Genetic screening of karyotypes and azoospermic factors for infertile men who are candidates for ICSI.

The overall incidence of chromosomal abnormalities was 8%. There was no single distinctive feature of semen parameters predictive of the existence of a chromosomal anomaly. All of the patients with obstructive azoospermia had normal karyotypes and AZF. Three patients out of 13 (23%) with nonobstructive azoospermia without 47,XXY had AZF deletions, as well as two (5%) of 43 with severe oligozoospermia. Ninety-two couples underwent 112 ICSI cycles for which a pregnancy rate of 58% was achieved. Five patients with abnormal karyotypes underwent 6 cycles of ICSI that resulted in 1 successful pregnancy. Two patients with AZF deletions achieved pregnancies. One ICSI-derived male had the same AZF deletion as his father, and 1 female baby had no risk of AZF deletion. The authors recommend karyotyping, excluding those with obstructive causes, prior to ICSI for genetic counseling.

Effects of cryopreservation on meiotic spindles of oocytes and its dynamics after thawing: clinical implications in oocyte freezing--a review article.

Embryo freezing has been a successful practice, but oocyte cryopreservation formerly achieved poorer results. This was mainly due to low rates of survival, fertilization, and development. The major dissimilarities for oocytes to embryos are the character of the plasma membrane, the presence of cortical granules, at the metaphase of meiosis II with the spindle system. In addition, the oocytes must be fertilized by sperm at the appropriate time. To improve the survival rate, a refined slow freezing method with increased sucrose concentration would dehydrate oocytes more sufficiently. Vitrification is another approach to prevent ice crystal formation. Intracytoplasmic sperm injection is used to overcome possible zona hardening from the release of cortical granules. The microtubules of meiotic spindles are vulnerable to the thermal changes and would depolymerize. Cryopreserved oocytes exhibited serious disturbances of the microtubules immediately after thawing. Fertilization of oocytes with disorganized spindles could lead to chromosomal aneuploidy, digyny, and arrest of cleavage. After incubation, the microtubules would repolymerize in a time-dependent way. Normal fertilization and development of cryopreserved oocytes improved after appropriate incubation and timing of insemination, compatible with recovery of the spindles. With the improvement of survival, fertilization, and cleavage, oocyte cryopreservation would gain an imperative role.

Vitrification of mouse oocytes using closed pulled straws (CPS) achieves a high survival and preserves good patterns of meiotic spindles, compared with conventional straws, open pulled straws (OPS) and grids.

BACKGROUND: We modified the loading of pulled straws into a new closed system, called closed pulled straws (CPS) for holding oocytes for vitrification. The morphological survival, dynamics of meiotic spindles, and fertilization in vitro of vitrified oocytes using CPS were compared with conventional straws, open pulled straws (OPS), and grids. METHODS: Surviving oocytes were stained for spindles and chromosomes after 1, 2 and 3 h incubations, and compared with controls. The capacity of fertilization and embryonic cleavage were examined in vitro. RESULTS: The survival rates of the CPS (79%) and straw (77%) groups were significantly higher (P < 0.05) than the OPS (63%) and grid (39%) groups. At a 1h incubation, vitrified oocytes of four groups had significantly fewer normal spindles than controls (P < 0.05). The straw group was inferior to the others in spindle morphology (P < 0.05). After a 3 h incubation, recovery of vitrified oocytes with normal spindles was significantly improved in all groups (P < 0.05). The percentages of fertilization and blastocyst formation of vitrified oocytes after a 1 h incubation was significantly lower than controls (P < 0.05), but they were improved after 2 or 3 h incubations (P < 0.05). CONCLUSIONS: Oocytes vitrified using CPS, OPS or grids could lessen spindle injuries and expedite recuperation. The survival using OPS or grids is lower. Sufficient culture time for recovery of meiotic spindle would be imperative for fertilization events of vitrified oocytes. CPS has the advantages of achieving a high survival and preserving good spindles.

Elevated E2: oocyte ratio in women undergoing IVF and tubal ET. Correlation with a decrease in the implantation rate.

OBJECTIVE: To investigate the influence of various estradiol (E2): oocyte ratios on reproductive outcome in women undergoing in vitro fertilization and tubal embryo transfer (IVF-TET). STUDY DESIGN: Two hundred seven women undergoing 251 IVF-TET cycles were recruited in this retrospective study. All the women received a flare-up gonadotropin releasing hormone agonist (GnRHa) protocol to achieve ovarian hyperstimulation. Oocyte retrieval was performed 34-36 hours after human chorionic gonadotropin (hCG) injection, followed by TET two days later. RESULTS: An E2: oocyte ratio > or = 350 pg/mL had a higher E2 level (2,213 +/- 2,258 vs. 1,553 +/- 972 pg/mL, P < .05) and fertilization rate (77 +/- 23 vs. 64 +/- 23%, P < .001) but a lower oocyte number (4.8 +/- 4.7 vs. 7.6 +/- 4.8, P < .001) than in those with a ratio < 350 pg/mL. The pregnancy (17.9% vs. 32.8%, P = .03) and implantation (5.3% vs. 12.9%, P = .008) rates were significantly decreased in cycles with an E2: oocyte ratio > or = 350 pg/mL as compared to those with a ratio < 350 pg/mL. CONCLUSION: IVF-TET cycles with an elevated E2: oocyte ratio correlated with lower pregnancy and implantation rates. The poor reproductive outcome possibly was due to the relatively high E2 concentration, which might have a detrimental effect on endometrial receptivity.

Open pulled straws for vitrification of mature mouse oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws.

Vitrification of oocytes has been applied recently for humans, but remains elusive. The microtubules of oocytes are vulnerable to cryoprotectants and thermal changes. Using mouse oocytes, the effects of vitrification in open pulled straws (OPS) were investigated on survival, the meiotic spindle, and chromosomes and compared with conventional straws. Mature oocytes were allocated to four groups for exposure to cryoprotectants, vitrification in conventional straws, or vitrification in OPS. They were diluted in stepwise sucrose solutions. Oocytes without treatments were used as controls. The surviving oocytes were stained for meiotic spindles and chromosomes. After dilution, all of the oocytes exposed to cryoprotectants survived. Vitrification sometimes resulted in lysis so that survival using OPS (62%) was significantly (P < 0.05) smaller than that using conventional straws (81%). Oocytes exposed to cryoprotectants or vitrified exhibited serious disturbances of microtubules immediately post-dilution. After 1 h incubation, the microtubules could repolymerize so that the OPS group had significantly (P < 0.05) more normal spindles (78%) than did the conventional straw group (21%). The former also tended to have more compact chromosomes (87%) than did the latter (78%). OPS for vitrification of oocytes achieve more rapid cooling, warming, and dilution and so reduce spindle injury. However, the lower survival rate in OPS needs improvement.

Cryopreservation of mature human oocytes by vitrification with ethylene glycol in straws.

OBJECTIVE: To examine the effect of vitrification with ethylene glycol (EG) for mature human oocytes in straws. DESIGN: Prospective, randomized, in vitro experiments. SETTING: Reproductive unit of a university hospital. PATIENT(S): Immature oocytes from 110 patients undergoing intracytoplasmic sperm injection (ICSI). INTERVENTION(S): The immature oocytes were incubated to reach metaphase II (MII). The MII oocytes were treated with EG-based cryoprotectants and vitrified in straws. They were diluted in sucrose solutions, inseminated by ICSI, and cultured in vitro. MAIN OUTCOME MEASURE(S): Survival, fertilization, and embryo cleavage. RESULT(S): The survival rates were greater for oocytes pretreated with 1.5 M of EG (65% for 0 minute, 93% for 5 minutes, and 96% for 10 minutes). The oocytes vitrified in 60 and 90 seconds had a greater rate of fertilization than those vitrified in 120 seconds. There were no differences in survival and fertilization for vitrified oocytes diluted by three or four steps. The cleavage rates to the six- to eight-cell stage were comparable with controls. However, no blastocyst formation was observed in vitrified oocytes. CONCLUSION(S): Vitrification of human oocytes with EG in straws achieves a high rate of survival, fertilization, and early cleavage of embryos. Further studies should be conducted for the improvement of blastocyst formation.

Schedule to inject in vitro matured oocytes may increase pregnancy after intracytoplasmic sperm injection.

To ascertain the value of using immature oocytes in an intracytoplasmic sperm injection (ICSI) program, the authors designed a schedule, at 5 p.m. on day 1 (the day of oocyte retrieval) and at 8 a.m. and 2 p.m. on day 2, to recognize and inject the in vitro matured (IVM) oocytes. For the 1,166 oocytes retrieved in 107 ICSI cycles, 128 (11.0%) were at the stage of metaphase I (MI) and 113 (9.7%) at germinal vesicle. Routine ICSI for metaphase 11 oocytes was performed at 2 p.m. on day 1 (initial ICSI). In culture medium of human tubal fluid with 15% maternal serum, 85.1% (205/241) immature oocytes progressed to maturation in which 16.4% (21/128) of MI oocytes matured at 5 p.m. of day 1. The rate of normal fertilization for IVM oocytes (58.5%) was not significantly different from that of initial ICSI (64.0%). One patient received a transfer of two fertilized IVM oocytes alone that were injected at 5 p.m. of day 1, maturing from the MI stage, and achieved a normal pregnancy. The fertilized IVM oocytes were replaced along with the embryos from initial ICSI for 40 cycles that led to 14 (35%) clinical pregnancies. In 43 fertilized IVM oocytes donated for research, we observed that cleavage (95.3%) to the 2- to 4-cell stage was not distinct from that of initial ICSI (94.6%); however, the percentage of embryos of grade I and II morphology was significantly smaller (24.4% vs. 62.5%). Only five (11.6%) developed to blastocysts in vitro. Twenty-one fertilized IVM oocytes were frozen for future transfer. A schedule to inject IVM oocytes in ICSI cycles may generate more accessible embryos for fresh transfer or cryopreservation to increase the chance of pregnancy, although the embryo quality was relatively poor.

Transmission of de novo mutations of the deleted in azoospermia genes from a severely oligozoospermic male to a son via intracytoplasmic sperm injection.

OBJECTIVE: To investigate the transmission of microdeletions in the deleted in azoospermia (DAZ) genes to a male offspring via intracytoplasmic sperm injection (ICSI). DESIGN: Case report. SETTING: Reproductive unit of a university teaching hospital. PATIENT(S): A 29-year-old, severely oligozoospermic male with microdeletions of the DAZ genes in Yq interval 6 and his son, who was conceived via ICSI. INTERVENTION(S): DNA screening for the microdeletions in Yq interval 6 with 24 sequence tagged sites with the use of polymerase chain reaction amplification for the patient, the patient's father, and the patient's son. Paternity identification was performed using nine hypervariable short tandem repeats. MAIN OUTCOME MEASURE(S): Deletion mapping of Yq interval 6 from sequence tagged sites and electropherogram of short tandem repeats for DNA fingerprinting. RESULT(S): The son had the same microdeletions of the DAZ genes as the patient, and the patient's father had normal DAZ genes. The paternity of the patient, the patient's father, and the patient's son was verified. CONCLUSION(S): De novo DAZ microdeletions in an infertile male can be transmitted to a male offspring via ICSI. DNA screening tests for DAZ genes before ICSI may help in the genetic counseling of patients with idiopathic azoospermia or severe oligozoospermia.

In vitro fertilization pregnancy achieved with epididymal caput sperm of a patient with obstructive azoospermia.

A successful epididymal sperm aspiration, in vitro fertilization and embryo transfer in a woman whose husband had previously undergone extensive attempts at recanalization of the spermatic ducts is reported. A twin pregnancy was achieved using sperm obtained from the husband's epididymal caput. This case demonstrates that pregnancy can be achieved by this reproductive technique in cases of obstructive azoospermia, even after extensive surgery.

Co-culture with Vero cell monolayer maintains the motility of asthenozoospermic semen samples.

The clinical effectiveness of co-culture with Vero (Green monkey kidney) cell monolayer in maintaining the motility and viability of fresh asthenozoospermic semen (18 samples) and frozen-thawed semen with poor motility (motility fraction < 50%) (15 samples) in a 24-h period was evaluated. Co-culture with Vero cell monolayer in human tubal fluid (HTF) medium for 24 h resulted in a statistically better maintenance of motility percentage (P < 0.005), mean amplitude of lateral head displacement (ALH) (P < 0.005), and mean track speed (VCL) (P < 0.05) than culture in HTF medium alone. However, these motility parameters (motility percentage, ALH, VCL) declined soon after removal of spermatozoa from the monolayer. Co-culture with Vero cell monolayer also maintained the viability percentage of these sperm samples (52% of the original value) after the 24-h period compared with culture in HTF medium alone (22% of the original) (52% versus 22%, P < 0.05). It is concluded that Vero cell monolayer is effective in the maintenance of motility and viability of asthenozoospermic semen or frozen-thawed semen with poor motility. This co-culture system may be beneficial in enhancing the in-vitro performance of asthenozoospermic semen samples in the practice of assisted reproductive technology. However, its safety needs further evaluation.

First-trimester selective termination in multiple gestation by transvaginal ultrasound-guided intrathoracic injection of potassium chloride.

PATIENTS AND METHODS: Selective termination by transvaginal ultrasound-guided intrathoracic injection of potassium chloride (KC1) was performed between 8 and 10 weeks' gestation in eight women with multiple gestation after ovulation induction (6/8) or in vitro fertilization (IVF) (2/8). RESULTS: Four patients have delivered uneventfully at term, one delivered prematurely at 34 weeks' gestation with good neonatal outcome, and the other 3 patients lead a smooth pregnancy at present. No pregnancy loss or any major complication was found. CONCLUSIONS: Transvaginal ultrasound-guided intrathoracic injection of KC1 may be the procedure of choice for first-trimester selective termination with acceptable safety. However, more experience is needed to clarify the risk/benefit ratio associated with this procedure.

Acrosin activity of human sperm did not correlate with IVF.

To evaluate the predicting value of sperm acrosin activity in human, the acrosin activity index (AAI) was measured in 95 semen samples from patients participating in an IVF program. All patients had at least two mature oocytes. Of 95 patients, 84 had successful fertilization and 11 failed to fertilize all oocytes in vitro. The numbers of mature oocytes were similar between fertilization and nonfertilization groups. The mean AAI, measured using a commercially available (Accu-Sperm) acrosin activity assay, was greater in the fertilization group than in the nonfertilization group, but the difference was not significant. There was no correlation between AAI and the in vitro fertilization rate of mature oocytes. The relation between AAI and semen parameters also showed no significant difference. It would appear that measurement of AAI inaccurately reflects in vitro fertilizability of human sperm.

Successful treatment of two viable tubal pregnancies by two-step local injection.

Two viable tubal pregnancies were diagnosed by transvaginal ultrasound with a serum beta-hCG level of up to 3,004 mIU/mL in Case 1 and 16,676 mIU/mL in Case 2. Under transvaginal sonographic guidance, a local injection of potassium chloride (0.5 mL = 1.0 mEq) into the embryo was performed for the purpose of embryocide. In Case 1, a follow-up of serum beta-hCG levels showed an initial plateau and subsequent regression to negative, 49 days after the local injection. However, a persistent increase in serum beta-hCG levels was noted in Case 2 for two samples at intervals of two days during follow-up, 27,800 and 36,500 mIU/mL, in spite of the fact that no fetal cardiac activity was visible. Six days later, laparoscopy was done and methotrexate, 50 mg in 6 mL of normal saline, was injected into the ampullar mass of the right fallopian tube in two divided dosages. The serum beta-hCG levels then gradually decreased and returned to negative 60 days after the methotrexate injection. For a viable ectopic pregnancy, this new modality of two-step local injection, first with potassium chloride and then with supplemental methotrexate, separately by two procedures, may offer an additional choice of conservative treatment.

Binding and growth-stimulation of cervical cancer cell lines by prolactin.

Two cervical cancer cell lines CC7-T and Si-Ha were employed to observe the relationship between cervical cancer and prolactin. By immunocytochemical and indirect immunofluorescent assays using two prolactin monoclonal antibodies PRL-149 and PRL-151, both cell lines with added prolactin (10 ng/mL) were noted to be positive for PRL-151, but negative for PRL-149. The control cell lines from ovarian cancer and the myeloma lines were both stained negative. By using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, it was noted that CC7-T and Si-Ha grew better in the presence of various added concentrations of prolactin, ranging from 0.1 to 1,000 ng/mL, suggesting that prolactin may enhance the growth of cervical cancer. The degree of stimulation appears to depend on cell differentiation. However, prolactin levels in the cultured supernatant were undetectable by the enzyme immunoassay (EIA) method. We postulate that prolactin can bind and stimulate the growth of some cervical cancer cell lines, probably through the prolactin receptor rather than by autocrine regulation.

Pregnancy after transfer of frozen-thawed human embryos.

Cryopreservation of human embryos has been successfully applied in in vitro fertilization (IVF) and embryo transfer (ET) programs at the National Taiwan University Hospital since 1988. Our preliminary results with 120 frozen-thawed embryos in 31 transfer cycles showed that the survival rate of frozen embryos was 66%. Following transfer, the implantation rate and clinical pregnancy rate were 6.5% and 13%, respectively. Four clinical pregnancies and one preclinical pregnancy following a frozen-thawed embryo transfer were achieved. Two normal male babies have been delivered and another pregnancy is progressing without any problem.* Unfortunately, one pregnancy was terminated due to intrauterine fetal death discovered at the 10th week of gestation; chromosome abnormality (47, XX, +5) of the fetus was found. The single preclinical pregnancy showed an elevation of serum beta-human chorionic gonadotropin levels for three consecutive weeks following ET, but no definite gestational sac was visualized by transvaginal ultrasound.

The use of a long-acting gonadotropin-releasing hormone analog(D-Trp-6-LHRH) for improvement of ovarian stimulation in assisted conception programs.

To assess the efficacy of gonadotropin-releasing hormone analog (GnRHa) as an adjuvant in controlled ovarian stimulation in assisted conception programs, 114 infertile patients, who were treated by in vitro fertilization and embryo transfer (n = 61) or tubal embryo transfer (n = 53), were randomized sequentially to receive ovarian stimulation according to two protocols. In protocol 1 (n = 57), long-acting GnRHa (D-Trp-6-LHRH) microcapsules were administered intramuscularly at menstruation and ovarian stimulation using follicle-stimulating hormone (FSH) and human menopausal gonadotropin (hMG) was started 2 to 3 weeks later when the pituitary was completely suppressed. In protocol 2 (n = 57), patients received FSH and hMG from day 3 of the cycle without GnRHa pre-treatment. We found that premature luteinization did not occur in patients treated with protocol 1, and the number of cycles cancelled was also decreased. The days of ovarian stimulation and the amount of hMG required to achieve adequate follicular development were significantly higher in protocol 1 than that in protocol 2. Similarly, the mean serum estradiol levels on the day of human chorionic gonadotropin administration, number of large follicles (mean diameter greater than 10 mm), number of oocytes recovered and number of embryos obtained were also significantly higher in patients treated with protocol 1. The data suggest that the use of D-Trp-6-LHRH as an adjuvant in ovarian stimulation is associated with a lower incidence of cycle cancellation and an improvement in ovarian response in assisted conception programs.

Translaparoscopic tubal embryo transfer: preliminary experience at National Taiwan University Hospital.

Forty-six couples with various causes of infertility were treated by tubal embryo transfer. Oocyte retrievals were carried out under ultrasound guidance transvaginally and embryos were transferred 48 hours later by laparoscopy into fallopian tubes. Totally 52 cycles were stimulated and 45 retrievals were performed with an average of 8.1 oocytes per retrieval. The average fertilization rate was 54.8%. The implantation rate was 23.8% and the pregnancy rate was 53.5% per transfer. The pregnancy rate per transfer was comparable whether 3 or 4 embryos were transferred. Nine (39.1%) of the 23 pregnancies had multiple pregnancies. Six cases (26.1%) aborted in the first trimester and the remaining 17 were ongoing or term delivery.

Pregnancy following oocyte donation and tubal embryo transfer in patients with premature ovarian failure: report of two cases.

Two pregnancies in patients with premature ovarian failure utilizing donated oocytes, in-vitro fertilization (IVF) and tubal embryo transfer (TET), are reported. The recipients received cyclic hormone replacement therapy for six months to prepare the endometrium for implantation. An evaluation cycle was tested to document that the hormone milieu established by the hormone replacement protocol was similar to that of a natural ovulatory cycle. During the oocyte donation cycle, the recipient received incremental estrogen replacement treatment of flexible length during the follicular phase of the donor's stimulated cycle to synchronize the recipient's endometrium to the donor's embryo. Concurrently, the donor underwent controlled ovarian hyperstimulation and transvaginal ultrasound-guided oocyte retrieval. After fertilization of the donated oocytes with sperm from the recipient's husband and cleavage of the fertilized oocytes into the 2- to 4-cell stage, laparoscopic embryo transfer into the recipient's fallopian tube was performed. Case 1 received 4 embryos by the TET procedure. Pregnancy was confirmed by visualization of a gestational sac in the uterine cavity 3 weeks after TET, but miscarriage occurred at the tenth gestational week. In Case 2, the pregnancy was established after TET of 2 embryos. Estrogen and progesterone supplements were maintained until day 100 after TET. The patient delivered a healthy male baby, weighing 2,520 g at 38 weeks of gestation.

Treatment of patients with unexplained infertility: gamete intrafallopian transfer (GIFT) versus tubal embryo transfer (TET).

This study compares the results of 65 cycles of gamete intrafallopian transfer (GIFT), and 19 cycles of tubal embryo transfer (TET) in couples with unexplained infertility (UI). Oocyte retrievals were carried out by laparoscopy in GIFT and transvaginally in TET, in which the embryos were transferred by laparoscopy into the fallopian tubes 48 hours later. The mean age, duration of infertility, serum estradiol levels on the day of human chorionic gonadotropin administration, number of large follicles (mean diameter greater than 10 mm) and the number of oocytes recovered were similar between these two groups. From the 65 GIFT cycles, 20 clinical pregnancies resulted (30.8%). From the 19 cycles of TET, 10 conceptions occurred (52.6%). The implantation and pregnancy rates after TET were higher than that after GIFT, but the differences were not statistically significant. The data suggest that GIFT has a similar success rate to TET in couples with UI.

Human sperm chromosome analysis in primary infertility: a preliminary report.

Human sperm chromosomes can be analyzed using the zona-free hamster egg system. In order to familiarize ourselves with this new technique and to investigate the chromosome aberration frequency in fertile and possibly infertile men, we performed sperm chromosome analysis in 39 successful experiments. The subjects were divided into two groups. Group 1 was healthy men with proven fertility. Group 2 was husbands of couples with unexplained infertility. Semen samples were prepared freshly or preserved in TEST-yolk buffer for 12 to 72 hours. In group 1 using fresh samples, the mean penetration rate was 67.40 +/- 14.08% (mean +/- 2SD), the maturation index was 1.33 +/- 0.31. For samples treated with yolk buffer, the mean penetration rate was 91.34 +/- 7.42%, and the maturation index was 1.58 +/- 0.42. There was a statistically significant increase in penetration after yolk buffer treatment (p less than 0.001). In group 1 there were 89 haploid chromosomes, four were aneuploidy (4.5%), and three with structural aberrations (3.4%). In group 2, for fresh samples, the mean penetration rate was 71.34 +/- 27.96%, and the maturation index was 1.37 +/- 0.25. For samples treated with yolk buffer, the penetration rate was 86.33 +/- 25.18%, and maturation index 1.40 +/- 0.23. In group 2, there were 135 haploid chromosomes, 6 were aneuploidy (4.4%), and 4 with structural aberrations (3.0%). There was no statistically significant difference between group 1 and group 2 in the penetration rate, maturation index and frequency of chromosome abnormalities.