ICSI outcomes in men undergoing TESE for azoospermia and impact of maternal age.

This retrospective study compared clinical outcomes in men with obstructive and nonobstructive azoospermia after ICSI following testicular sperm extraction and the influence of maternal age. Fertilisation rates, embryo quality, pregnancy rates, miscarriage rates and live birth rates were evaluated. Men with obstructive azoospermia (OA) had significantly higher rates of diploid fertilisation and clinical pregnancy than men with nonobstructive azoospermia (NOA), but miscarriage rates and live birth rates were not significantly different. The higher rates of fertilisation, embryo quality and clinical pregnancy in men with OA were statistically significant when their female partners were <35 years but results were similar in both groups when female partners ≥35 years. Although the OA group had better overall quality embryos than the NOA group when maternal age was <35 years, embryologists can select the morphologically better embryos for transfer, eliminating the effect of embryo quality differences present in these two groups. Understanding more about factors that affect TESE/ICSI outcomes will not only help us predict patients' outcomes but it can help us educate and better counsel our patients.

Impact of male age on the outcome of assisted reproductive technology cycles using donor oocytes.

This study assessed the influence of the age of the male partner on the outcome of oocyte donation cycles. A total of 408 couples participating in 519 consecutive anonymous oocyte donation cycles were examined. Main outcome measures were fertilization rate, embryo quality, clinical pregnancy, implantation, miscarriage and live birth rates, as well as the total reproductive potential, which estimates the outcome from fresh and cryopreserved-thawed embryo transfers. A total of 241 cycles resulted in clinical pregnancy (48.5% of transfers). The mean embryo score for transferred embryos (ESTE) was higher in cycles resulting in pregnancy (P=0.003). Semen volume (P<0.001), sperm motility (P<0.001) and fertilization rate (P=0.04) decreased significantly with advanced male age, which did not correlate with mean ESTE or implantation rate. Fertilization rate was the only predictor of ESTE (B=16.066, P=0.012), whereas inseminated/retrieved egg ratio was the only predictor of implantation rate (B=0.555, P=0.039). Pregnancy was only predicted by ESTE (Exp(B)=1.023, P<0.001), which also was the only predictor of live birth (Exp(B)=1.017, P=0.009). There was no predictor of miscarriage (47 cycles, 9.1%) identified. Although semen volume, sperm motility and fertilization rate decreased with advanced male age, embryo quality, clinical pregnancy, implantation, miscarriage and live birth rates were not affected.

Impact of metformin therapy on ovarian stimulation and outcome in 'coasted' patients with polycystic ovary syndrome undergoing in-vitro fertilization.

This study was designed to determine if metformin therapy improves ovarian stimulation and IVF outcomes in coasted patients with clomiphene-resistant polycystic ovarian syndrome (PCOS). A retrospective data analysis was performed on women with clomiphene citrate-resistant PCOS treated with or without metformin, who underwent 72 cycles of IVF-embryo transfer with intracytoplasmic sperm injection (ICSI). In 59 cycles, patients were coasted to allow oestradiol concentrations to drop before human chorionic gonadotrophin administration. In patients undergoing coasting, the outcome of IVF with ICSI was compared in those who were treated and untreated. In patients treated with metformin, follicular fluid concentrations of testosterone and insulin were significantly lower (60.5 +/- 5 versus 79.1 +/- 6 ng/dl; P < 0.05 and 18 +/- 2.5 versus 22 +/- 2.4 micro IU/ml; P < 0.05 respectively), and the mean number of oocytes retrieved (22.3 +/- 2.4 versus 19.7 +/- 1.6) did not differ. The metformin-treated group showed an increase in the mean number of mature oocytes, oocytes fertilized and cleaving embryos (4-cell or greater by 72 h). However, in the group of patients undergoing coasting, maximum oestradiol concentrations and number of days of coasting were all lower in the metformin-treated group with increased clinical pregnancy rates (71 versus 30%, P < 0.05). Therefore, metformin use appears beneficial in improving IVF outcomes in clomiphene citrate-resistant PCOS patients.

Metformin treatment of patients with polycystic ovary syndrome undergoing in vitro fertilization improves outcomes and is associated with modulation of the insulin-like growth factors.

OBJECTIVE: To determine if metformin therapy improves in vitro fertilization (IVF) outcomes in patients with clomiphene-resistant polycystic ovarian syndrome (PCOS). DESIGN: Retrospective data analysis of selective groups of patients. SETTING: A private IVF unit. PATIENT(S): Forty-six women with clomiphene citrate-resistant PCOS underwent 60 cycles of IVF embryo transfer with intracytoplasmic sperm injection. INTERVENTION(S): In half of the cycles, patients received metformin (1000 to 1500 mg) daily, starting the cycle prior to gonadotropin treatment. MAIN OUTCOME MEASURE(S): Total number of follicles; serum estradiol (E2) on the day of hCG administration and the cycle's E2 maximum; total number of oocytes, mature oocytes, embryos, fertilization, and pregnancy rates; and follicular fluid levels of insulin-like growth factors (IGF-I, IGF-II) and IGF-binding proteins (IGFBP-1, IGFBP-3). RESULT(S): In patients treated with metformin, the total number of follicles on the day of hCG treatment was decreased (23 +/- 1.2 vs. 33 +/- 2.6) with no change in follicles > or = 14 mm in diameter (21 +/- 1.2 vs. 25 +/- 1.7). Metformin treatment did not affect the mean number of oocytes retrieved (22 +/- 1.9 vs. 20.3 +/- 1.5). However, the mean number of mature oocytes (18.4 +/- 1.5 vs. 13 +/- 1.5) and embryos cleaved (12.5 +/- 1.5 vs. 5.9 +/- 0.9) were increased after metformin treatment. Fertilization rates (64% vs. 43%) and clinical pregnancy rates (70% vs.30%) were also increased. Metformin led to modulation of preovulatory of follicular fluid IGF levels with increases of IGF-I (140 +/- 8 vs. 109 +/- 7ng/mL) and decreased of IGFBP-1 (133 +/- 8 vs.153 +/- 9ng/mL). CONCLUSION(S): Metformin use appears to improve IVF outcomes in patients with clomiphene citrate-resistant PCOS.

Cauterization of hydrosalpinges before in vitro fertilization is an effective surgical treatment associated with improved pregnancy rates.

OBJECTIVE: Our purpose was to determine whether proximal tubal cauterization is an effective method of reversing the decreased pregnancy rates seen in patients undergoing in vitro fertilization-embryo transfer with hydrosalpinges present. STUDY DESIGN: We studied a group of 94 patients with tubal factor infertility. Sixty patients had hydrosalpinges documented by either hysterosalpingography or laparoscopy, or both. Forty-five had surgical treatment of hydrosalpinges by salpingectomy or by proximal tubal cauterization. In vitro fertilization-embryo transfer was performed within 3 months after surgery. Pregnancy and implantation rates were compared. RESULTS: Patients with hydrosalpinx had significantly decreased clinical pregnancy and implantation rates per cycle (14% and 8%, respectively) compared with those of patients undergoing proximal tubal cauterization before the in vitro fertilization cycle (73% and 36%, respectively). These pregnancy and implantation rates are comparable with those found in patients with tubal factor infertility without hydrosalpinges (53% and 22%, respectively), as well as in salpingectomy-treated patients (46% and 24%, respectively). CONCLUSIONS: Proximal tubal cauterization is effective in reversing the adverse effects of hydrosalpinges.

Follicular fluid insulin-like growth factor-I and insulin-like growth factor-binding protein-1 and -3 vary as a function of ovarian reserve and ovarian stimulation.

PURPOSE: Follicular fluid concentrations of insulin-like growth factor (IGF)-I, IGF-II, IGF-binding protein (BP)-1, and IGFBP-3 in 57 women undergoing in vitro fertilization and embryo transfer were examined to determine whether levels reflected differences in patients' exposure to gonadotropin stimulation and a diminished ovarian reserve. METHODS: Preovulatory follicular fluid was obtained from both gonadotropin-stimulated and unstimulated cycles. Subjects were grouped according to normal or decreased ovarian reserve and whether or not they received gonadotropin stimulation. RESULTS: The mean follicular fluid concentrations of IGF-I and IGFBP-1 were significantly lower in the "decreased" ovarian reserve group compared with the "normal" ovarian reserve group, with no change in estradiol or IGF-II levels. This resulted in a decreased molar IGF-I: BP ratio and an increased molar IGF-II:IGFBP-1 ratio. In unstimulated cycles, mean follicular fluid concentrations of IGFs did not differ significantly compared with those in stimulated cycles, whereas concentrations of IGFBP-1 and IGFBP-3 were significantly lower, leading to higher molar ratios of the IGFs to the binding proteins. CONCLUSIONS: Follicular fluid IGF and binding proteins vary as a function of ovarian reserve and gonadotropin stimulation. This may reflect either differences in oocyte quality or a suboptimal follicular fluid environment.

Reversal of tubal sterilization using laparoscopically placed titanium staples: preliminary experience.

We tested the feasibility of performing outpatient laparoscopic surgery to reverse tubal sterilization using titanium staples to reapproximate the oviducts. A total of 14 women underwent the procedure which involved excision of the tubal eschar, stenting of the severed remnants, and circumferential stapling of the muscularis and serosa. Reapproximation was possible in all cases, with a measured tubal length post-anastomosis of 4.5 +/- 0.5 cm (range 3.0-7.0 cm). The length of operating time was 2.8 +/- 0.2 h (range 2.2-3.8 h), and all patients were discharged the same day. There were no operative complications, and no readmissions were necessary. Within 6 months of surgery there were six pregnancies including one spontaneous abortion and five ongoing pregnancies. Of those not conceiving within 8 months, seven (100%) demonstrated tubal patency on a follow-up hysterosalpingogram. We conclude the laparoscopic approach to tubal sterilization reversal is a viable alternative to open abdominal microsurgical approaches. Although preliminary, laparoscopic surgery promises to be cost effective, as it can be performed on an outpatient basis, may reduce operative time and minimizes the recuperative period of patients.

Outpatient Reversal of Sterilization with Laparoscopically Placed Titanium Staples

We assessed the feasibility of performing outpatient reanastomosis of the fallopian tubes using titanium staples. Twelve women underwent routine infertility evaluation followed by laparoscopic tubal anastomosis. After preparation of the fallopian tubes, cannulation was performed proximally by hysteroscope or distally with a 3F catheter stent. The mesosalpinx was approximated with titanium staples. Circumferential staples (3-4) were placed around the stented tube to approximate the tubal muscularis. Patency was documented intraoperatively by chromotubation. Mean operating time was 130 minutes (range 90-170 min). Patients were generally discharged within 6 hours of surgery. Bilateral anastomosis was performed in nine women and unilateral anastomosis in three due to inadequate tubal length. Preliminary follow-up with hysterosalpingogram at 6 months revealed 80% tubal patency, and four women are currently pregnant. We suggest that laparoscopic reversal of sterilization is a viable alternative to more traditional methods and can be performed in an outpatient setting.

The significance of endometrial filling defects detected on routine transvaginal sonography.

The objective of this study was to compare endometrial filling defects detected on transvaginal sonography performed without distending media with findings on hysterosalpingography. Forty-three infertile patients were studied in the follicular phrase. Of these, 17 patients had endometrial contour abnormalities detected on vaginal sonogram and 26 patients had no filling defects on ultrasonography. Ultrasonography of the endometrium and hysterosalpingography were performed on all patients as part of a routine workup for infertility. Patients with contour abnormalities detected on either hysterosalpingography or transvaginal ultrasonography underwent hysteroscopy. When a contour abnormality was detected on vaginal sonogram, hysterosalpingography confirmed a defect in 94% of the cases and hysteroscopy in 100% of the cases. Seven of 26 subjects (27%) with normal vaginal ultrasonograms had abnormal hysterosalpingograms; however, only two patients with a normal sonogram had abnormal hysteroscopic findings. Filling defects seen on hysterosalpingography that were not detected on sonography include synechiae, and cornual polyps. This study indicates that vaginal ultrasonography is an accurate tool in the identification of submucous fibroids and polyps. If the examination is performed in the follicular phase, use of distending medium is not necessary to detect major abnormalities of the endometrium. Synechiae are not, however, visible without distention of the endometrial cavity.

High dosages of gonadotropins are associated with poor pregnancy outcomes after in vitro fertilization-embryo transfer.

OBJECTIVE: To compare the pregnancy outcomes during IVF-ET when different dosages of hMG are used after follicular phase suppression with leuprolide acetate (LA). DESIGN: Retrospective chart review. SETTING: Hospital-based IVF-ET program. PATIENTS: From January 1990 to December 1992, 264 cycles reached ET after LA downregulation and gonadotropin stimulation. RESULTS: Higher doses of gonadotropins, as measured by both average daily dose and total dose per cycle, were associated with lower clinical pregnancy rates. This effect was independent of age, basal FSH level, endometrial thickness, maximal E2 levels, number of eggs retrieved, and embryos transferred. CONCLUSION: High dosages of exogenous gonadotropins are associated with lower pregnancy rates in IVF-ET.

Phosphorylation of synthetic insulin receptor peptides by the insulin receptor kinase and evidence that the preferred sequence containing Tyr-1150 is phosphorylated in vivo.

Three peptides were synthesized corresponding to potential autophosphorylation sites of the beta subunit of the human insulin receptor. These were peptide 1150 corresponding to amino acids 1142-1153 of the pro-receptor, peptide 960 corresponding to amino acids 952-961 of the proreceptor, and peptide 1316 corresponding to amino acids 1313-1329 of the proreceptor. Peptide 1150 served as a better substrate for the insulin receptor tyrosine protein kinase than either of the other peptides or than the Src peptide (corresponding to the sequence surrounding the autophosphorylation site at Tyr-416). Microsequencing of the phosphorylated peptide 1150 indicated that Tyr-1150 rather than Tyr-1146 or Tyr-1151 was phosphorylated in the in vitro reaction. The insulin receptor was then isolated from 32P-labeled IM-9 cells that had been exposed to insulin. Tryptic digestion of the beta subunit revealed one peptide whose phosphorylation was dependent upon insulin and occurred exclusively on Tyr. This peptide was selectively immunoprecipitated by an antipeptide antibody directed to the Tyr-1150-containing sequence. We conclude that Tyr-1150 is preferentially phosphorylated by the purified receptor kinase and that one of the autophosphorylation reactions elicited by insulin in intact cells occurs in a sequence that contains this residue.

Increasing the cAMP content of IM-9 cells alters the phosphorylation state and protein kinase activity of the insulin receptor.

The effect of 8-bromo-cAMP and forskolin on the phosphorylation state and protein kinase activity of the insulin receptor was evaluated in cultured IM-9 lymphoblasts. 8-Bromo-cAMP (1 mM) or forskolin (10 microM) enhanced the phosphorylation of the insulin receptor purified from 32P-labeled cells by affinity chromatography on wheat germ agglutinin-agarose and immunoprecipitation with monoclonal antibody. In the absence of insulin, phosphorylation of the beta subunit of the receptor was increased approximately 2-fold by raising intracellular cAMP. Phosphoamino acid analysis of the beta subunit following treatment of cells with forskolin revealed an increase in phosphoserine and phosphothreonine residues. In contrast, the insulin-stimulated phosphorylation of the receptor occurred on serine, threonine, and tyrosine residues and was diminished by prior exposure of cells to forskolin. Pulse-chase experiments indicated that forskolin did not enhance the turnover of phosphate on the receptor of cells previously exposed to insulin. Furthermore, extracts from forskolin-treated cells did not differ from control extracts in their capacity to dephosphorylate 32P-labeled receptor isolated from cells treated with insulin. The insulin-dependent tyrosine protein kinase activity of the receptor isolated from forskolin-treated cells was approximately 50% as active as the receptor isolated from either control or insulin-treated cells. This was assessed using both histone and a peptide synthesized in accordance with the deduced amino acid sequence of a potential autophosphorylation site of the human receptor (Thr-Arg-Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg-Lys) as substrates for the protein kinase reaction. These results suggest that agents that raise intracellular cAMP increase phosphorylation of the insulin receptor on serine and threonine residues, reduce insulin-mediated receptor phosphorylation on tyrosine, serine, and threonine residues, and inhibit the insulin-dependent tyrosine protein kinase activity of the receptor. Thus cAMP may attenuate insulin action by altering the state of phosphorylation of the insulin receptor.

Phosphorylation of exogenous substrates by the insulin receptor-associated protein kinase.

A glycoprotein-enriched fraction derived from 3T3-L1 adipocyte membranes by Triton X-100 extraction and chromatography on wheat germ agglutinin-agarose contains an insulin-activable protein kinase that catalyzes the phosphorylation of tyrosine residues in proteins (Petruzzelli, L. M., Ganguly, S., Smith, C.J., Cobb, M. H., Rubin, C.S., and Rosen, O. M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6792-6796). The best peptide substrates for the enzyme are angiotensin II, and a synthetic peptide related to the amino acid sequence surrounding the site of tyrosine phosphorylation in the transforming protein kinase of Rous sarcoma virus. Kinetic analysis of the phosphorylation reaction with angiotensin II showed that insulin decreased the Km from 5.0 to 2.6 mM, and increased the Vmax from 0.6 to 2.2 pmol/min/10 fmol of insulin binding capacity. For the src-related peptide, the addition of insulin decreased the Km from 1.6 to 1.2 mM and increased the Vmax from 0.17 to 2.2 pmol/min/10 fmol of insulin binding capacity. Angiotensin III inhibitor, proctolin, beta-lipotropin (61-65) and Tyr-Arg were poorer substrates for the protein kinase. Protein substrates for the insulin-stimulated protein kinase include anti-src IgG, tubulin, casein, and histone H2b. The data suggest that the substrate specificity of the insulin-activable protein kinase is similar to that reported for the src and epidermal growth factor receptor protein kinases.