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Eggs can be 'forever'. A longstanding goal in assisted reproductive technology (ART) has been realized at last, namely, cryopreservation of oocytes by vitrification technology. This breakthrough heralds benefits for infertility treatment, fertility preservation, and even postponement of reproduction but, as so often with ARTs, new waves of technology draw ethical and societal concerns in their wake., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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 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., Conclusion: 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.

Objective: To examine the results of a 3-year trial using blastocyst cryopreservation to limit multiple pregnancy and optimize overall pregnancy per cycle., Design: Retrospective clinical evaluation of pregnancy rates after freezing and thawing human blastocysts., Setting: Tertiary-care academic center., Patient(s): Seven hundred fifty-three different patients treated in 783 IVF cycles with blastocysts frozen from July 2000 to June 2003., Intervention(s): 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., Main Outcome Measure(s): Pregnancy rate per cycle with thaw., Result(s): 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., Conclusion(s): 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.

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.

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., (Copyright 2001 American Cancer Society.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A controlled comparison of the efficacy and reliability of sperm retrieval by testicular fine needle aspiration (TFNA), percutaneous testicular needle biopsy (PercBiopsy) and microsurgical epididymal sperm aspiration (MESA) was performed in nine patients with obstructive azoospermia. During a planned MESA procedure, sperm retrieval was attempted on the same testis with TFNA and PercBiopsy. Spermatozoa were obtained from all patients using MESA and PercBiopsy. Spermatozoa were retrieved using TFNA from 6/9 (67%) men. The mean number of epididymal spermatozoa retrieved with MESA (15 x 106) was significantly higher (P = 0.003) than that retrieved percutaneously from the testis. The mean number of spermatozoa obtained by PercBiopsy was 0.116 x 10(6) while TFNA recovered 0.014 x 106 spermatozoa (P = 0.025). MESA is the optimal choice to retrieve the greatest number of spermatozoa with highest motility for assisted reproduction and subsequent cryopreservation. However, percutaneous testicular retrieval does not require microsurgical expertise and is less invasive. Our results suggest that the optimal percutaneous procedure for sperm retrieval from the testis involves percutaneous testicular needle biopsy with an automatic biopsy gun.

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.

In-vitro fertilization patients (n = 15) at risk of ovarian hyperstimulation syndrome (OHSS) (oestradiol > or =4500 pg/ml on the day of human chorionic gonadotrophin administration and 25 or more follicles of intermediate or large size) underwent aspiration of all follicles and cryopreservation of all fertilized oocytes at the pronuclear stage. Patients were monitored for up to 2 weeks post-retrieval. Subsequent transfer of cryopreserved-thawed embryos was performed in programmed cycles using exogenous oestrogen and progesterone for endometrial preparation. Two patients (13%) developed OHSS necessitating hospitalization and vaginal aspiration of ascitic fluid. Two other patients (13%) developed moderate OHSS requiring ascitic fluid vaginal aspiration in the office setting, with dramatic improvement of the condition. Subsequent transfer of cryopreserved-thawed embryos yielded a clinical pregnancy rate of 58% per transfer and ongoing or delivery rates of 42 and 67% per transfer and per patient respectively. By eliminating pregnancy potential with cryopreservation of all prezygotes and examining the pregnancy potential with subsequent cryopreserved-thawed transfers, it is concluded that OHSS is reduced, but not eliminated for patients at risk. Subsequent transfer of cryopreserved-thawed prezygotes in a programmed cycle with exogenous steroids yields an excellent pregnancy rate.

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 detect simultaneously multiple gene expression in mouse and human individual embryos by reverse transcriptase-polymerase chain reaction., Design: Transcripts involved in the insulin-like growth factor (IGF) system were detected in mouse and human preimplantation embryos., Setting: An academic teaching hospital., Main Outcome Measure(s): Transcripts of the IGF family genes., Result(s): In the mouse, genes are expressed differentially and messenger RNA transcripts of maternal origin in nonfertilized ova decline gradually until the initiation of the embryonic genome transcription. Insulin-like growth factor-binding protein-2 (IGFBP)-2, -3, -4, and beta-actin transcripts appear to be initiated at the two- to four-cell stage, whereas IGFBP-1, -5, and -6 transcripts are initiated at later stages. Transcription, once initiated, appears to continue through to the blastocyst stage. In humans, almost all genes of the IGF system were expressed in preimplantation embryos. This is the first report of the assessment of IGF family transcripts in individual embryos, and introduces a novel method for research and clinical diagnosis of preimplantation embryos.

Objectives: To provide fertility for men with nonobstructive azoospermia., Methods: A retrospective review of treatment results at a university infertility center was undertaken. Sixteen couples entered an attempted in vitro fertilization (IVF)-intracytoplasmic sperm injection (ICSI) cycle for treatment of nonobstructive azoospermia. Each man was azoospermic, and the male factor diagnosis of nonobstructive azoospermia was made on testis biopsy for 14 men and on clinical grounds for 2 men. Sperm were retrieved by testicular biopsy on the day of oocyte retrieval. Results of testicular examinations, serum follicle-stimulating hormone levels, and testicular histology as well as evaluation of the success rates of sperm retrieval, fertilizations, and pregnancies were made., Results: Sperm were extracted from testis biopsies in 10 of 16 (62%) testicular sperm extraction (TESE) attempts. For cycles in which sperm were retrieved, normal fertilizations were achieved for 51 of 98 (52%) mature oocytes injected with testicular sperm in 10 couples. Biochemical pregnancies were achieved for 6 of 16 (38%) couples, with clinical pregnancies during 5 of 16 (31%) attempts at sperm retrieval, and ongoing pregnancy and subsequent live delivery for 4 of 16 (25%) attempts. CONCLUSIONS; Pretreatment clinical parameters are unable to predict which men with nonobstructive azoospermia will have spermatozoa retrieved by TESE. When sperm are found, clinical pregnancies can occur for half (5/10) of these couples using TESE with ICSI, with ongoing pregnancy and delivery for 4 of 10 (40%). Many men with nonobstructive azoospermia will have retrievable sperm with testis biopsy that are suitable for ICSI; however, 6 of 16 (38%) couples will not have sperm retrieved with TESE and may undergo an unnecessary IVF procedure.

Objective: To study the effects of different nuclear maturational status (prophase I [PI] versus metaphase II [MII]) 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 MII 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, MII, PI oocytes matured in vitro, PI oocytes failing to mature, and MII oocytes cultured for additional 24 hours. The variability in the levels of these transcripts increased during the vitro culture., Conclusions: The level of c-mos and cyclin-B1 transcripts were not different in PI versus MII oocytes, therefore, differences seen in the clinical outcome of PI and MII 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 the suppression and flare regimens of gonadotropin-releasing hormone agonist (GnRH-a) in ovarian hyperstimulation in women with variable basal gonadotropin values in an in vitro fertilization (IVF) program., Study Design: A retrospective study comparing the initiation of GnRH-a in the midluteal phase of the preceding cycle (suppression protocol) and follicular phase of the stimulated cycle (flare protocol) in women with basal follicle-stimulating hormone (FSH) values < 15 mIU/mL and > or = 15 mIU/mL., Results: The pregnancy rate per initiated cycle and implantation rate for women with basal FSH levels > or = 15 mIU/mL were 20.4% and 9.8% in flare GnRH-a cycles and 11.7% and 3.5%, respectively, in suppression GnRH-a cycles. Comparing the percent differences in clinical pregnancy and implantation rates between both protocols for women with different basal FSH values, pregnancy outcome was significantly greater in the flare protocols in women with values > or = 15 mIU/mL (P < .001). Individualization of the stimulation protocol by retrospective sorting of women undergoing IVF with respect to their basal gonadotropin levels significantly improved clinical pregnancy (P < .05) and implantation rates (P < .05) and reduced the cancellation rate (P < .05)., Conclusion: The flare regimen with GnRH-a is a useful alternative for controlled ovarian hyperstimulation in women with elevated basal FSH values (> or = 15 mIU/mL) undergoing IVF.

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.

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.

The present study was conducted to determine if the cryopreservation of immature human oocytes has a deleterious effect on the meiotic spindle following maturation in vitro. Oocytes were obtained in excess from in-vitro fertilization patients and divided into four groups. Groups 1 (n = 98) and 2 (n = 80) consisted of immature oocytes cryopreserved before or after maturation in vitro respectively. Groups 3 (n = 37) and 4 (n = 9) served as non-frozen controls and included oocytes matured in vitro and in vivo respectively. The meiotic spindle was identified after incubation in anti-tubulin monoclonal antibody (1 h, 37 degrees C) and fluorescein-conjugated goat anti-mouse immunoglobulin G (IgG) (1 h, RT). Chromosomes were counterstained with 4',6'-diamidino-2-phenylindole. Following cryopreservation, group 1 oocytes demonstrated a 63% survival rate and 68% maturation rate in vitro. In all, 58% of the oocytes in group 2 survived the thaw. The number of oocytes with normal spindles in group 1 (81.0%) was not significantly different from control groups 3 (83.8%) and 4 (88.9%), while the number of group 2 oocytes with normal structures (43.5%) was significantly lower than groups 1 (P = 0.0004), 3 (P = 0.0002), and 4 (P = 0.025). These results suggest that cryopreservation of the prophase I human oocyte does not significantly increase abnormalities in the resulting meiotic spindle.

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 16 mg oral 6-alpha-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 250 mg 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 nonmicromanipulated embryos does not appear to have a significant effect on pregnancy or implantation rates.

Objective: To examine the results of 7 years of thawed ET during natural or controlled cycles using exogenous steroids., Design: Retrospective evaluation to compare implantation and pregnancy rates with two protocols for transfer of cryopreserved-thawed pre-embryos., Setting: Tertiary care academic center., Patients: From January 1987 to December 1993, 521 patients who were < 40 years of age underwent 628 thawed embryo transfers., Main Outcome Measure: Pregnancy and implantation rates per thawed embryo transfer cycle., Results: A total 1,987 pre-embryos survived the thawing process and were used in 628 thaw-transfer cycles. Transfer was performed in a natural cycle 2 days after the LH peak or on day 17 of a programmed cycle using a GnRH-agonist and hormone replacement therapy protocol; 182 pregnancies were established (182/628; 29%). Similar pregnancy rates were seen in the natural cycle (112/398; 28%) and the programmed cycle (70/230; 30%). The implantation rates were similar in the two methods of transfer cycles (11.9% versus 10.3%, natural versus programmed cycle). There were no significant differences in clinical or ongoing pregnancy rates in a natural or programmed cycle, correcting for the number of cryopreserved-thawed pre-embryos transferred. Patient's age at the time of freezing and the number of cryopreserved-thawed pre-embryos transferred are more important determinants of pregnancy than the type of cycle in which transfer occurs., Conclusion: Transferring cryopreserved-thawed pre-embryos in a natural or programmed cycle yields similar pregnancy results.

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 0.2 M 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 determine the potential for in vitro maturation, fertilization, and cleavage after cryopreservation of immature, prophase I human oocytes., Design: Immature oocytes obtained in excess of the number required by the patient were randomized and cryopreserved at the prophase I stage or cultured as control. After thawing and maturation in vitro, test and control oocytes were inseminated with husband's sperm and evaluated for fertilization and cleavage in vitro., Setting: In vitro fertilization program., Patients: Consenting patients undergoing controlled ovarian hyperstimulation for the purposes of IVF., Main Outcome Measures: Rates of maturation to metaphase II, fertilization, and cleavage were compared between control and cryopreserved oocytes., Results: Upon thaw, 58.5% (72/123) of prophase I oocytes were viable. Control oocytes demonstrated a 74.8% (98/131) maturation rate to metaphase II, a 56.5% (52/92) fertilization rate, and an 11.5% (6/52) blastocyst rate. Cryopreserved oocytes showed a 83.3% (60/72) rate of maturation, a 57.7% (30/52) fertilization rate, and a 3.3% (1/30) blastocyst rate. No significant differences were noted between any of these parameters., Conclusions: These results demonstrate that prophase I oocytes from stimulated IVF cycles are able to survive cryopreservation and resume meiosis to achieve full nuclear maturation post-thaw. In addition, cryopreserved oocytes retain the same capacity for fertilization and development as control oocytes.

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 of 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.

All IVF cycles in which subsequent transfers of thawed pre-embryos occurred were studied. Both age and basal (cycle day 3) FSH level are important determinants of the chance for cryopreservation and the performance of cryopreserved pre-embryos. Although there was no age or FSH level above which pregnancy with frozen pre-embryos was not possible, the chances clearly decline. Thus, consideration to transferring larger numbers of pre-embryos fresh should be given to women in the fifth decade and those with basal FSH > 15 IU/L.

Problem: 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., Results: 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 was < 1 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., Conclusion: 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.

Whether one carefully and meticulously records detailed observations in the laboratory, or jumps feet first into new and exciting areas of investigation, each scientist contributes to the advances being made in the control of human reproduction. The study of spermatozoa, oocytes, sperm-oocyte interaction, preembryo growth and development, and early pregnancy rewards us with breakthroughs in innovative technology and promotes paths of better understanding. One cannot search through the massive amounts of currently available literature without desiring to try "this" technique, expound on "that" one, take someone else's work a little further, or look at one's own results from a different angle. Presented here are some informative data, most published within the past year. Oocyte maturational competence and integrity, sperm function and quality, preembryo culture conditions, preembryo morphology, and ovarian age all emerge as factors important for interpreting a patient's chance of achieving pregnancy.

When frozen-thawed pre-embryos were transferred in a natural menstrual cycle, a clinical pregnancy rate of 22% per transfer cycle was obtained. The pregnancy rates were higher when serum progesterone levels at thaw were less than or equal to 3.4 ng/ml than when they were greater than or equal to 3.5 ng/ml. This was reflected in a significantly higher oestradiol/progesterone ratio at thaw in the conception cycles than in the non-conception cycles.