Application of intracytoplasmic sperm injection in assisted reproductive technologies.

Intracytoplasmic sperm injection (ICSI) entails the mechanical insertion of a chosen spermatozoon directly into the cytoplasm of an oocyte. Due to the consistent fertilization and pregnancy outcome, ICSI is routinely used to treat azoospermic patients where spermatozoa are retrieved by epididymal aspiration or testicular biopsy. Since male subfertility has been associated with a higher incidence of genomic defects, ranging from numerical chromosomal abnormalities to Yq microdeletions, concerns have been raised as to the risk of transmitting genetic defects to the offspring. Screening for such defects can provide invaluable information for appropriate counselling prior to ICSI treatment. In order to address these concerns, a follow-up of the children born after ICSI treatment was conducted.

Fertility preservation in breast cancer patients: IVF and embryo cryopreservation after ovarian stimulation with tamoxifen.

BACKGROUND: Breast cancer chemotherapy commonly causes premature ovarian failure and infertility. Because increased estrogen levels are thought to be potentially risky in breast cancer patients, natural cycle IVF (NCIVF) has been used to preserve fertility and treat infertility in these women. METHODS: Twelve women with breast cancer received 40-60 mg tamoxifen for 6.9 +/- 0.6 days beginning on days 2-3 of their menstrual cycle (15 cycles), and had IVF (TamIVF) with either fresh embryo transfer (six cycles) or cryopreservation (nine cycles). They were compared to a retrospective control group (n = 5) who had natural cycle IVF (NCIVF, nine cycles). RESULTS: Cycle cancellation was significantly less frequent in TamIVF, compared with NCIVF (1/15 versus 4/9, P < 0.05). Compared with NCIVF, TamIVF patients had a greater number of mature oocytes (1.6 +/- 0.3 versus 0.7 +/- 0.2, P = 0.03) and embryos (1.6 +/- 0.3 versus 0.6 +/- 0.2, P = 0.02) per initiated cycle. TamIVF resulted in the generation of embryo(s) in every patient (12/12) while only three out of five patients had an embryo following NCIVF. Two out of six patients in TamIVF, and 2/5 in NCIVF conceived. One patient in the TamIVF group delivered a set of twins. After a mean follow up of 15 +/- 3.6 months (range 3-54), none of the patients had a recurrence of cancer. CONCLUSIONS: Tamoxifen stimulation appears to result in a higher number of embryos and may provide a safe method of IVF and fertility preservation in breast cancer patients.

Testicular sperm extraction combined with intracytoplasmic sperm injection in the treatment of men with persistent azoospermia postchemotherapy.

BACKGROUND: Men who remain azoospermic long after undergoing chemotherapy have generally been considered sterile. The authors report their experience with testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI) applied to azoospermic men who previously received chemotherapy for a variety of indications. METHODS: Among 231 cycles in 198 patients who underwent TESE-ICSI for nonobstructive azoospermia from 1995 to 2000, 20 TESE procedures in 17 patients who previously received chemotherapy were identified. All TESE procedures were performed with microsurgical control under local anesthesia with sedation or general anesthesia. The pretreatment hormonal profile, histology of testicular biopsies, and outcomes of TESE-ICSI in this subgroup of patients were analyzed. RESULTS: The mean patient age was 37.4 years (range, 28-54 years), and the mean follicle-stimulating hormone level was 21.8 mIU/mL (range, 7.1-43.1 mIU/mL). The mean age for female partners was 33.5 years (range, 22-43 years). Six patients had received chemotherapy for Hodgkin lymphoma (34%), four patients had received chemotherapy for testicular neoplasm (24%), two patients had received chemotherapy for non-Hodgkin lymphoma (12%), two patients had received chemotherapy for leukemia (12%), one patient had received chemotherapy for Wilms tumor (6%), one patient had received chemotherapy for mediastinal germ cell tumor (6%), and one patient had received chemotherapy for nephrotic syndrome (6%). Three patients (18%) received additional radiation therapy. The mean interval from chemotherapy to TESE was 16.3 years (range, 6-34 years). All patients had at least two semen analyses to confirm azoospermia. A total of 20 attempts of TESE-ICSI were performed (mean, 1.2 attempts per patient). Testicular histology revealed Sertoli cell-only pattern in 76% of patients. The remaining 24% of patients had hypospermatogenesis as their most advanced spermatogenic pattern. Among the men with Sertoli cell-only pattern, 23% had sperm retrieved by TESE. Sperm retrieval was accomplished in 9 of 20 attempts (45%), with biochemical pregnancy after sperm retrieval in 4 of 9 couples (45%) and clinical pregnancy in 3 of 9 couples (33%). Live deliveries were achieved in 2 of 9 couples (22%). Two healthy boys and one girl were delivered. No correlation was noted between the outcome of TESE-ICSI and the underlying conditions that were treated with chemotherapy nor with the chemotherapeutic agents used. CONCLUSIONS: Using TESE-ICSI, sperm retrieval leading to pregnancy and the delivery of healthy children is possible for men with long-standing azoospermia after chemotherapy. The prognosis for sperm retrieval was not influenced clearly by the chemotherapy regimen or the disease treated. Diagnostic biopsy also was of limited value in predicting the outcome of sperm retrieval. Despite prolonged nonobstructive azoospermia after undergoing chemotherapy, men no longer should be considered sterile in the era of advanced assisted reproductive techniques.

Endocrine function and oocyte retrieval after autologous transplantation of ovarian cortical strips to the forearm.

CONTEXT: In reproductive-age women, one of the common adverse effects of chemotherapy and radiotherapy is premature ovarian failure. In addition, a significant number of women experience early menopause due to oophorectomy performed for benign indications. OBJECTIVE: To develop an ovarian transplantation technique to preserve endocrine function in women undergoing sterilizing radiotherapy and/or chemotherapy, or oophorectomy. DESIGN AND SETTING: Case study of 2 patients in New York who received autologous ovarian transplantation (patient A, November 1999; patient B, April 2000) to the forearm prior to pelvic radiotherapy or after oophorectomy. PARTICIPANTS: Patient A is a 35-year-old woman with stage IIIB squamous cell cervical carcinoma and patient B is a 37-year-old woman with recurrent benign ovarian serous cysts. MAIN OUTCOME MEASURES: Follicular development evident by ultrasound examination; cyclical production of estradiol and progesterone; restoration of serum follicle-stimulating hormone, luteinizing hormone, and testosterone levels to nonmenopausal range; and disappearance of menopausal symptoms. RESULTS: Menopause was confirmed immediately after the transplantation in both patients by serum follicle-stimulating hormone measurements (patient A, 47 mIU/mL; patient B, 50.7 mIU/mL). In patient A, follicle development was noted by physical and ultrasound examinations approximately 10 weeks after the transplantation. The mean (SE) follicle-stimulating hormone and luteinizing hormone levels decreased to 8.6 (0.4) mIU/mL and 12.8 (0.8) mIU/mL, respectively. The peripheral estradiol levels showed cyclical variation (mean [SE], 115 [9.2] pg/mL [422 (33.8) pmol/L), and during the 18-month follow-up, a dominant follicle developed each month. The estradiol levels from the right cubital vein were consistent with ovarian vein measurements (mean [SE], 1069 [269] pg/mL [3924 (987.5) pmol/L]). Percutaneous oocyte aspirations yielded a mature oocyte. In patient B, ovarian function was demonstrated by ultrasound visualization of a 9-mm follicle by 6 months after transplantation. Thereafter, the patient had spontaneous menstruation every 25 to 28 days. Ovulation was further confirmed by midluteal progesterone measurements (range, 7-10.1 ng/mL; mean [SE], 8.5 [0.9] ng/mL). Patient B's ovarian graft was still functional 10 months after the transplantation. CONCLUSIONS: Subcutaneous ovarian transplantation appears to be a relatively simple, novel technique to preserve endocrine function in women undergoing sterilizing cancer therapy or surgery.

Preliminary findings in germinal vesicle transplantation of immature human oocytes.

Transplanting a germinal vesicle (GV) from an aged woman's oocyte into a younger ooplasm has been proposed as a possible way to reduce the incidence of oocyte aneuploidy which is considered to be responsible for age-related infertility. In this study, we have assessed the efficiency of each step involved in nuclear transplantation-specifically cell survival, nuclear-cytoplasmic reconstitution, and the capacity of the reconstituted oocytes for in-vitro maturation. In addition, we have evaluated the fertilizability and karyotypic status of the manipulated oocytes by intracytoplasmic sperm injection (ICSI) and fluorescent in-situ hybridization technique respectively. Nuclear transplantation was accomplished with an overall efficiency of 73%. Due to the limited availability of materials, most nuclear transplantation procedures were performed between sibling oocytes. The maturation rate of 62% following reconstitution was comparable with that of control oocytes, as was the incidence of aneuploidy among the reconstituted oocytes. The ICSI results of the reconstituted oocytes yielded a survival rate of 77%, a fertilization rate of 52%, and a satisfactory early embryonic cleavage. Furthermore, in a limited number of observations where the nucleus of an aged oocyte was transferred into a younger ooplasm, there was an appropriate chromosomal segregation. These findings demonstrate that human oocytes reconstituted with GV nuclei are able to undergo maturation, fertilization, and early embryo cleavage, and maintain a normal ploidy. Although in-vitro maturation seems to be a limiting step, this technique would allow us to investigate further the nuclear-ooplasmic relationship during meiotic maturation.

Autologous endometrial coculture in patients with in vitro-fertilization (IVF) failure: correlations of outcome with leukemia inhibiting factor (LIF) production.

PROBLEM: To determine if LIF produced by autologous endometrial co-culture (ECC) was associated with outcome in 46 patients with a history of multiple IVF failures. METHOD OF STUDY: The conditioned media (CM) from ECC cells exposed or non-exposed to human embryos was analyzed for LIF. RESULTS: Exposure or non-exposure to an embryo did not result in differing levels of LIF in the CM. LIF levels were significantly greater in the CM than in the serum controls (LIF was not found in the serum controls). Embryos grown on ECC demonstrated a significant improvement in number of blastomeres and fragmentation when compared to embryos grown in conventional media without ECC (6.7 +/- 1.3 vs. 5.6 +/- 1.2 blastomeres and 17.6% +/- 9.3 vs. 26.4% +/- 9.8 fragmentation; P < 0.05). When LIF levels were detectable in the CM, the embryos grown in ECC were of improved quality as compared to the embryos grown only in conventional media and demonstrated a non-significant increase in pregnancy rates (60 vs. 48%, P = 0.50). CONCLUSIONS: We have demonstrated a significant improvement in embryo quality with ECC. The cells in the ECC express LIF. The presence of LIF in the CM was associated with embryonic development and clinical pregnancy.

Shared oocyte donation: society's benefits.

OBJECTIVE: To assess the efficacy of oocyte donation when a cohort of oocytes is shared between two phenotypically matched recipients. DESIGN: A retrospective analysis of a program using shared anonymous oocyte donation. SETTING: Academic infertility center. PATIENT(S): Recipient women with partial or complete ovarian failure; oocyte donors who have been properly screened. INTERVENTION(S): Each oocyte donor was phenotypically matched with two potential recipients. The cohort of donated oocytes were divided between these two recipients if eight or more mature oocytes were obtained at retrieval. Recipients underwent hormone replacement therapy consisting of down-regulation with a GnRH agonist, transdermal estradiol, and intramuscular progesterone in a dose determined by a previous preparatory cycle. MAIN OUTCOME MEASURE(S): Pregnancy and delivery rates for all transfers originating from a cohort of oocytes obtained by retrieval of a single donor; pregnancy and delivery rates per recipient; rate of conversion of a shared donation cycle to a single recipient. RESULT(S): A total of 249 donor cycles permitted 241 retrievals. Each recipient received 8.3 +/- 3.5 oocytes per donation. There were 424 fresh ETs and 48 frozen ETs performed. For fresh ETs, clinical pregnancy and ongoing or delivery rates per recipient were 56.8% and 49.7%, respectively. For frozen ETs, these rates were 50% and 39.5%. Implantation rates were 31.8% and 26.1% for fresh and frozen ET, respectively. When analyzed per donor retrieval, clinical pregnancy and ongoing or delivery rates were 109.5% and 95.4%. These high pregnancy rates per donor reflect the numerous fresh and frozen ETs that can result from one donor's retrieval. Conversion of a donation cycle from two recipients to one recipient occurred for 26 of 241 cycles (10.8%). CONCLUSION(S): Shared anonymous oocyte donation provides a very high pregnancy rate per donor retrieval that is not achievable with unshared donation. In addition, there is a diminished risk exposure of donors per total completed recipient transfers. We support shared oocyte donation as the most efficient use of the precious resource of human oocytes.

Human zona pellucida micromanipulation and monozygotic twinning frequency after IVF.

To assess the association of zona pellucida micromanipulation and subsequent development of monozygotic twins, cases of assisted embryo hatching (AH) and intracytoplasmic sperm injection (ICSI) were identified and related to treatment type, implantation and zygosity data. Embryology records from all patients undergoing in-vitro fertilization (IVF) at this centre from January 1995 to March 1998 were reviewed. In this study, 3546 transfer cycles were completed, with clinical pregnancy established in 1911 (54% per transfer) patients undergoing a single IVF cycle. These pregnancies occurred in 1674 (88%) IVF cycles, 120 (6%) donor oocyte cycles (DER), and 117 (6%) frozen embryo transfer (FET) cycles. During the study period, 23 cases of monozygotic (MZ) twins were identified, representing an overall frequency of 1.2%. Chorionicity was determined by transvaginal ultrasound at 7 weeks when the number of embryos transferred was less than the number of fetal heart-beats, or when >1 fetal heartbeat per gestational sac was seen. Zygosity was confirmed by placental evaluation at delivery, and corroborated the antenatal diagnosis in all cases. Among IVF study patients the frequency of MZ twinning was not statistically different between zona manipulated and zona intact subgroups. While this investigation is the largest to date describing the relationship between MZ twins and zona procedures, studies with even greater statistical power are needed to clarify it more precisely, particularly in DER and FET settings. A greater overall frequency of MZ twinning for IVF patients may be a function of the higher number of embryos transferred in IVF, rather than discrete zona manipulations.

Interleukin-1 levels in the supernatant of conditioned media of embryos grown in autologous endometrial coculture: correlation with outcome after in vitro fertilization.

PROBLEM: To determine if interleukin (IL)-1 produced by autologous endometrial coculture (AECC) was associated with outcome in patients with a history of multiple in vitro fertilization (IVF) failures. METHOD OF STUDY: The conditioned media (CM) from AECC cells exposed or non-exposed to human embryos was analyzed for IL-1. RESULTS: Embryos grown on AECC demonstrated a significant improvement in number of blastomeres and fragmentation (frag) when compared to embryos grown in conventional media without ECC (6.4 +/- 1.3 vs. 5.5 +/- 1.2 blastomeres and 14.6 +/- 9.3%, vs. 18.4 +/- 9.8% frag; P< 0.008 and 0.003, respectively). When IL-1alpha and IL-1beta were undetectable in the CM, the embryos grown in ECC were of improved quality as compared to the embryos grown only in conventional media. Conversely, IL-1ra levels in the CM were positively associated with embryo quality. Exposure or non-exposure to an embryo did not result in differing levels of IL-1alpha, IL-1beta, or IL-1ra in the CM. IL-1beta levels were negatively associated with clinical pregnancy outcome (3.3 pg/mL (pregnant, n = 12) vs. 27.1 pg/mL (not pregnant, n = 17); P = 0.008, Mann Whitney U-test). IL-1alpha and IL-1ra levels were not associated with outcome. CONCLUSIONS: We have demonstrated a significant improvement in blastomere number and frag with ECC. The presence of IL-1beta in the CM was negatively associated with embryonic development and clinical pregnancy. The presence of IL-1alpha in the CM was negatively associated with embryonic development and the presence of IL-1ra in the CM was positively associated with embryonic development. Whether IL-1beta itself interferes with successful outcome after embryo transfer or if it is a marker for undetected endometritis in the biopsy specimens remains to be determined.

Expression of apoptosis-related genes in human oocytes and embryos.

PURPOSE: The objective was to study whether apoptosis occurs in human embryogenesis. METHODS: Human viable, arrested, and nonviable embryos and immature, and nonfertilized oocytes donated by our patients were used to detect apoptosis by Tunel labeling, annexin staining, and single-cell reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: DNA fragmentation and phosphotidylserine translocation, the two markers for apoptosis, were detected frequently in fragmented human embryos derived from in vitro fertilization-embryo transfer (IVF-ET). Using RT-PCR, apoptotic genes also were detected in these embryos. The frequencies of gene expression in viable embryos, arrested embryos, nonviable embryos, immature oocytes, and non-fertilized oocytes were: 7/8, 5/5, 5/6, 0/6, 0/3, for Bax; 8/8, 5/5, 7/7, 0/4, 0/5 for Fas; 2/8, 0/2, 0/3, 0/5, 0/3 for BCL-2; 0/8, 1/3, 0/2, 0/3, 0/2 for Fas-ligand; and 8/8, 17/17, 21/21, 24/24, 15/15 for actin, respectively. CONCLUSIONS: Our preliminary data did not show a significant difference in the expression frequency of all studied genes between viable embryos and nonviable or arrested embryos. However, the expression of Bax and Fas was noticeably higher in nonviable embryos than in viable embryos as judged by the intensities of amplicons visualized after ethidium bromide staining. In addition, BCL-2 was only detected in viable embryos. Whether embryos quality is related to the regulation of BCL-2, Bax, and Fas expressions requires further study.

ICSI and its outcome.

Since its introduction in 1992, intracytoplasmic sperm injection (ICSI) has become a popular assisted fertilization technique proved very efficient in treating male factor infertility. Many healthy children have been born worldwide from this procedure, and their physical and mental development appears to be within the normal limits. However, because of the peculiarity of the technique and the poor characteristics of the spermatozoa used, concern about the safety of ICSI still exist. In this article, we analyze the in vivo development of embryos conceived after ICSI as well as the obstetric outcome, occurrence of chromosomal abnormalities, and rate of congenital malformations in neonates born as a result of this treatment. A total of 2435 couples were studied in whom the male partners were presumed to be the cause of repeated failed attempts at in vitro fertilization (IVF) or had semen parameters that were unacceptable for conventional IVF treatment. Pregnancies resulting from 3573 ICSI cycles were analyzed; pregnancy outcome data were obtained from the records of obstetrician-gynecologists and/or pediatricians. The overall clinical pregnancy (fetal heartbeat) rate was 44.8% with a resultant delivery rate of 39.2% per ICSI cycle (n = 1388). In 37 of the 77 miscarriages for which cytogenetic data were available, an autosomal trisomy was found in each and 29 additional pregnancies were terminated because of a chromosomal abnormality revealed by prenatal diagnosis. There was an equal distribution of vaginal deliveries and cesarean sections (n = 682 and n = 658, respectively). Of the 2059 neonates resulting from ICSI treatment, 38 (1.8%) presented with congenital abnormalities (22 major and 16 minor). When the frequency of miscarriages and congenital malformations was analyzed in terms of semen origin, the outcome was no different between ICSI and IVF. The course of pregnancies and occurrence of congenital malformations following treatment by ICSI are within the ranges obtained following conventional IVF.

Recycling of a single human blastomere fixed on a microscopic slide for sexing and diagnosis of specific mutations by various types of polymerase chain reaction.

OBJECTIVE: To investigate the suitability of recycling single blastomeres to assess multiple genetic variables for preimplantation genetic diagnosis. DESIGN: Prospective randomized study. SETTING: An academic medical center. PATIENT(S): Patients undergoing IVF-ET. INTERVENTION(S): Blastomeres were disaggregated from donated embryos obtained from patients. MAIN OUTCOME MEASURE(S): Polymerase chain reaction (PCR) amplification products. RESULT(S): Fifty-eight blastomeres individually fixed on slides were separated into four groups. Sequential PCRs (group I, n = 30), primed in situ labeling (PRINS) before five sequential PCRs (group II, n = 10), staining with hematoxylin before performing five sequential PCRs (group III, n = 11) and preamplification of whole DNAs by degenerate oligonucleotide primer (DOP) before performing PCR were executed. The amplification efficiencies of five sequential PCRs were 100%, 100%, 96.6%, 83.3%, 56.7% for group I; 100% 100%, 100%, 80%, 40% for group II; 54.5%, 36.4%, 18.2%, 9.1% for group III; and 100%, 100%, 100%, 100%, 100% for group IV. CONCLUSION(S): Blastomeres fixed for PRINS can be recycled for PCR to obtain more genetic information. Hematoxylin staining appears to increase the incidence of failed amplification. Preamplification of whole genomic DNAs by DOP-PCR appears to facilitate diagnosis with high efficiency.

Sperm integrity is critical for normal mitotic division and early embryonic development.

The human zygote relies on the paternal gamete to provide the centrosome component essential for the first mitotic division. It is not known whether normal centrosome function requires an intact spermatozoon, or whether donation of an isolated paternal centrosome component can result in normal zygotes and embryos. To explore this possibility, mature human oocytes were microinjected with either intact or dissected spermatozoa. Fertilization and cleavage rates were documented; nuclear and cytoskeletal changes were observed with fluorescent immunocytochemistry; and chromosomal normality was assessed with fluorescent in-situ hybridization. A pilot study was performed to identify cytoskeletal features suggestive of centrosome function. Unfertilized oocytes and tripronucleate (3PN) zygotes from in-vitro fertilization or intracytoplasmic sperm injection were assessed to confirm the sequence of the landmarks of human fertilization. Oocytes injected with mechanically-dissected spermatozoa appear to be capable of normal pronuclear formation and embryonic cleavage, but do not undergo normal mitotic division. Although decondensed, apposed nuclei are noted in combination with diffuse cytoskeleton assembly, no spindle was detected in any zygote resulting from the injection of a dissected spermatozoon. Analysis of selected embryos resulting from dissected sperm injection revealed chromosomal mosaicism in the majority of specimens. The lack of a bipolar spindle, in combination with chromosomal mosaicism, suggests abnormalities of the mitotic apparatus when sperm integrity is impaired following dissection.

First unaffected pregnancy using preimplantation genetic diagnosis for sickle cell anemia.

CONTEXT: Sickle cell anemia is a common autosomal recessive disorder. However, preimplantation genetic diagnosis (PGD) for this severe genetic disorder previously has not been successful. OBJECTIVE: To achieve pregnancy with an unaffected embryo using in vitro fertilization (IVF) and PGD. DESIGN: Laboratory analysis of DNA from single cells obtained by biopsy from embryos in 2 IVF attempts, 1 in 1996 and 1 in 1997, to determine the genetic status of each embryo before intrauterine transfer. SETTING: University hospital in a large metropolitan area. PATIENTS: A couple, both carriers of the recessive mutation for sickle cell disease. INTERVENTIONS: Standard IVF treatment, intracytoplasmic sperm injection, embryo biopsy, single-cell polymerase chain reaction and DNA analyses, embryo transfer to uterus, pregnancy confirmation, and prenatal diagnosis by amniocentesis at 16.5 weeks' gestation. MAIN OUTCOME MEASURE: DNA analysis of single blastomeres indicating whether embryos carried the sickle cell mutation, allowing only unaffected or carrier embryos to be transferred. RESULTS: The first IVF attempt failed to produce a pregnancy. Of the 7 embryos analyzed in the second attempt, PGD indicated that 4 were normal and 2 were carriers; diagnosis was not possible in 1. Three embryos were transferred to the uterus on the fourth day after oocyte retrieval. A twin pregnancy was confirmed by ultrasonography, and subsequent amniocentesis revealed that both fetuses were unaffected and were not carriers of the sickle cell mutation. The patient delivered healthy twins at 39 weeks' gestation. CONCLUSION: This first unaffected pregnancy resulting from PGD for sickle cell anemia demonstrates that the technique can be a powerful diagnostic tool for carrier couples who desire a healthy child but wish to avoid the difficult decision of whether to abort an affected fetus.

Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men.

The evident ability of the intracytoplasmic sperm injection (ICSI) procedure to achieve high fertilization and pregnancy rates regardless of semen characteristics has induced its application with spermatozoa surgically retrieved from azoospermic men. Here, ICSI outcome was analysed in 308 cases according to the cause of azoospermia; four additional cycles were with cases of necrozoospermia. All couples were genetically counselled and appropriately screened. Spermatozoa were retrieved by microsurgical epididymal aspiration or from testicular biopsies. Epididymal obstructions were considered congenital (n = 138) or acquired (n = 103), based on the aetiology. Testicular sperm cases were assessed according to the presence (n = 14) or absence (n = 53) of reproductive tract obstruction. The fertilization rate using fresh or cryopreserved epididymal spermatozoa was 72.4% of 911 eggs for acquired obstructions, and 73.1% of 1524 eggs for congenital cases; with clinical pregnancy rates of 48.5% (50/103) and 61.6% (85/138) respectively. Spermatozoa from testicular biopsies fertilized 57.0% of 533 eggs in non-obstructive cases compared to 80.5% of 118 eggs (P = 0.0001) in obstructive azoospermia. The clinical pregnancy rate was 49.1% (26/53) for non-obstructive cases and 57.1% (8/14) for testicular spermatozoa obtained in obstructive azoospermia, including three established with frozen-thawed testicular spermatozoa. In cases of obstructive azoospermia, fertilization and pregnancy rates with epididymal spermatozoa were higher than those achieved using spermatozoa obtained from the testes of men with non-obstructive azoospermia.

Autologous endometrial co-culture in patients with repeated failures of implantation after in vitro fertilization-embryo transfer.

PURPOSE: Our purpose was to evaluate the effect of coculture on preembryo development and clinical outcome. METHODS: Enrolled patients underwent a luteal-phase endometrial biopsy. The tissue was then enzymatically digested (collagenase) and the stromal and glandular cells were separated by differential sedimentation rates. These cells were cultured to confluence, released, and then cryopreserved until the patient's in vitro fertilization (IVF)-embryo transfer (ET) cycle. All normally fertilized oocytes were then placed on the co-cultured cells until transfer on day 3. Preembryo development on co-culture was compared to that in the patient's noncocultured previous cycle. Implantation and clinical pregnancy rates were compared to those in a control group of patients undergoing IVF during the study period who were matched for age, stimulation protocol, number of oocytes retrieved, and preembryos transferred. RESULTS: Twenty-nine women underwent 31 cycles of IVF-ET. On day 3 the overall mean number of blastomeres per preembryo on co-culture compared to that in the patient's previous cycle was 6.3 +/- 1.8 vs. 5.6 +/- 1.2 (P = 0.04). The average percentage of cytoplasmic fragments on co-culture compared to the previous cycle was 16 +/- 9% vs. 19 +/- 9% (P = 0.32). At transfer, after preembryo selection, the mean number of blastomeres per preembryo on co-culture compared to that in the patient's previous cycle was 6.8 +/- 1.6 vs. 6.6 +/- 1.3 (P = 0.5). The implantation and clinical pregnancy rates between co-culture and the matched control group were 15% (14/93) vs. 13% (16/124) (P = 0.79) and 29% (9/31) vs. 25% (10/40) (P = 0.45). CONCLUSIONS: There was a significant improvement in the average number of blastomeres per preembryo on co-culture compared to that in the patient's previous noncoculture cycle. The overall implantation and clinical pregnancy rates between co-culture and a matched control group were not significantly different.

Expression of inhibin/activin subunits and their receptors and binding proteins in human preimplantation embryos.

PURPOSE: Our purpose was to study the role of inhibin/activin during embryogenesis. METHODS: Transcripts of inhibin/activin subunits (alpha, beta A, beta B), activin receptors (types I and II), and follistatin were detected by a reverse transcriptase-polymerase chain reaction in human reproductive cells and preembryos cultured alone or co-cultured with human endometrial cells. RESULTS: Transcripts of alpha, beta A, beta B subunits were all detected in granulosa luteal cells, but only beta A units were detected in endometrial stromal and decidualized cells. In human preimplantation embryos, none of these subunits were detected in embryos from the four-cell to the morula stage and only beta A subunits were detectable in blastocyst embryos. Activin receptors were detectable in all of the studied embryos and cells. Transcripts of beta A, activin receptors, and follistatin were differentially expressed in human preimplantation embryos cultured in vitro and their expressions were significantly enhanced with the presence of endometrial stromal cells. CONCLUSIONS: Our data suggest that there is a possible endometrium-embryo interaction via endometrial activins and preimplantation embryo receptors and that the embryonic expressions of these activins, their receptors, and binding proteins are dependent on embryonic stage.

Human endometrial stromal cells improve embryo quality by enhancing the expression of insulin-like growth factors and their receptors in cocultured human preimplantation embryos.

OBJECTIVE: To demonstrate the mechanism by which human endometrial stromal cells improve embryo quality in coculture. DESIGN: Randomized study. SETTING: Academic research center. PATIENT(S): Patients undergoing IVF-ET. INTERVENTION(S): Donated human embryos were cultured randomly either alone (group A) or with human endometrial stromal cells (group B), and the embryonic expression of insulin-like growth factors (IGFs) and their receptors was detected by reverse transcriptase polymerase chain reaction after culture. MAIN OUTCOME MEASURE(S): The embryo frequency distribution of groups A and B before and after culture and the embryonic transcripts of the IGF family genes of the two study groups after culture were compared. RESULT(S): The embryo frequency distribution of the day 3 embryonic stages in groups A and B was not different. However, after culture, a statistically significant difference in blastocyst formation was observed between groups A and B. A significant increase in the expression of IGF-1, IGF-2, the IGF-1 receptor, and the insulin-receptor also was noted. Among the embryos that reached the blastocyst stage, the expression of IGF-1 and the IGF-1 receptor also was significantly different in the two study groups. CONCLUSION(S): Human endometrial stromal cells enhanced the expression of IGFs and their receptors in cocultured human embryos, which may be essential for improving embryo quality.

Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia: testicular histology can predict success of sperm retrieval.

PURPOSE: We present treatment results of testicular sperm extraction with intracytoplasmic sperm injection for men with nonobstructive azoospermia and reevaluate the role of testicular histology on open diagnostic testicular biopsy as a predictor of sperm retrieval success. MATERIALS AND METHODS: We evaluated 75 men diagnosed with nonobstructive azoospermia. Cases were categorized into 3 groups of hypospermatogenesis, maturation arrest or Sertoli-cell-only based on the most advanced pattern of spermatogenesis seen on histology. A total of 81 testicular sperm extractions with intracytoplasmic sperm injection were performed for these 75 men. The main outcome measures reviewed included sperm retrieval, fertilization and pregnancy rates with intracytoplasmic sperm injection. Sperm retrieval success rates for men in the 3 histological categories were compared. RESULTS: Spermatozoa were successfully retrieved during 47 of 81 (58%) testicular sperm extraction attempts, with subsequent fertilization of 268 of 439 (61%) injected metaphase II oocytes using intracytoplasmic sperm injection. Clinical pregnancies were obtained in 26 of 47 (55%) cycles when sperm were retrieved, with ongoing pregnancies or live deliveries for 20 of 47 (43%). Of 39 men with hypospermatogenesis on diagnostic biopsy 31 (79%) had successful sperm retrieval, compared to 9 of 19 (47%) with maturation arrest and 5 of 21 (24%) with a pure Sertolicell-only pattern. CONCLUSIONS: Critical examination of the most advanced pattern of spermatogenesis from open diagnostic testis biopsy allows prediction of sperm retrieval success with testicular sperm extraction. In this study population spermatozoa were retrieved in 58% of attempts. When this testicular sperm was used with intracytoplasmic sperm injection, clinical pregnancy rate was 55% for men with nonobstructive azoospermia.