% per embryo transferred. CONCLUSION(S): Ultrarapid embryo freezing by vitrification of eight-cell stage embryos is a reliable method, as evidenced by high rates of embryo survival and pregnancy, making it a superior alternative to the conventional slow-cooling method.

Impact of recombinant follicle-stimulating hormone and human menopausal gonadotropins on in vitro fertilization outcome.

OBJECTIVE: To investigate possible differences between using recombinant FSH (rFSH) and hMG for ovarian stimulation in IVF/intracytoplasmic sperm injection (ICSI) cycles. DESIGN: Parallel group design. Prospective, randomized clinical study. SETTING: A tertiary care infertility clinic. PATIENT(S): A total of 578 patients of our IVF/ICSI routine were recruited. INTERVENTION(S): Treatment with hMG was used for 282 patients (282 cycles), whereas 296 patients (296 cycles) were treated with rFSH. The number of cycles leading to an embryo transfer were 248 and 259, respectively. MAIN OUTCOME MEASURES: Primary: clinical pregnancy rate. Secondary: treatment days, total dose of gonadotropin administered, number of oocytes retrieved, number of mature oocytes, and embryo quality. RESULT(S): Of the cycles with embryo transfer, the pregnancy rates were 30.1% and 32.3% in the rFSH and the hMG groups, respectively. This difference is not statistically significant (P=0.798). Treatment with rFSH resulted in a significantly higher number of recovered oocytes compared with the hMG group but was also associated with a higher number of ampoules needed to reach the criterion for hCG administration. No significant differences were found with regard to the number of mature oocytes, the number of treatment days, and the embryo quality. CONCLUSION(S): In terms of the clinical pregnancy rate, no significant differences between the two stimulation regimens can be stated.

Detrimental effect of hydrosalpinx fluid on the development and blastulation of mouse embryos in vitro.

OBJECTIVE: To study the effects of human hydrosalpinx fluid on mouse embryo blastulation rate. DESIGN: Comparison of mouse embryo blastulation rate in media containing increasing concentrations of hydrosalpinx fluid. SETTING: Tertiary care center. PATIENT(S): Women undergoing laparoscopic evaluation or treatment for infertility noted to have hydrosalpinx or paratubal cyst. INTERVENTION(S): Exposure of mouse embryos to hydrosalpinx or paratubal cyst fluid collected during laparoscopy. MAIN OUTCOME MEASURE(S): Blastulation rate of mouse embryos. RESULT(S): Culture of mouse embryos at 0% (controls), 0.3%, 0.6%, and 0.9% hydrosalpinx fluid concentrations demonstrated significantly lower blastulation rate at each level compared with the controls. CONCLUSION(S): Hydrosalpinx fluid is highly embryotoxic.

Gene regulation of interleukin-1 beta, interleukin-1 receptor type I, and plasminogen activator inhibitor-1 and -2 in human granulosa-luteal cells.

OBJECTIVE: To investigate the regulation of messenger ribonucleic acid (mRNA) levels of interleukin-1 beta (IL-1 beta), interleukin-1 (IL-1) receptor type 1, and plasminogen activator (PA) inhibitor-1 and -2 in cumulus cells, granulosa-luteal cells, and macrophage-depleted granulosa-luteal cells obtained from human preovulatory follicles. DESIGN: Prospective longitudinal study. SETTING, PATIENTS: Patients undergoing assisted reproductive technologies (ART) in the Department of Gynecology and Obstetrics, Stanford University, Stanford, California. INTERVENTIONS: Cumulus cells and granulosa-luteal cells were collected by ultrasound-guided transvaginal aspiration at the time of ART. MAIN OUTCOME MEASURES: Northern blot analysis of mRNA levels of IL-1 beta, IL-1 receptor type 1, PA inhibitor-1 and -2 in cumulus cells, granulosa-luteal cells and macrophage-depleted granulosa-luteal cells, and indirect immunocytochemical analysis of the IL-1 system and macrophages in granulosa-luteal cell preparations were performed. RESULTS: Interleukin-1 beta mRNA levels in uncultured cumulus cells were less than those of uncultured granulosa-luteal cells with no differences in IL-1 receptor type 1 mRNA levels between these two cell types. Granulosa-luteal cell IL-1 receptor type 1 mRNA levels were expressed constitutively throughout 24 hours of culture with no effect by hCG, whereas IL-1 beta mRNA levels increased within 6 hours, and then remained elevated for 24 hours with no effect by hCG. Interleukin-1 beta significantly increased granulosa-luteal cell mRNA levels of IL-1 beta (over twofold), IL-1 receptor type 1 (over twofold), PA inhibitor-1 (approximately 1.4-fold), and PA inhibitor-2 (approximately 1.6-fold). In contrast, IL-1 beta had no effect on IL-1 beta and IL-1 receptor type 1 mRNA levels in macrophage-depleted granulosa-luteal cells. Granulosa-luteal cells, not macrophages, account for the majority of the immunocytochemical staining for IL-1 beta and IL-1 receptor type 1 in follicular aspirates. CONCLUSIONS: These studies suggest that the IL-1 system is regulated in human granulosa-luteal cells during the periovulatory period. Furthermore, the augmentation of PA inhibitor-1 and -2 mRNA levels by IL-1 beta suggests a potential role for IL-1 beta in remodeling of the human ovary during the periovulatory period.

The effect of interleukin-1 beta (IL-1 beta) on the regulation of IL-1 receptor type I messenger ribonucleic acid and protein levels in cultured human endometrial stromal and glandular cells.

Because we hypothesize that the interleukin-1 (IL-1) system may be important in the dialogue between mother and embryo during the implantation process, we have analyzed the effect of IL-1 beta, a secretory product of the human embryo and human endometrium, on the mRNA and protein levels of IL-1 receptor type I (IL-1R tI) in the human endometrium. For this purpose, endometrial epithelial cells (EEC) and stromal cells (ESC) were isolated and cultured with progesterone (3.18 micrograms/mL) and epidermal growth factor (20 ng/mL) for 8 days in the presence or absence of hrIL-1 beta (20 pg/mL). EEC from proliferative and secretory endometrium expressed high levels of IL-1R tI mRNA compared to ESC, and these levels were not modulated by IL-1 beta. However, prostaglandin E2 levels peaked on day 4 in EEC treated with progesterone, epidermal growth factor, and IL-1 beta (208.7 +/- 92 ng/10(7) cells), whereas no prostaglandin E2 was detectable in cells not treated with IL-1 beta, indicating that these cells responded to IL-1 beta. With regard to ESC from secretory endometrium, IL-1 beta increased its own receptor mRNA levels (4 +/- 0.5-fold increase) after 8 days in culture. However, when ESC were isolated from proliferative endometrium, an up-regulation of IL-1R tI (3.5 +/- 0.5-fold increase) was observed on days 6 and 8 of culture regardless of the presence or absence of IL-1 beta. Immunoreactive IL-1R tI was identified in cultured EEC and ESC, and patterns similar to those of mRNA were observed. The constitutive presence of IL-1R tI in EEC, which was not affected by IL-1 beta, and the up-regulation of IL-1R tI mRNA by its ligand IL-1 beta in ESC isolated during the luteal phase suggest a role for the IL-1 system in human implantation.

Embryonic implantation in mice is blocked by interleukin-1 receptor antagonist.

We have investigated the relevance of interleukin-1 receptor type I (IL-1R tI) in the implantation process in vivo in a murine model. Indirect immunofluorescence experiments demonstrate that IL-1R tI is located in mouse endometrial lumenal epithelium with increased intensity in the periimplantation period, whereas IL-1 beta staining is located in the mouse placenta. PMSG/human CG (hCG)-stimulated and mated 12-week-old B6C3F-1 female mice were randomly allocated to three groups: A, control noninjected; B, buffer-injected animals; and C, animals injected ip with 20 micrograms recombinant human IL-1 receptor antagonist (rhIL-1ra) every 12 h beginning on pregnancy day 3. Injections were continued until day 9, and animals were killed 12 h after the last injection. Pregnancy rates in the three groups were: noninjected, 58.8% (10 of 17); buffer-injected, 73.7% (14 of 19); rhIL-1ra-injected, 6.7% (1 of 15), P = 0.0001155, Fisher exact test. To rule out the possibility that pregnancy failure was due to an embryotoxic effect of rhIL-1ra, 2-cell mouse embryos (n = 276) were flushed from the same group of animals used for in vivo experiments and cultured with increasing concentrations of rhIL-1ra: 0 microgram/ml (n = 91), 1 microgram/ml (n = 36), 50 micrograms/ml (n = 36), 100 micrograms/ml (n = 52), and 200 micrograms/ml (n = 61) rhIL-1ra. The percentages of 2-cell mouse embryos reaching the blastocyst stage after 72 h in culture were 85.7%, 91.6%, 94.4%, 96%, and 85.2%, respectively. We further cultured these blastocysts for 5 days on fibronectin-coated plates with or without 200 micrograms/ml rhIL-1ra. In both groups, hatching, attachment to fibronectin, outgrowth, and migration were documented to be similar. Furthermore, our longitudinal morphological study of embryonic implantation in control and rhIL-1ra-injected mice shows that the blockade of IL-1R tI interferes with the attachment of mouse blastocysts to maternal endometrium in vivo. In summary, we demonstrate that blockade of maternal endometrial IL-1R tI with IL-1ra prevents implantation in the mouse by interfering with embryonic attachment, without adverse effects on blastocyst formation, hatching, fibronectin attachment, outgrowth, and migration in vitro.

Immunocytochemical localization of transforming growth factor-alpha and epidermal growth factor receptor in human fallopian tubes and cumulus cells.

PROBLEM: Transforming growth factor-alpha (TGF-alpha) has been shown to be a potent stimulant of oocyte maturation and embryonic development. The role of maternal growth factors and their mechanism of action in early mammalian development is not well understood. METHOD: In this study, the presence of TGF-alpha and epidermal growth factor receptor (EGF-R) in human cumulus cells and fallopian tubes was investigated by immunocytochemical techniques. RESULTS: The fallopian tube showed intense staining for TGF-alpha in the apical region of the epithelial cells, and the cumulus cells showed intense staining for EGF-R on cell membranes. CONCLUSION: The presence of TGF-alpha in the fallopian tube epithelium and its receptor on cumulus cells suggest a paracrine mechanism between maternal growth factors and the developing embryo.

Zona opening with 308 nm XeCl excimer laser improves fertilization by spermatozoa from long-term vasectomized mice.

Spermatozoa from long-term vasectomized mice have greatly reduced fertilizing ability in vivo and in vitro, which makes this a useful animal model for male factor infertility. The purpose of this study was to evaluate the 308 nm XeCl excimer laser for opening the zona pellucida to enhance the fertilization rate with spermatozoa from vasectomized males. Inseminating zona-intact (control) oocytes with 5 x 10(6) spermatozoa/ml resulted in only 6% fertilization and 33.3% development to the blastocyst stage; zona-opened oocytes showed significant improvement with 31.5% fertilization, 90% cleavage to the 2-cell stage, and 72.2% blastocyst formation. Out of the 130 oocytes in the experimental group, zona ablation was performed successfully on 127 and only three were damaged. These results suggest that laser micromanipulation for assisted fertilization potentially offers a simplified and precise method for mechanical zona cutting.

Regulation of plasminogen activator inhibitor-1 and -2 messenger ribonucleic acid levels in human cumulus and granulosa-luteal cells.

Plasminogen activators and their inhibitors have been implicated in the process of fibrinolysis, tissue remodeling, and ovulation. Epidermal growth factor (EGF), a paracrine hormone found in the human ovary, increases plasminogen activator (PA) activity and the gene expression of PA and plasminogen activator inhibitor (PAI) in human endothelial cells and human cell lines. Gonadotropins also increase PA activity and gene expression in rat preovulatory granulosa cells. We have now analyzed the gene expression of PAI-1 and PAI-2 in uncultured human cumulus cells (CC), uncultured granulosa-luteal cells (GLC), and cultured GLC obtained from preovulatory follicles of patients undergoing assisted reproductive technologies. We also studied the effects of hCG and EGF on PAI-1 and PAI-2 mRNA levels in cultured GLC; GLC were cultured in serum-free medium for various times within 24 h with or without hCG and for 6 h with or without hCG, EGF, or EGF plus hCG. Total RNAs from CC and GLC were extracted, and blot hybridizations with 32P-labeled PAI-1, PAI-2, or 28S ribosomal RNA cDNA probes were performed. Both CC and GLC expressed PAI-1 and PAI-2 genes. In GLC, steady state levels of PAI-1 mRNA levels steadily increased within 24 h of culture, whereas PAI-2 levels peaked at 6 h of culture. PAI-1 mRNA levels were not affected by hCG or EGF at 6 h of culture, but PAI-2 mRNA levels were significantly increased by EGF at 6 h of culture. These studies demonstrate that human GLC PAI-1 and PAI-2 mRNA levels are differentially regulated and suggest that EGF may be involved in modulation of the human ovarian PA system during the periovulatory period.

Fluorescence staining of nuclear deoxyribonucleic acid allows for accurate assessment of the hamster egg penetration assay.

To improve the assessment of sperm penetration during the hamster penetration assay, we compared the Hoechst 33342 and 33258 DNA-specific fluorescent stains with the standard acetolacmoid stain. The fluorescence stains produced distinct staining of the DNA within the egg cytoplasm and nucleus, and this allowed for accurate and fast assessment of sperm penetration.

Local anesthesia with conscious sedation for laparoscopic intrafallopian transfer.

Local anesthesia with conscious sedation is well accepted by patients and provides scheduling flexibility, cost containment, patient safety, and ease of recovery. We believe the technique should be offered to selected patients undergoing intrafallopian transfer. By adhering to specific guidelines for surgical technique and monitoring, the procedure is a safe and acceptable alternative to general anesthesia for laparoscopic intrafallopian transfers.

A new cervical introducer for embryo transfer with soft open-end catheters.

Fine open-end ET catheters offer several benefits and are now commonly used in IVF-ET procedures. However, they are not always easy to thread into the uterine cavity. We describe a metallic cervical cannula that allows the use of the popular Tomcat catheter in the majority of patients in whom we were unable to achieve a successful ET with the Tomcat alone.

Human granulosa contain messenger ribonucleic acids encoding insulin-like growth factor-binding proteins (IGFBPs) and secrete IGFBPs in culture.

OBJECTIVE: To determine if luteinizing human granulosa cells contain messenger ribonucleic acid (mRNAs) encoding insulin-like growth factor-binding protein (IGFBPs) and if cultured granulosa secrete IGFBPs into conditioned medium (CM). DESIGN: Northern analysis, using IGFBP-specific complementary deoxyribonucleic acid probes, was used to detect granulosa-derived IGFBP mRNAs. Western ligand blot analysis of CM was used to detect IGFBPs secreted by granulosa cultures with and without human chorionic gonadotropin (hCG). SETTING: Granulosa cells were obtained from the In Vitro Fertilization (IVF) Program at Stanford University, a private teaching institution. PATIENTS, PARTICIPANTS: Patients undergoing IVF for tubal disease. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Transcripts of IGFBP mRNA and IGFBPs secreted into CM were detected by autoradiography of Northern and Western ligand blots, respectively. RESULTS: Transcripts of IGFBP-3, IGFBP-2, and IGFBP-1 mRNA were detected in human luteinizing granulosa. Cultured granulosa secreted IGFBPs with molecular weights corresponding to IGFBP-3, IGFBP-2, and IGFBP-1, and the latter two IGFBPs increased with 10 ng/mL hCG. A 24 kd IGFBP was noted, which may be newly characterized IGFBP-4. CONCLUSIONS: These data show that luteinizing human granulosa cells express mRNAs encoding three IGFBPs, secrete IGFBPs into culture medium, and that production of at least two of the IGFBPs is hCG-dependent, further supporting a role for the IGF system in human folliculogenesis.