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Purpose: The aim of this study was to report the successful fertility treatment of men with Klinefelter syndrome using testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). Methods: A total of 42 men with Klinefelter syndrome who underwent 54 TESE procedures were identified. Before TESE, patients with serum testosterone levels less than 15.6 nmol/liter were treated with an aromatase inhibitor. Sperm retrieval rates and results of ICSI, including fertilization and clinical pregnancy, were collected. Results: Mean pretreatment FSH and testosterone levels were 33.2 IU/liter and 9.8 nmol/liter. During medical therapy, the mean testosterone level rose to 17.0 nmol/liter (P < 0.01). Spermatozoa were found during 39 microdissection TESE procedures, on the day before, or day of oocyte retrieval during a programmed in vitro fertilization cycle. The sperm retrieval rate was 72% (39 of 54) per TESE attempt, and 29 of the 42 different men (69%) had adequate sperm found for ICSI. Thirty-three...

Objective To determine whether the presence of a Y microdeletion confers any adverse effects on in vitro fertilization or intracytoplasmic sperm injection (IVF/ICSI) outcome. Design Retrospective case–control study. Setting Academic infertility center. Patient(s) A total of 17 patients with Y microdeletions who attempted IVF/ICSI cycles at our center between March 1996 and March 2002 were studied. Study patients were analyzed in two groups: those who underwent testicular sperm extraction (TESE) and those for whom ejaculated sperm was used. Intervention(s) The two patient study groups were matched to controls treated at the same time who had either nonobstructive severe oligozoospermia or azoospermia with normal Y chromosomes. Controls were matched for age of the female partner, sperm concentration, and number of embryos transferred. Main outcome measure(s) Fertilization and clinical pregnancy rates. Result(s) Sperm was only obtained from patients with azoospermic factor (AZF)c microdeletions (and from one patient with a partial AZFb microdeletion). A trend toward lower fertilization rates in patients with Y microdeletions was noted, which did not reach statistical significance. Clinical pregnancy rates per cycle and per transfer were similar to those for controls. Conclusion(s) Patients with AZFc microdeletions seem to have IVF/ICSI outcomes comparable to those of controls with normal Y chromosomes.

Purpose To develop an accurate mutation analysis procedure for retinoblastoma gene (RB1) mutation, which is sensitive at the single-cell level, and to use in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) to achieve pregnancies without retinoblastoma. Design Case report. Methods Twelve day 3 embryos, obtained by IVF with intracytoplasmic sperm injection, underwent single-cell DNA testing via polymerase chain reaction and restriction enzyme analysis to detect the presence of a paternal RB1 mutation. Embryos were diagnosed as being unaffected and were transferred to the uterus on day 5. Main outcome measures Achieving a healthy pregnancy and delivery, assessed by clinical presentation, fundus photography, and RB1 molecular analysis. Results A singleton pregnancy was achieved, and a child without retinoblastoma was born. The absence of the paternal RB1 mutation was confirmed on a sample of peripheral blood from the newborn. Conclusion We are first to report a successful human liveborn, delivered after IVF with preimplantation genetic diagnosis for retinoblastoma. The successful result indicates that preimplantation genetic diagnosis exists for this genetic disease and may represent a viable alternative to prenatal diagnosis with the subsequent option of terminating an affected pregnancy.

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.

To analyze the effectiveness of autologous endometrial coculture by the cycle day of the endometrial biopsy.Retrospective study.University-based IVF center.Two hundred eight patients with multiple IVF failures.Embryos were split and randomly allocated to growth on autologous endometrial coculture or conventional media.Embryo quality and pregnancy outcome.The overall clinical pregnancy rate was 41.8%. Embryos grown on autologous endometrial coculture were of higher quality (more blastomeres and less fragmentation) than embryos grown with conventional media. Early luteal biopsies (5 days after LH surge) for autologous endometrial coculture did not demonstrate an improvement in embryo quality as compared to the significant improvement demonstrated with later luteal endometrial biopsies (or =5 days after LH surge). The date of the biopsy was predictive of pregnancy outcome when using autologous endometrial coculture (44.7% [or =5 days after LH surge] vs. 18.8% [5 days after LH surge], P=.012).We have demonstrated an improvement in embryo quality when using autologous endometrial coculture. The improvement in embryo quality and higher pregnancy rates were limited to biopsiesor =5 days after the LH surge. This suggests that mid/late luteal phase endometrium contains factors that enhanced embryo growth and subsequent implantation.

Purpose: To evaluate the effect of autologous endometrial coculture in patients (less than 36 years old) with a history of a single IVF failed cycle associated with poor quality embryos. Methods:Design: Controlled clinical study. Setting: University-based in vitro fertilization center. Patients: Twenty-six patients with a history of a single prior failed IVF-ET with poor preembryo quality. Intervention(s): Autologous endometrial coculture. Main outcome measures: Preembryo blastomere numbers and cytoplasmic fragmentation rates were compared between the treatment and previous cycle. Clinical pregnancy rates were analyzed. Results: Twenty-six women with an average age of 32.8 ± 2.9 years underwent treatment. On Day 3 the overall mean number of blastomeres per preembryo on coculture compared to conventional medium in a previous cycle was 6.1 ± 1.8 vs. 5.1 ± 1.3 (P = 0.01; Wilcoxon test). The average percentage of cytoplasmic fragments on coculture compared to the conventional medium in a previous cycle was 14% ± 10 vs. 22% ± 13 (P = 0.003; Wilcoxon test). At transfer the mean number of blastomeres per preembryo on coculture was 7.4 ± 1.8 compared to 6.7 ± 1.5 on conventional medium in a previous cycle (P = 0.02; Wilcoxon test). The clinical pregnancy rate (positive fetal cardiac activity) per patient was 88.5%. The delivery rate was 73.1% (19/26). Conclusions: There was an improvement in the preembryo quality for preembryos on autologous endometrial coculture compared to noncocultured preembryos from the same patient in a previous cycle. An excellent delivery rate was subsequently found.

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.

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. Cancer 2001;92:1632–7. © 2001 American Cancer Society.

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.

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.

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 nonfertilized 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.

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.

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-1α and IL-1β 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-1α, IL-1β, or IL-1ra in the CM. IL-1 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-1α 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-1β in the CM was negatively associated with embryonic development and clinical pregnancy. The presence of IL-1α 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- 1 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.

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.

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.

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.

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 ...

Purpose: Our purpose was to study the role of inhibin/activin during embryogenesis. Methods: Transcripts of inhibin/activin subunits (α, β A , β 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 α, β A , β B subunits were all detected in granulosa luteal cells, but only β 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 β A subunits were detectable in blastocyst embryos. Activin receptors were detectable in all of the studied embryos and cells. Transcripts of β 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.

Purpose: Our purpose was to evaluate the effect of co-culture 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.

The impact of intracytoplasmic sperm injection (ICSI) on cryopreserved zygotes and embryos was evaluated by comparing embryo survival and implantation between embryos derived from ICSI and those derived from standard insemination procedures. The study included patients whose excess zygotes and embryos were cryopreserved between September 1993 and December 1994 and who subsequently underwent a frozen embryo transfer. Embryo survival, clinical pregnancy rates per transfer and pregnancy outcome were compared. Three hundred and thirty eight cryopreservation cycles, during which 1471 embryos were cryopreserved, were included in this study. Of those, 961 were derived from oocytes fertilized by insemination in vitro and 510 were derived from oocytes fertilized by ICSI. A total of 690 of the embryos (451 in the insemination group and 239 in the ICSI group) have since undergone a thaw cycle. The embryo survival rates were similar between the two groups (70.5 and 73.2%, insemination and ICSI respectively) and were not significantly affected by the stage at cryopreservation. There was no significant difference in pregnancy rates per transfer (31.8 and 32.3%), the preclinical pregnancy loss rate (16.7 and 23.8%), or the clinical miscarriage rate (16.7 and 23.8%) between the insemination and the ICSI groups respectively. It is concluded that ICSI does not have an adverse impact on the survival and successful implantation of cryopreserved and thawed embryos.

Genetic abnormalities, including partial deletions of the Y-chromosome, are commonly detectable in men with non-obstructive azoospermia (NOA). NOA can be treated using testicular sperm extraction (TESE) with intracytoplasmic sperm injection (ICSI). Recent studies have shown that the presence of deletions involving the AZFc region do not appear to affect the chance of retrieving spermatozoa or have a significant impact on fertilization or pregnancy rates with ICSI. We investigated the effect of Y-chromosome partial deletions on the chance of sperm retrieval with TESE. Eighty attempts at sperm retrieval were performed using TESE on men who were previously evaluated for Y-chromosome partial deletions. Y-chromosome analysis was performed using a polymerase chain reaction (PCR)-based technique with 35 sequence-tagged-sites. Of the 80 men, nine (11%) had partial Y-chromosome deletions detected. Two azoospermic men with AZFc deletions had successful sperm retrieval, ICSI and a subsequent clinical pregnancy. Seven men had deletions involving the AZFb region (three men had isolated AZFb deletions, one had AZFa, AZFb and AZFc deleted, and three had AZFb and AZFc deleted). None of the seven men had spermatozoa extracted by TESE, a result that is significantly different from the overall 64% (47/73) sperm retrieval rate achieved at our centre (P = 0.001). Two men with AZFb deletions had cells consistent with round spermatids identified and injected into oocytes without effecting any normal fertilizations. Although preliminary, these results suggest that the presence of an AZFb deletion is a significantly adverse prognostic finding for TESE. Men with AZFb deletions should be apprised of these results before attempting TESE-ICSI. Alternatives such as donor insemination or adoption should be considered or therapy delayed until improved results with round spermatid injections are likely.

Electroejaculation has been successfully used for sperm procurement in anejaculatory men desiring fertility. However, electroejaculates typically have normal sperm numbers but poor motility, morphology, and functional deficiencies. Here we report the pregnancy outcome of a series of couples undergoing combined electroejaculation and in-vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI). In all, 13 couples underwent a total of 18 cycles. The aetiologies of anejaculation included history of retroperitoneal lymph node dissection for testicular cancers, spinal cord injury and psychogenic causes. ICSI was performed on 192 oocytes, resulting in a fertilization rate of 75.5%. A total of 15 embryo transfers were performed using a total of 51 embryos. Clinical pregnancy rate, as defined by positive fetal heart rate(s) using vaginal sonography, was 55.6% per retrieval; implantation rate was 33.3% per embryo. These rates appear to be similar to those obtained in standard IVF for non-male factor infertility, or ICSI for male factor infertility. The use of ICSI for electroejaculates undoubtedly provides these couples with the highest chance of pregnancy.

Objective: To compare the endocrine responses of patients who first received hMG plus FSH, then were treated in a subsequent cycle with FSH alone. Design: Retrospective study. Setting: An academic research environment. Patient(s): Ninety-six women with pituitary down-regulation who underwent two sequential IVF treatments, the first with combined hMG and FSH and the second with FSH alone. Main Outcome Measure(s): Duration of stimulation, serum estradiol level on the day of hCG administration, amount of gonadotropin used, number of oocytes retrieved, number of oocytes fertilized, and selected preembryo morphologic features. Result(s): No difference in the mean duration of stimulation was observed between the treatment cycles among patients who received hMG and FSH (11.9 days) followed by FSH alone (11.7 days). The mean number of oocytes retrieved, the mean number of oocytes fertilized, the percentage of preembryo fragmentation, and the preembryo cell number at transfer did not differ significantly between the stimulation protocols. The cumulative amount of gonadotropin used during stimulation was slightly greater in the cycles stimulated with FSH alone, but this difference was not significant (29.4 ampules of hMG plus FSH versus 31.8 ampules of FSH alone). Serum estradiol levels measured on the day of hCG administration during stimulation with hMG and FSH (1,382 pg/mL) were higher than those measured during stimulation with FSH alone (1,149 pg/mL). Conclusion(s): Follicular response and preembryo quality were not significantly different when patients were treated first with hMG and FSH and then with FSH alone in a subsequent cycle. Similarities in ovarian response and preembryo characteristics, as well as differences in estradiol patterns seen in each stimulation setting, should be anticipated when patients receive these protocols.

PROBLEM: Insulin-like growth factors (IGFs) and insulin have been demonstrated to stimulate oocyte maturation and embryo development. Therefore, the expression of IGFs and their receptors may be an important intrinsic factor for embryo growth and may be a potential marker for embryo quality. METHOD OF STUDY: Thirty donated day 3 embryos were cultured in vitro for an additional 3 days to observe their developmental potential and were semiquantitatively analyzed for the expression of IGF-I, IGF-II, IGF-IR, IGF-IIR, and insulin-R. RESULTS: Our results show that the activity of these gene expressions correlates well with the morphological assessment and that high and more gene expressions were often associated with embryos of high growth potential. CONCLUSION: The IGF system may indeed play an important role in human embryogenesis ; IGF gene expressions can be a good indicator of embryonic developmental stage and/ or growth potential; finally, the IGF system can serve as a marker for embryo quality.

We have analysed the use of a programmed cycle of administration of exogenous steroids without prior suppression with a gonadotrophin-releasing hormone agonist (GnRHa) for the transfer of cryopreserved-thawed pre-embryos. From July 1992 to June 1994, 199 cycles (162 patients) were studied. Pre-embryos had been previously cryopreserved at the pronuclear stage using 1.5 M 1,2-propanediol as a cryoprotectant. Preparation of the endometrium was achieved in a step-up regime with transdermal oestradiol patches (0.1 to 0.4 mg). Progesterone in oil (50 mg i.m.) was started on cycle day 13. Pre-embryos were thawed on day 14 and transferred on day 15 after evidence of survival and cleavage. The mean (+/- SD) age of patients undergoing transfer was 35.4 +/- 4.3 years. The mean number of pre-embryos thawed was 4.7 +/- 1.8 with a mean of 3.3 +/- 1.4 pre-embryos being transferred. Eight of the cycles demonstrated follicular development >16 mm prior to thaw and transfer; however, these patients did not demonstrate a luteinizing hormone surge. Mean endometrial thickness on day 13 was 10.8 +/- 2.1 mm. Overall pregnancy rate was 29.2% (57/195). The ongoing or delivery rate was 16.1% (32/195). The rate of preclinical losses per transfer was 6.2% (12/195). Overall, the implantation rate was 6.2% (47/757). Thus, the use of a programmed cycle for cryopreserved embryo transfer yields favourable pregnancy outcome and offers practical advantages to patients. Prior suppression with a GnRHa is not necessary for endometrial preparation.

Objective To study the effects of different nuclear maturational status (prophase I [PI] versus metaphase II [Mil]) and in vitro culture on the kinetics of maternal messenger ribonucleic acid (mRNA) in human oocytes. Design Molecular biology on excess oocytes obtained from our clinical IVF program. Interventions The oocytes, classified as either PI or Mil at collection, were used as such or cultured in vitro for an additional 24 hours. The relative levels of c-mos and cyclin-B1 were measured using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). Results The mean levels of c-mos and cyclin-B1 transcripts were indistinguishable between the PI, Mil, PI oocytes matured in vitro, PI oocytes failing to mature, and Mil oocytes cultured for additional 24 hours. The variability in the levels of these transcripts increased during the in vitro culture. Conclusions The levels of c-mos and cyclin-B1 transcripts were not different in PI versus Mil oocytes, therefore, differences seen in the clinical outcome of PI and Mil oocytes may be unrelated to levels of these gene products. C-mos and cyclin B1 mRNA were maintained in vitro, thus degradation of maternal RNA is not activated in excess during the 24-hour culture.

Objective To evaluate binding characteristics of a specific zona pellucida (ZP) protein 3 (ZP3) antiserum to human oocytes in order to determine its usefulness as a clinical marker for human ZP integrity and function and its correlation with IVF outcome. Design Prospectively designed, blinded, internally controlled study. Setting Tertiary care academic center. Patients Patients undergoing IVF therapy who had either total failed fertilization or partial fertilization were studied. Interventions Metaphase II oocytes showing absence of pronuclear formation were salt stored 48 hours after insemination and bisected into matching hemizonae using micromanipulation. One hemizona was incubated with AS ZP3–6 (an antiserum generated against a synthetic ZP3 peptide derived from an amino acid sequence that is highly conserved in the structure of ZP3), whereas the matching hemizona was incubated with AS ZP3–7, an antiserum detecting exclusively mouse ZP3 (internal, negative control). Antibody binding was visualized using the peroxidase-antiperoxidase method and diaminobenzidine as color reagent. Results A total of 104 unfertilized oocytes were evaluated. Analysis of variance showed a significant interaction between gamete factor groups (sperm and oocyte) and antiserum factor. Patients with oocyte factor had significantly lower mean staining scores for the AS ZP3–6-treated hemizonae than patients with sperm factor. Conclusions These results demonstrate that anomalies of human ZP3 can be identified with AS ZP3–6 and that these ZP abnormalities correlate with fertilization failure during IVF treatment. Thus, this newly developed biomarker may be of clinical significance in the identification of oocyte defects that are associated with fertilization disorders and may help in the decision-making process in the IVF-assisted fertilization setting.

Patients with cancer might want to avoid infertility and premature menopause as sequelae of chemotherapy and other treatments. Most patients with cancer do not have time for ovarian stimulation as a prelude to in vitro fertilization (IVF). An alternative approach, which could also be useful for single women who do not want to use donor sperm and for children, is to cryopreserve oocytes, but this also requires several weeks of ovarian stimulation. Ovarian function has been preserved experimentally after orthotopic transplantation of frozen-thawed ovarian tissue and heterotopic grafting of fresh human ovarian cortical pieces. This study intended to restore fertility by subcutaneously transplanting frozen, banked ovarian tissue. One ovary was removed from a woman aged 30 years with breast cancer before starting gonadotoxic chemotherapy. Cortical pieces were cryopreserved using a slow freezing technique and dimethylsulfoxide as a cryoprotectant. The tissue was transplanted beneath the abdominal skin 6 years later, after the patient had entered menopause. There were an estimated 11,000 follicles that could potentially last for 12 months. All 15 ovarian cortical pieces were transplanted in the lower abdominal wall. Tissue was suppressed with a combination of follicle-stimulating hormone and human menopausal gonadotropin before every cycle of ovarian stimulation. Oocyte maturity was induced by recombinant human chorionic gonadotropin. Oocytes were retrieved 36 to 40 hours later and intracytoplasmic sperm injection was carried out. A lump was felt at the transplant site 85 days after transplantation, and ovarian endocrine function resumed (Fig. 1). Eight percutaneous oocyte retrievals were carried out over 8 months, 6 of them after ovarian stimulation. Eight of 20 oocytes taken from transplanted tissue were suitable for IVF. One of them fertilized normally and developed into a high-grade 4-cell-stage embryo 24 hours later that was transferred to the patient's uterus. This experience indicates that endocrine function and fertility could be restored by long-term cryopreservation of ovarian tissue.

Objective To determine the efficacy and factors affecting outcome of intracytoplasmic sperm injection (ICSI) in patients with severe male factor infertility. Design Prospectively designed clinical trial of patients selected to participate in the study based upon the following inclusion criteria: previous total failed fertilization or unsuitable sperm parameters for conventional IVF. Setting Tertiary care academic center. Patients Ninety-two consecutive couples undergoing IVF therapy augmented with ICSI during April through December 1994 were studied. Main Outcome Measures Fertilization and ongoing implantation and pregnancy rates (PRs). Results A total of 1,163 preovulatory oocytes were manipulated, yielding a diploid fertilization rate of 60.9%; the oocyte damage rate was 13.2%. The transfer rate was 95% with 43.1% of cycles having excess embryos that were cryopreserved. Overall, the clinical and ongoing PRs per transfer were 31.9% and 26.8%, respectively. None of the sperm parameters of the original semen analysis correlated with ICSI outcome. Female age did not affect fertilization results but had a significant impact on PR ( Conclusions Intracytoplasmic sperm injection offers a new and powerful therapeutic option to treat couples with severe male factor infertility associated with a variety of sperm abnormalities. An adequate female age is a pivotal factor determining a successful outcome.

Objective To determine the effect on pregnancy rate (PR) of low-dose glucocorticoid treatment in cycles without micromanipulation. Design Randomized, prospective, double-blinded, placebo-controlled trial. Setting One university-based tertiary infertility center and two private infertility centers. Patients All patients receiving standard stimulation IVF-ET or transfer of cryopreserved embryos at the participating facilities from January to September 1993 were asked to participate in this study. Patients having micromanipulation were excluded from this study. Interventions Participating patients were randomized to either 16mg oral 6- α -methylprednisolone for four evenings starting the evening of retrieval or the evening before thawing cryopreserved embryos or to placebo administered in an identical fashion. Both groups were treated with 250mg oral tetracycline four times per day starting with initiation of the study medication and continuing for 4 days. Cryopreservation and stimulation cycles were managed according to pre-established protocols for all patients. A clinical pregnancy was confirmed by an appropriately rising hCG titer and a gestational sac on ultrasound. Results A total of 206 stimulation patients and 61 cryopreservation patients were randomized and had an ET. Patient characteristics were similar between groups. The clinical pregnancy and implantation rates between placebo and glucocorticoid groups were 35.9% versus 40.8% and 12.8% versus 11.7% for stimulation cycles and 30.3% versus 25% and 9.9% versus 7.4% for cryopreservation cycles, respectively. None of these differences were statistically significant. Conclusions Glucocorticoid plus antibiotic treatment at these doses for transfers of nonmicro-manipulated embryos does not appear to have a significant effect on pregnancy or implantation rates.

Objective To evaluate the cryopreservation of immature human oocytes obtained from unstimulated ovarian tissue. Design Immature prophase I oocytes were obtained from unstimulated follicles and were either cryopreserved or cultured as controls. Cryopreservation was performed in a programmable freezing machine using one of two protocols. Method I (n=133) used a one-step addition of cryoprotectant followed by a slow freeze and thaw protocol. With method II (n=95), the cryoprotectant was added in a stepwise manner with cryopreservation performed in the presence of 0.2M sucrose followed by rapid freezing and thawing. Setting Basic research center at a medical school. Patients Patients undergoing oophorectomy for nonovarian pathology. Main Outcome Measures Rates of survival and maturation to metaphase II were compared between control oocytes and oocytes cryopreserved with methods I and II. Results With method I, a survival rate of 15.6% was obtained with 58.3% of surviving oocytes reaching metaphase II after culture compared with 50.0% of nonfrozen control oocytes. Method II produced a survival rate of 43.3% with 27.3% maturing to metaphase II. Maturation of control oocytes for method II was 46.4%. Although the survival rate with method II was significantly higher than with method I, the rate of in vitro maturation to metaphase II showed no difference. Conclusions These results demonstrate that human prophase I oocytes obtained from unstimulated antral follicles are capable of meiotic maturation after cryopreservation.

Objective To develop a mathematical formula that accurately predicts the probability of a singleton, twin, and higher-order multiple pregnancy according to implantation rate and number of embryos transferred. Design A total of 12,003 IVF cycles from a single center resulting in ET were analyzed. Using mathematical modeling we developed a formula, the Combined Formula, and tested for the ability of this formula to accurately predict outcomes. Setting Academic hospital. Patient(s) Patients undergoing IVF. Intervention(s) None. Main Outcome Measure(s) Goodness of fit of data from our center and previously published data to the Combined Formula and three previous mathematical models. Result(s) The Combined Formula predicted the probability of singleton, twin, and higher-order pregnancies more accurately than three previous formulas (1.4% vs. 2.88%, 4.02%, and 5%, respectively) and accurately predicted outcomes from five previously published studies from other centers. An online applet is provided (https://secure.ivf.org/ivf-calculator.html). Conclusion(s) The probability of pregnancy with singletons, twins, and higher-order multiples according to number of embryos transferred is predictable and not random and can be accurately modeled using the Combined Formula. The embryo itself is the major predictor of pregnancy outcomes, but there is an influence from "barriers," such as the endometrium and collaboration between embryos (embryo–embryo interaction). This model can be used to guide the decision regarding number of embryos to transfer after IVF.

Objective To examine the results of a 5-year trial using cryopreservation to limit multiple pregnancy and optimize overall pregnancy per cycle. Design Retrospective clinical evaluation of pregnancy rates (PRs) per cycle after freezing pronuclear stage human oocytes. Setting Tertiary care academic center. Patients Six hundred seventeen patients treated in 776 IVF-ET cycles from January 1987 to December 1991 (less oocyte donation cycles). Main Outcome Measure Pregnancy rate per cycle after transfer of pre-embryos developed from thawed pronuclear stage oocytes. Results Three thousand seven hundred thirty-one oocytes were frozen. Of these, 2,039 were thawed. One thousand three hundred seventy-seven survived thawing (68%), and 1,370 were transferred after passing through syngamy to at least the first cleavage (68%). Of patients with thawing, 359 of 401 (90%) (449 of 505 cycles [89%]) received intrauterine transfer. One hundred thirty-three separate clinical pregnancies were established from 128 different cycles (128/449; 29%); 5 cycles had two thaws, each of which resulted in pregnancy. This PR is less than the overall fresh PR observed in patients who had excess pronucleate oocytes frozen (279/776; 36%) but is remarkably similar when adjusted for the number of pre-embryos transferred per cycle. The age of the patient at the time of cryopreservation and the number and quality of pre-embryos ultimately available for transfer were important factors in the establishment of pregnancy. The mode of ovarian stimulation and duration of cryostorage did not prove meaningful. Conclusions Cryopreserved pronucleate oocytes that survive freezing, thawing, and progress through syngamy demonstrate a similar potential for implantation and pregnancy when compared with fresh conceptuses, the cumulative effect of which is an enhanced total PR per cycle.

World Congress of In Vitro Fertilization and Embryo Transfer. Norfolk, Virginia, April 5-10, 1987. 20. Edwards RG: In vitro fertilization: past and future. Ann Biol Clin 1987;45:321-329 21. Gordts S, Roziers P, Campo R, Noto V: Survival and pregnancy outcome after ultrarapid freezing of human embryos. Fertil Steril 1990;53:469-472 22. Barg PE, Barad DH, Feichtinger W: Ultrarapid freezing (URF) of mouse and human preembryos: A modified approach. J Vitro Fert Embryo Transfer 1990;7:355-357 23. Feichtinger W, Hochfellner C, Ferstl U: Clinical experience with ultra-rapid freezing of embryos. Hum Reprod 1991;6: 735-736

Since there has been no reported use of the YAG laser to micromanipulate oocytes, our purpose was to study whether (1) a YAG laser could be used to open the zona pellucida of hamster oocytes; (2) human sperm could reach the ooplasm and (3) under sperm penetration assay conditions, sperm would bind and penetrate the ooplasm.A YAG 100 laser was used at 10 W and 0.4-sec pulse width to open eight of eight ooplasm oocytes. The opening in the zonae was 0.25 to 1.0 rad (10 to 40 microns). For the initial eight oocytes and two parallel controls, the coarse appearance of the ooplasm was unchanged after 3 days. Next, in 11 of 12 manipulated oocytes, the sperm clustered at the opening of the zona. When 16 more oocytes were opened and exposed to sperm in sperm penetration assay conditions, each ooplasm bound sperm. There was no penetration noted. Each manipulation time was1 min. To clarify the laser effect, oocytes were exposed to laser energy then utilized as the interactive surface in the sperm penetration assay. It was found that only 20% bound sperm with no penetration.While the time factor compares favourably with other methods of zona opening, further study needs to be performed to minimize effect to the exposed oocyte.

The results of predictive fertilization bioassays (hemizona assay, hamster ova-human sperm penetration assay), in vitro fertilization treatment, fertile donor cross-match tests with either sperm or oocytes, and oocyte micromanipulation for assisted fertilization were used to establish a pathophysiologic diagnosis in cases of recurrent failed fertilization in vitro. Disorders of sperm function manifested at the level of zona binding, zona penetration, oolemma fusion, and pronuclear decondensation as well as oocyte anomalies were considered to represent the specific gamete defects that led to abnormal sperm-oocyte interactions (i.e., failed fertilization). Our findings show that the sequential application of bioassays can elucidate specific sperm or oocyte defects that characterize functional abnormalities among sperm populations.

Three hundred and twenty-seven consecutive in-vitro fertilization (IVF) cycles in which luteal-phase leuprolide had been given were ranked according to the number of pre-ovulatory oocytes obtained (1-5, 6-10, greater than 10). Excess pre-embryos were cryopreserved at the pronuclear stage and later transferred into monitored natural cycles on the day after ovulation. The results indicate that the retrieval of large numbers of pre-ovulatory oocytes (greater than 10) has a small negative impact on oocyte quality as judged by fertilization rates (4% lower). However, implantation was not impaired compared to lower levels of retrieval in either the original IVF or subsequent cryo-thaw cycles. Overall, despite the small reduction in fertilization rate, the retrieval of many pre-ovulatory oocytes has produced a 'take-home baby rate' per stimulation cycle of 28.3% when 6-10 pre-ovulatory oocytes were retrieved and 41.5% when greater than 10 were retrieved: even higher rates are anticipated when the remaining cryopreserved pre-embryos are ultimately thawed.

Women with ovarian failure transferred with donated oocytes provide a unique in vivo model for the elucidation of the window of implantation and efficiency of reproduction in the human. Throughout 52 ovum donation cycles, the temporal window of endometrial receptivity was tested by replacing 2- to 12-cell embryos between days 16 and 24 of hormonally and histologically defined cycles. Of 37 transfers within days 17 to 19, 15 (40.5%) conceptions occurred. Twelve (32.4%) have reached viability. Of 11 patients transferred on days greater than or equal to 20, none conceived. Likewise, no pregnancies were achieved with 4 transfers on cycle day 16. Analysis of multiple embryo transfers within the suggested window of endometrial receptivity (days 17 to 19) revealed 14 of 24 (58.3%) to be conception cycles. considering only transfers with two or more embryos, at least one of which is of high quality (grades 1 to 2), yielded a 63.2% pregnancy rate. The results indicate a very high efficiency for in vitro fecundity provided optimal conditions are attained. The concepts leading to success in the ovum donation model should set the course for continued research toward improving results in other forms of assisted reproduction.

The aim of this study was to report the successful fertility treatment of men with Klinefelter syndrome using testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI).A total of 42 men with Klinefelter syndrome who underwent 54 TESE procedures were identified. Before TESE, patients with serum testosterone levels less than 15.6 nmol/liter were treated with an aromatase inhibitor. Sperm retrieval rates and results of ICSI, including fertilization and clinical pregnancy, were collected.Mean pretreatment FSH and testosterone levels were 33.2 IU/liter and 9.8 nmol/liter. During medical therapy, the mean testosterone level rose to 17.0 nmol/liter (P0.01). Spermatozoa were found during 39 microdissection TESE procedures, on the day before, or day of oocyte retrieval during a programmed in vitro fertilization cycle. The sperm retrieval rate was 72% (39 of 54) per TESE attempt, and 29 of the 42 different men (69%) had adequate sperm found for ICSI. Thirty-three in vitro fertilization cycles yielded embryos for transfer in the 39 (85%) cycles with sperm retrieved. Eighteen clinical pregnancies have resulted in 21 live births [18 of 39 (46%)]. All children had a normal karyotype.TESE/ICSI is a successful intervention for the majority of patients with azoospermia and Klinefelter syndrome. Sperm retrieval and ICSI success in men with Klinefelter syndrome are comparable with other men with nonobstructive azoospermia treated at our center.

A simple evaluatory formula for reporting pregnancy rates involving cryopreserved material has so far remained elusive. It is highly desirable to have a method which would allow not only an evaluation of cryotechnology per se, but also an evaluation of the role of cryotechnology in enhancing the total reproductive potential of a single cycle. In this paper, nine separate formulae are described which can be used for the evaluation of cryopreservation. It is concluded that none of the existing formulae used alone expresses the total potential of cryopreservation, and that while each formula provides information regarding some aspect of the role of cryopreservation, the most comprehensive evaluation requires the answer to five different equations. The other four formulae, while often used, are regarded as providing information which is unsubstantiated or does not allow complete evaluation.

Multiple gestation is now recognized as a major problem associated with both assisted reproductive technologies (ART) and also with ovulation induction therapies. Although some countries are beginning to adopt measures to address this issue, either through legislation or the development of clinical guidelines, there is a clear need to ensure recognition and a consistent approach to this problem worldwide. In particular, there is a need to educate both healthcare professionals and the lay population that multiple gestations are not a desirable outcome for the infertile couple.

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.

The value of cryopreserving prezygotes, pre-embryos or blastocysts for future thaw and transfer is an important consideration of every IVF program. The convergence of two factors, a higher pregnancy rate and a lower multiple gestation rate, can be managed effectively through the establishment of a successful cryopreservation programme. In this article, freezing and thawing results from pronuclear oocytes, pre-embryos, and blastocysts are compared.

To examine the results of a 3-year trial using blastocyst cryopreservation to limit multiple pregnancy and optimize overall pregnancy per cycle.Retrospective clinical evaluation of pregnancy rates after freezing and thawing human blastocysts.Tertiary-care academic center.Seven hundred fifty-three different patients treated in 783 IVF cycles with blastocysts frozen from July 2000 to June 2003.Two thousand, two hundred fifty-nine blastocysts were frozen in cycles in which only blastocysts were cryopreserved (cycles with pronuclear stage oocytes or pre-embryos also cryopreserved were excluded from the analysis). Of these, 628 (27.6%) were thawed in 218 cycles.Pregnancy rate per cycle with thaw.Four hundred seventy-nine (76.3%) blastocysts survived thawing, and 440 (92.0%) were transferred after exhibiting evidence of survival (most commonly, blastocoele reexpansion). In cycles with a thaw, 211 (96.8%) of 218 underwent intrauterine transfer. An average of 2.09 blastocysts was transferred per replacement. One hundred twenty-five (59.2%) clinical pregnancies were established, which included 23 sets of twins and 5 triplet gestations. Two sets of monozygotic twins were identified after the replacement of a single thawed blastocyst (1.6%). The age of the patient at the time of cryopreservation (37 years) was an important factor in the establishment of clinical and ongoing pregnancy. The mode of ovarian stimulation, replacement method, and whether blastocysts were frozen on day 5 or day 6 of development did not demonstrate clinical significance.Cryopreserved and thawed blastocysts demonstrated a similar potential for implantation when compared with fresh pre-embryos on day 3. On the basis of these results, the blastocyst stage of development appears to be optimal for clinical freeze-thaw trials.