Analysis of androgen receptor and anti-Müllerian hormone pathways in human granulosa cells under luteinizing hormone treatment.

BACKGROUND: The objective of this study was to determine the gene expression profiles of the androgen/androgen receptor (AR) and anti-Müllerian hormone (AMH)/ Sry-related high-mobility group box 9 (SOX9) pathways in granulosa-luteal cells from patients undergoing standard in vitro fertilization (IVF) with or without recombinant luteinizing hormone (rLH) therapy. METHODS: Levels of reproductive hormones in the pre-ovulatory follicular fluid and the expression levels of LHR (luteinizing hormone receptor), AR, SOX9, AMH, AR-associated protein 54(ARA54)and ARA70 were determined in granulosa-luteal cells by real-time reverse-transcription PCR. The effects of androgen and rLH treatments on AR and AMH expression levels were also tested in vitro using HO23 cells. RESULTS: We collected 35 an 70 granulosa cell samples from patients cycled with and without rLH supplementation, respectively. The clinical outcomes were similar in patients who received rLH therapy and those who did not, though the pre-ovulatory follicular fluid levels of androstenedione, testosterone, and estradiol were significantly higher and progesterone was lower in the rLH supplementation group. Moreover, granulosa-luteal cell mRNA levels of LHR, AR, AMH, and SOX9 were significantly higher in the rLH supplementation group relative to the group that did not receive rLH supplementation. In addition, we observed significant correlations between LHR and AR mRNA expression and among AR, AMH, and SOX9 mRNA expression in granulosa-luteal cells from patients undergoing standard IVF treatment. CONCLUSIONS: Increased expression of LHR, AR, AMH, and SOX9 is characteristic of granulosa-luteal cells from IVF/ intracytoplasmic sperm injection (ICSI) patients receiving rLH supplementation.

VEGF, IVF clinical variables and ovarian hyperstimulation syndrome: a prospective study of the preimplantation luteal phase.

OBJECTIVE: To explore the relationships between vascular endothelial growth factor (VEGF) levels in the preimplantation luteal phase and ovarian response, embryonic development, pregnancy outcome and occurrence of ovarian hyperstimulation syndrome (OHSS) in women undergoing in vitro fertilization (IVF). STUDY DESIGN: The level of total VEGF in peritoneal fluid and serum was determined in 61 consecutive women undergoing day 3 tubal embryo transfer via laparoscopy. A MEDLINE search of the literature for 1996 to 2003 was conducted to review the relationship between VEGF levels and clinical variables at different time points during the IVF treatment cycle. RESULTS: No correlation existed between total VEGF levels and patient age, estradiol production, number of oocytes retrieved, embryo development or pregnancy outcome. There was no significant difference between the 12 patients who developed OHSS and the 49 who did not. A literature review yielded inconclusive results. CONCLUSION: A single measurement of total VEGF obtained from the serum or peritoneal fluid during the preimplantation luteal phase adds little to the clinical assessment of IVF response or to predicting the risk of OHSS. Large-scale studies at different times during the IVF cycle are needed to better define its potential use as a biomarker.

Effects of retinoic acid on the inner cell mass in mouse blastocysts.

This is the first evidence that excess retinoic acid has a direct cellular response from proliferation to cell death (apoptosis) and affects in vitro development in mouse inner cell mass.

Quantified short-term outcome of uterine artery embolization with gelatin sponge particles and lipiodol for symptomatic myoma.

OBJECTIVE: To investigate and quantify clinical outcomes and spectral Doppler analyses of uterine arteries in patients with myoma undergoing uterine artery embolization (UAE) with gelatin sponge particles and lipiodol. DESIGN: Prospective observational study. SETTING: Tertiary medical center. PATIENT(S): Forty premenopausal women with symptomatic myoma. INTERVENTION(S): Uterine artery embolization with gelatin sponge particles and lipiodol. MAIN OUTCOMES MEASURE(S): Hemoglobin, hematocrit, CA-125, pictorial blood loss assessment, visual analogue pain scale, questionnaire for symptoms, tumor volume, and spectral Doppler analyses of uterine arteries. RESULT(S): The mean follow-up period was 8.1 months (range, 6-12). Menstrual flow improved in 29 of 35 patients (83%) and decreased significantly by 78.4%. Menstrual pain improved in 27 of 35 patients (77%) and decreased significantly by 70%. Hematocrit and CA-125 improved significantly. The mean percentage reductions of uterine and myomal volumes were 40.2% and 54.9%, respectively. The mean peak systolic velocity of the uterine arteries decreased by 52%. The major complication rate was 2.56%. There was no correlation between tumor volume reduction and clinical outcome. CONCLUSION(S): Uterine artery embolization with gelatin sponge particles and lipiodol had satisfactory short-term outcomes, comparable to those associated with polyvinyl alcohol particles. Quantified and semiquantified measurements provided objective assessment of clinical outcomes. Serum CA-125 might play a role in clinical follow-up. Reduction of tumor volume is not predictive of UAE efficacy.

Impact of duration of cryopreservation of spermatozoa obtained through testicular sperm extraction on intracytoplasmic sperm injection.

Cryopreservation of testicular spermatozoa is feasible for patients suffering obstructive or nonobstructive azoospermia. A stndardized intracytoplasmic sperm injection procedure using frozen-thawed testicular tissue gives rise to fertilization and cleavage rates, which appear not to be affected by the duration of the period of cryostorage.

Transfer of frozen-thawed blastocysts that underwent quarter laser-assisted hatching at the day 3 cleaving stage before freezing.

OBJECTIVE: To examine the pregnancy potential of frozen-thawed blastocysts that underwent quarter laser-assisted hatching (AH) at the cleaving stage before freezing and to compare clinical and embryo characteristics between the groups that succeeded in and failed to achieve pregnancy. DESIGN: Prospective observational study. PATIENT(S): Thirty-four of 112 patients with frozen blastocysts requiring transfer of thawed embryos between January 2000 and December 2001. SETTING: Assisted reproductive technology unit in a tertiary medical center. INTERVENTION(S): Embryos of patients undergoing blastocyst transfers routinely underwent quarter AH using a nontouch 1.48-microm diode laser. Blastocysts not transferred were cryopreserved using a six-step freezing protocol with glycerol as the cryoprotectant. MAIN OUTCOME MEASURE(S): Postthaw embryo survival and zona pellucida (ZP) maintenance, implantation rate, and clinical pregnancy rate per transfer of thawed blastocysts. RESULT(S): A total of 118 frozen blastocysts was thawed. Of these, 89 (75.4%) embryos survived and were transferred in 35 cycles. Assisted hatching-manipulated ZP tolerated the freeze-thaw procedures without shape distortion in surviving embryos. Eleven (31.4%) clinical pregnancies with 15 intrauterine gestational sacs occurred, for an implantation rate of 16.9%. Those who succeeded in pregnancy tended to have more embryos available before freezing, more original blastocysts of good quality for freezing, and more surviving blastocysts after thawing for transfer than did those who failed to achieve pregnancy. CONCLUSION(S): An acceptable clinical pregnancy rate was obtained from transfer of thawed blastocysts that underwent laser AH on the ZP at the day 3 cleaving embryo stage in fresh cycles in selected patients. Embryo characteristics before freezing played major roles in determining implantation potential of thawed embryos.

Transfer of nonselected transferable day 3 embryos in low embryo producers.

OBJECTIVE: To examine the implantation potential of embryos from assisted reproductive technology cycles with low embryo production and to assess the effects of clinical variables and embryo scores (ES) on pregnancy outcome. DESIGN: Prospective clinical study. SETTING: Assisted reproductive technology unit in a tertiary medical center. PATIENT(S): From July 1998 to December 2001, 280 cycles in 229 infertile couples produced a limited number of one, two, or three embryos 3 days after oocyte retrieval and underwent fresh embryo transfer (ET). INTERVENTION(S): Embryos with two or more blastomeres were scored and transferred. MAIN OUTCOME MEASURE(S): ES and implantation rate per ET. RESULT(S): Of 863 fresh ET cycles during the study period, 32.4% (280) were low embryo producers. Among them, there were no significant differences in average ES of individual embryos in single, dual, or triple ET or in embryos obtained from patients with low or high E2 responses, or young or old age. Embryos derived from conventional IVF had a better ES than those derived from intracytoplasmic sperm injection. The clinical pregnancy rate was strongly correlated with the cumulative ES. Implantation rates were similar among and between groups, with an average rate of 15.9%. CONCLUSION(S): Embryos of low embryo producers had an inherently low implantation potential that appeared to be unrelated to the number of embryos transferred, female age, ovarian E2 genesis, or fertilization method. The cumulative ES can serve as a predictor of pregnancy.

Significance of mRNA levels of connexin37, connexin43, and connexin45 in luteinized granulosa cells of controlled hyperstimulated follicles.

OBJECTIVE: To evaluate the mRNA levels of connexins in different sizes of luteinized follicles. DESIGN: Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to determine the connexin mRNA levels in the granulosa cells of 91 luteinized follicles. SETTING: Academic tertiary care medical center and research unit of university. PATIENT(S): Ninety-one female patients on controlled ovarian hyperstimulation (COH). INTERVENTION(S): Sonoguided aspiration to collect the oocytes and the granulosa cells simultaneously. MAIN OUTCOME MEASURE(S): The RT-PCR data are normalized by comparing the intensity of the connexins to the intensity of internal controls (beta-actin). The follicles are grouped according to the size and the mRNA levels of the connexins. The correlations among the size of the follicles, the outcome of oocytes, and the mRNA levels of the connexins are compared by Fisher's exact test and Mann-Whitney test. RESULT(S): The mRNA levels of the connexins are low in the follicles equal or larger than 5.5 mL. High cx43 levels are linked to good prognosis of oocytes. CONCLUSION(S): The luteinized granulosa cells from the large follicles are relatively quiescent in the connexin mRNA expression. In addition to the volume, the mRNA levels of cx43 may serve as a marker to predict the outcome of oocytes.

Modified laser-assisted zonal opening method for human embryo biopsy in preimplantation genetic diagnosis: preliminary experience.

OBJECTIVE: To test the safety and efficacy of a modified laser-assisted zonal opening method for human embryo biopsy. STUDY DESIGN: The embryo was treated with a modified method to create an ample perivitelline space between the zona pellucida and underlying blastomeres. This was done to protect the blastomeres from damage by laser treatment of the zona pellucida. Subsequently, the zona pellucida was completely perforated using a diode laser, and the targeted blastomere was aspirated. In vitro embryo development of the 40 biopsied embryos was compared with that of 322 corresponding control embryos without embryo biopsy. RESULTS: Forty targeted blastomeres were successfully extracted from 40 human embryos. The incidence of embryo development to the blastocyst stage was not different between the biopsied and nonbiopsied groups (55% vs. 54.3%). Furthermore, the percentage of top-scoring blastocysts from biopsied embryos was also similar to that of control embryos (77.3% vs. 78.9%). CONCLUSION: These preliminary results demonstrate that the modified technique for human embryo biopsy in preimplantation genetic diagnosis is simple and safe.

Comparison of the offspring sex ratio between cleavage stage embryo transfer and blastocyst transfer.

OBJECTIVE: To compare the sex ratio of offspring born after cleavage stage embryo transfer and blastocyst transfer. MATERIALS AND METHODS: In this retrospective study of embryo transfer (ET), we included 473 offspring from 446 deliveries during the period January 2002 to December 2007. Statistical analysis was performed on the sex ratio of offspring resulting from day 3 cleavage stage embryo transfer and from sequential blastocyst culture transfer. RESULTS: In total, 446 patient deliveries were included in this analysis. There were 251 singleton pregnancies, 109 twin pregnancies, and four triplet pregnancies. The total number of offspring was 473, of which 118 resulted from day 3 ETs, and 355 resulted from blastocyst ETs. At our center, the influence on the sex ratio of cleavage stage ET and blastocyst-stage ET showed a bias towards males in both cases. The overall female to male ratio for offspring resulting from day 3 ETs was not significantly higher than the same ratio for offspring resulting from blastocyst ETs (p = 0.24; odds ratio, 0.762). The female to male ratio for either singleton births or multiple deliveries was also not significantly different between day 3 ETs and blastocyst ETs. CONCLUSION: The sex ratio was influenced by cleavage stage ET and blastocyst-stage ET. In both cases, there was a bias towards males. In addition, when blastocyst ET was compared with day 3 ET, there was no further increase in the percentage of male offspring.

Significantly superior response in the right ovary compared with the left ovary after stimulation with follicle-stimulating hormone in a pituitary down-regulation regimen.

OBJECTIVE: To compare the differences between the right and left ovarian responses in patients undergoing standard in vitro fertilization (IVF) treatment. DESIGN: Prospective, observational study. SETTING: A university hospital in Taiwan. PATIENT(S): Seven hundred eighty-nine consecutive patients undergoing IVF or intracytoplasmic sperm injection. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The number of oocytes retrieved, fertilization rates, number of grade 1 zygotes and number of embryos produced, embryo arrest-free rate, and mean number of top-quality embryos on the day of transfer. RESULT(S): The number of follicles > or = 16 mm in diameter on day 2.5 versus 2.2 of human chorionic gonadotropin (hCG) administration, the number of follicles > or = 10 mm in diameter on hCG day 6.5 versus 6.1, the number of oocytes (3.6 vs. 3.0), the percentage fertilization (79.9 vs. 74.6%), and the mean number of mature oocytes and grade 1 zygotes (2.3 vs. 1.8 and 1.79 vs. 0.93, respectively), and the mean available number of top-quality embryos on transfer day 1.39 versus 0.73 were all statistically significantly greater in the right compared with the left ovary; however, the pregnancy and implantation rates were similar. CONCLUSION(S): There are statistically significant differences between the right and left ovarian responses in IVF patients with healthy ovaries, and the right ovarian responses are superior to the left ovarian responses.

Comparison of in vitro fertilization versus intracytoplasmic sperm injection in extremely low oocyte retrieval cycles.

OBJECTIVE: To compare the results of IVF and intracytoplasmic sperm injection (ICSI) in extremely low oocyte retrieval cycles. DESIGN: Case-control study. SETTING: An IVF laboratory in a university hospital in Taiwan. PATIENT(S): Between January 2003 and December 2007, infertile couples with one, two, or three oocytes per retrieval were divided into two groups: 56 IVF cycles based on 28 demographic characteristics from the ICSI group. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Fertilization rate, cycle cancellation rate, clinical pregnancy rate, and implantation rate per transfer. RESULT(S): The normal fertilization rate was significantly higher after ICSI than after standard IVF (86.4% vs. 69.3%). The cycle cancellation and complete fertilization failure rates were comparable between the two groups. The clinical pregnancy rate per transfer (38.5% vs. 21.6%), implantation rate (23.5% vs. 11.1%), and live birth rate per transfer (30.8% vs. 13.7%) had a favorable trend, but no statistically significant differences, in the ICSI group compared with the IVF group. CONCLUSION(S): A higher normal fertilization rate can be achieved by ICSI in comparison with conventional IVF in extremely low oocyte retrieval cycles.

Comparison of the efficacy of two vaginal progesterone formulations, Crinone 8% gel and Utrogestan capsules, used for luteal support in blastocyst stage embryo transfers.

OBJECTIVE: To compare the efficacy of two vaginal progesterone formulations, Crinone gel and Utrogestan capsules, for luteal phase support in blastocyst stage embryo transfers. MATERIALS AND METHODS: We analyzed 460 consecutive cycles in patients undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI)-blastocyst transfer (BT) treatment at our institution between January 2004 and December 2007. Patients applied either Crinone 8% vaginal gel (90 mg, once daily) or Utrogestan vaginal capsules (200 mg, four times daily) for luteal supplementation. Progesterone was administered from the day of oocyte retrieval to pregnancy confirmation. Clinical pregnancy and implantation rates were the main outcome measures. RESULTS: The clinical pregnancy rate (58.7% vs. 44.3%) and implantation rate (32.04% vs. 23.89%) were significantly higher in the Crinone group compared with the Utrogestan group after IVF/ICSI-BT treatment. CONCLUSION: Luteal phase support with Crinone 8% vaginal gel (90 mg daily) resulted in better clinical pregnancy and implantation rates than Utrogestan vaginal capsules (200 mg, four times daily) in IVF/ICSI-BT cycles.

Zona-free versus laser zona-assisted hatching blastocyst transfer: a comparison of outcomes.

In unselected IVF patients, zona-free and laser zona-assisted hatching produced a comparable effect on blastocyst transfer.

Comparison of the offspring sex ratio between fresh and vitrification-thawed blastocyst transfer.

There was a significant difference in the sex ratio between offspring from fresh and from vitrification-thawed blastocyst transfer. The live birth sex ratio influenced by the blastocyst-stage embryo transfer correlated with top-quality embryo morphology.

Increased progesterone/estradiol ratio on the day of HCG administration adversely affects success of in vitro fertilization-embryo transfer in patients stimulated with gonadotropin-releasing hormone agonist and recombinant follicle-stimulating hormone.

OBJECTIVE: We investigated the influence of premature luteinization in in vitro fertilization using a long protocol of gonadotropin-releasing hormone agonist (GnRHa) and recombinant follicle-stimulating hormone (rFSH), taking ovarian response into account in the definition of premature luteinization. MATERIALS AND METHODS: A total of 339 cycles of controlled ovarian hyperstimulation with rFSH and GnRHa were performed in 311 infertile couples. Premature luteinization was defined as a progesterone (P) to estradiol (E2) ratio of > 1 on the day of human chorionic gonadotropin (hCG) administration. The P/E2 ratio is calculated as: P (ng/mL) 1,000/E2 (pg/mL). Clinical outcomes were compared for the prematurely luteinized and non-prematurely luteinized groups. RESULTS: The mean number of retrieved oocytes, recovered mature oocytes, embryos and top quality embryos were significantly higher in the non-prematurely luteinized group than in the prematurely luteinized group. Although fertilization rates and implantation rates were similar between the two groups, the clinical pregnancy rate was higher in the non-prematurely luteinized group than in the prematurely luteinized group. CONCLUSION: Premature luteinization, defined as late follicular P/E2 ratio of > 1 in long GnRHa cycles with rFSH stimulation, adversely affected ovarian responses and clinical outcomes. It seems unrelated to preovulatory luteinizing hormone (LH) elevation and LH/hCG content of gonadotropins and could be associated with poor ovarian response and the presence of dysmature follicles.

Expression of androgen receptor co-regulators in the testes of men with azoospermia.

OBJECTIVE: To elucidate the physiologic and pathologic roles of androgen receptor (AR) and co-regulators in human testes with obstructive azoospermia or nonobstructive azoospermia. DESIGN: Prospective laboratory and clinical study. SETTING: Infertility clinic at Chang Gung Memorial Hospital. PATIENT(S): Twenty-seven men with obstructive azoospermia and 24 men with nonobstructive azoospermia. INTERVENTIONS: None. MAIN OUTCOME MEASURE(S): Expression of AR and AR co-regulators was determined in testicular specimens and were analyzed using reverse transcription polymerase chain reaction (PCR) and immunohistochemical staining techniques. RESULT(S): Most of the AR co-regulators were expressed at similar levels in specimens obtained from men with nonobstructive and obstructive azoospermia. However, the levels of expression of ARA54 mRNA were significantly lower and ARA55 mRNA was significantly higher in specimens from men with nonobstructive than obstructive azoospermia. In specimens from men with obstructive and nonobstructive azoospermia, AR immunostaining was detected in Sertoli, Leydig, and peritubular myoid cells. ARA55 immunostaining was detected in peritubular myoid and endothelial cells of blood vessels. Interestingly, nuclear ARA54 immunostaining was detected in the late stage germ cells of specimens from men with obstructive azoospermia but in the somatic cells of specimens from men with nonobstructive azoospermia. CONCLUSION(S): These results demonstrated that the decreased expression of ARA54 and increased expression of ARA55 is a feature of nonobstructive azoospermia. In addition, the differential localization of ARA54 may play an important role in testicular development and spermatogenesis in humans.

Concomitant pelvic organ prolapse surgery with TVT procedure.

The aim of this study is to evaluate the efficacy and feasibility of concomitant pelvic reconstructive surgery with tension-free vaginal tape (TVT) procedure to treat pelvic organ prolapse women with urodynamic stress incontinence (USI) or occult USI. Seventy-five women with pelvic organ prolapse and diagnosed as USI or occult USI were enrolled in this study. All patients with USI or occult USI underwent TVT treatment under general anesthesia, combined with transvaginal total hysterectomy (VTH), anterior-posterior colporrhaphy (APC), and/or right sacrospinous ligament suspension (SSS) reconstructive surgeries. The subjective assessment was evaluated by using a visual analog scale (VAS) score and a urinary symptomatic questionnaire. The objective assessment was carried out with a 1-h pad test, cough stress test, and urodynamic examination. Of the 75 patients, 35 patients with grade III uterine prolapse underwent VTH and APC, 30 patients with grade IV uterine prolapse underwent VTH, SSS, and APC, and the other 10 patients who had previous hysterectomy with total vaginal vault prolapse underwent SSS and APC. The mean follow-up interval was 25 months (12-42 months). The mean hospitalization was 5.9 days and the mean catheterization time was 3.8 days. The subjective success rate for the treatment of urine incontinence was 88%, and the objective complete cure rate was 84%. The rate of postoperative complications with persistent urinary urgency, de novo detrusor overactivity, dysfunctional voiding, and tape erosion were 50, 8, 12, and 1.3%, respectively. There were no bladder perforations during the TVT procedure and no perioperative complications requiring conversion to laparotomy. Pelvic organ prolapse women with USI or occult USI can be treated by reconstructive surgeries combined with a TVT procedure to treat and prevent postoperative USI.

Expression of inhibin-activin subunits, follistatin and smads in granulosa-luteal cells collected at oocyte retrieval.

PURPOSE: To examine the expression of inhibin-activin subunits, follistatin, and Smads 2, 3 and 4 in granulosa-luteal cells at the time of oocyte retrieval. METHODS: Quantitative reverse transcriptase-polymerase chain reaction was performed to quantify the mRNA expression of the investigated genes: inhibin alpha-subunit, inhibin-activin betaA- and betaB-subunits, follistatin, and Smads 2, 3 and 4. RESULTS: alpha-, betaA- and betaB-subunits of inhibin-activin, and follistatin were all well expressed in granulosa-luteal cells obtained from either dominant or cohort follicles. alpha-subunits strongly correlated with Smad2 (p < 0.001). betaA-subunit significantly correlated with Smad4, p < 0.001. betaB-subunit significantly correlated with Smad2, p = 0.002, and follistatin expression strongly correlated with the expression of Smads 2 and 4 (p < 0.001 and = 0.007 respectively). CONCLUSIONS: We found, for the first time that inhibin-activin betaB-subunit mRNA was well expressed in human granulosa-luteal cells obtained from either dominant or cohort follicles. alpha-, betaA- and betaB-subunits and follistatin correlated with Smads to varied degrees, suggesting the active roles of the above genes at the time of oocyte retrieval.