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The objective of this study was to investigate the impact of severe oligoasthenoteratozoospermia (OAT) on pregnancy outcome. For this purpose 279 consecutive intracytoplasmic sperm injection (i.c.s.i) cycles were retrospectively evaluated and compared to 436 consecutive IVF cycles performed during the same time frame. Group A (n = 62) included ICSI patients with severe OAT; group B (n = 217) included patients who underwent ICSI for other indications; and group C (n = 436) included couples who underwent standard IVF. The mean age of female patients and mean number of embryos transferred were comparable in all groups. No difference was observed regarding implantation, clinical pregnancy, delivery and miscarriage rates between all three groups, but fertilization rate was significantly lower in group A than in groups B and C. It is concluded that couples undergoing ICSI with severe male infertility (OAT) have a slightly reduced fertilization rate but their chances of delivery and pregnancy loss are similar to those of other patients undergoing clinical ICSI and IVF with non-male infertility.

Recent evidence indicates that mammalian gametogenesis and preimplantation development may be adversely affected by both assisted reproductive and stem cell technologies. Thus, a better understanding of the developmental regulation of the underlying epigenetic processes that include DNA methylation is required. We have, therefore, monitored the expression, by PCR, of the mRNAs of DNA methyltransferases (DNMTs), methyl-CpG-binding domain proteins (MBDs), and CpG binding protein (CGBP) in a developmental series of amplified cDNA samples derived from staged human ovarian follicles, oocytes, preimplantation embryos, human embryonic stem (hES) cells and in similar murine cDNA samples. Transcripts of these genes were detected in human ovarian follicles (DNMT3A, DNMT3b1, DNMT3b4, DNMT1, MDBs1–4, MeCP2, CGBP), germinal vesicle (GV) oocytes (DNMT3A, DNMT3b1, DNMT1, MDBs1–4, MeCP2, CGBP), mature oocytes (DNMT3A, DNMT3b1, DNMT1, CGBP), and preimplantation embryos (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD2, MDB4, CGBP). Differential expression of DNMT3B gene transcripts in undifferentiated (DNMT3b1) and in vitro differentiated human ES cells (DNMT3b3) further demonstrated an association of the DNMT3b1 transcript variant with totipotent and pluripotent human cells. Significantly, whilst the murine Dnmt3L gene is both expressed and essential for imprint establishment during murine oogenesis, transcripts of the human DNMT3L gene were only detected after fertilisation. Therefore, the mechanisms and/or the timing of imprint establishment may differ in humans. Mol. Reprod. Dev. 67: 323–336, 2004. © 2004 Wiley-Liss, Inc.

BACKGROUND The study objective was to determine if ovarian function would be restored following fresh and cryopreserved extrapelvic autologous ovarian transplantation and if vascular endothelial growth factor (VEGF) administration would augment the success rate. METHODS Sixteen regularly cycling female cynomolgus monkeys underwent bilateral oophorectomy and were randomly assigned to one of three treatment groups: (i) sham transplant group (n = 5) underwent transplantation of pieces of adipose tissue; (ii) fresh autologous ovarian transplantation without VEGF administration (n = 6) and (iii) fresh autologous ovarian transplantation with 1 microg of VEGF (n = 5) administered at the transplantation site daily for 14 days after transplantation. The ovarian tissue from the sham transplanted group was cryopreserved. This material was later thawed and transplanted in four out of five of the sham operated group. RESULTS Five out of six (83%) of the primates in our transplantation group without VEGF had functioning ovarian transplants while two of five (40%) primates in our transplantation group with VEGF administration had functioning ovarian transplants. The cryopreserved group had two of four primates (50%) with functioning transplants. Ovarian stimulation yielded multiple follicles and one metaphase II oocyte from the fresh and one metaphase II oocyte from the cryopreserved group. CONCLUSIONS The success of ovarian transplantation in these non-human primates with menstrual cycles bodes well for the development of ovarian transplantation protocols for women at risk of ovarian failure.

Purpose: To determine if the removal of cytoplasm from metaphase II human donor oocytes damages the meiotic spindle apparatus. Materials and Methods: Cryopreservation of metaphase II human oocytes was performed using a fast-freeze, fast-thaw protocol. Upon thaw, oocytes were incubated for 3–4 h and then used for cytoplasmic donation (test oocytes). Oocytes thawed but not used for donation served as controls. Test and control oocytes were fixed using a microtubule-stabilizing buffer. Tubulin was localized using antitubulin monoclonal antibody. Chromosomes were identified by counterstaining with DAPI. Results: Forty-four oocytes had cytoplasm removed (test group) while 12 were not used for the procedure (controls). Twenty-three oocytes survived the donation procedure. Rates of normal spindle structure for the control and test groups were 21/23 (91.3%) and 12/12 (100%), respectively. Conclusion: The removal of cytoplasm from a metaphase II human donor oocyte does not appear to significantly increase the damage to chromosome alignment or to the spindle structure.

Spermatozoa-zona pellucida binding selects for human spermatozoa with progressive motility, normal morphology and functional competency. We postulated that this gamete interaction would also act to select against spermatozoa with chromosomal numerical aberrations. Spermatozoa from 41 men participating in the intracytoplasmic sperm injection (ICSI) programme were evaluated for the incidence of aneuploidy of chromosomes 18, X and Y. The hemizona assay was utilized to determine whether zona-bound spermatozoa from these patients have a reduced incidence of aneuploidy compared with those selected by motility only in a standard swim-up procedure. Using multicolour fluorescence in-situ hybridization (FISH) with DNA probes specific for chromosomes 18, X and Y, the disomy rates for chromosomes 18, X, Y and XY were found to be 0.31, 0.27, 0.29 and 0. 14% respectively in the swim-up motile fraction, and 0.31, 0.33, 0. 32 and 0.19% respectively in the pellet fraction. Analysing the zona-bound spermatozoa, the disomy rates for chromosome 18, X, Y and XY were found to be 0.02, 0.15, 0.12 and 0.07% respectively. The zona-bound spermatozoa had a significantly lower frequency of aneuploidy than the swim-up motile fraction or the pellet fraction (P < 0.0001). The incidence of chromosome 18 aneuploidy, including both chromosome 18 disomy and nullisomy, in the swim-up motile fractions was significantly increased in patients with an abnormal or borderline hemizona index compared with those with a normal hemizona index (P < 0.05). We also found that a high incidence of sperm aneuploidy was associated to a certain extent with low fertilization rate, and with failure to achieve pregnancy through ICSI. This study suggests that the human zona pellucida has the capacity to select against aneuploid spermatozoa by an as yet undetermined mechanism.

Purpose: To determine whether embryos resulting fromoocytes matured in vitro have a higher incidence of nuclearand/or genetic abnormalities compared to embryos resultingfrom oocytes matured in vivo.

Purpose: Our purpose was to examine the timing ofimplantation and early embryo development following uterinetransfer of oocytes/embryos previously subjected to zonapellucida micromanipulation. Methods: A total of 68 singleton pregnancies resulting fromIVF and embryo transfer with/without micromanipulation.Patients were divided into four groups according to the typeof micromanipulation technique: assisted hatching, embryobiopsy, intracytoplasmic sperm injection, and nomicromanipulation (control group). Serial serum β-hCG levels weremeasured between 10 and 25 days after fertilization andlog-transformed. Linear regression analyses were performedand extrapolated to hCG = 10 mIU/ml (hCG10) to estimatedetectable implantation. The slopes of the regression lineswere used to estimate the rising speed of hCG, an indirectsign of embryo development. Results: There were no significant differences among groupswith respect to hCG10, the slopes or intercepts of theregression lines. Conclusions: Various oocyte/embryo microsurgicalprocedures used in ART involving zona pellucida manipulationdo not appear to affect the timing of implantation or earlyembryo development.

The objective of this study was to investigate the role of the spermatozoon (paternal effects) on implantation and pregnancy outcome in in-vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). Male individuals of three types were analysed: infertile men with oligoasthenoteratozoospermia (OAT), infertile men with normozoospermia and fertile men (donors). Female counterparts were judged to have comparable egg quality within two groups studied, i.e. infertile women with pure mechanical (tubal) infertility and recipients of donor eggs. There were significantly higher differences in implantation and pregnancy rates in groups using donor spermatozoa and donor egg recipients. Analyses of key set groups revealed a trend toward a poorer implantation and pregnancy outcome when comparing OAT versus normozoospermic patients within IVF, but not within ICSI treatments, in couples with tubal infertility. In couples who were recipients of donor eggs, no differences were observed between OAT patients treated by ICSI and normozoospermic patients treated with IVF. No significant differences were observed in miscarriage rates within any groups studied. In conclusion, the poorer results observed in OAT patients undergoing IVF may be secondary to spermatozoal effects due to a high insemination concentration. Overall, there does not seem to be a significant effect of severe male infertility (OAT) on implantation and pregnancy outcome. However, this does not preclude that specific sperm aberrations may exert a negative effect on embryogenesis and therefore on implantation potential following assisted or in-vivo reproduction.

The objective of this study was to determine if assisted hatching improved the rates of implantation, clinical pregnancy and ongoing pregnancy for in-vitro fertilization (IVF) patients aged > or =36 years. On the day of oocyte aspiration, consenting patients were randomized according to whether all embryos underwent the hatching procedure (hatched; n = 41) or all embryos remained unhatched (controls; n = 48). Patients in both groups were treated with methylprednisolone and doxycycline starting on the day of oocyte retrieval and continuing for 4 days. The hatching procedure was performed approximately 55 h after insemination on all potential embryos for transfer and employed the release of acidified acid Tyrode's medium against the zona pellucida to create an opening approximately 20 microm in diameter. No significant differences were noted in the mean age, number of oocytes aspirated and number of embryos transferred between the hatched and control groups. In addition, no significant differences were observed in the rates of implantation (11.1 versus 11.3%), clinical pregnancy (39.0 versus 41.7%) and ongoing pregnancy (29.3 versus 35.4%) between the hatched and control groups respectively. These results suggest that assisted hatching may have no significant impact on IVF success rates in the patient population studied.

Purpose: The objectives of this study were (1) to investigate intracytoplasmic sperm injection (ICSI) outcome according to its indications, i.e., a history of failed or poor fertilization and unsuitable sperm parameters for conventional IVF, and (2) to examine the impact of a female's age, sperm concentration, motility, morphology, presence of antisperm antibodies, and hemizona assay (HZA) results on overall outcome. Methods: Two hundred seventy-nine ICSI cycles performed in 207 couples were retrospectively evaluated. Results: Clinical pregnancy and delivery rates were 36.8 and 29.8% for patients with prior failed fertilization, 23.2 and 17.8% for patients who had prior poor fertilization, and 28.6 and 21.3% for patients with unsuitable sperm parameters. The differences among all groups were found to be insignificant. There was a significant, negative correlation between a female's age and pregnancy results. No difference was found in the three basic sperm parameters between those patients who produced and those who did not produce a pregnancy, but the fertilization rate was significantly higher in patients with more adequate sperm parameters. Although there was a trend toward a better fertilization rate in patients with a hemizona index (HZI) greater than 30 (indicative of a superior sperm-zona pellucida binding capacity) than in those with a HZI less than 30, the difference was not significant. There were no differences in fertilization rate according to the presence or absence of antisperm antibodies. Conclusions: Fertilization history in a conventional IVF cycle has no effect on success rates following ICSI, and there is no correlation among the basic sperm parameters, the presence of antisperm antibodies, and pregnancy rates.

Purpose:Our purpose was (1) to determine if in vitro maturation of unstimulated oocytes could be improved with the addition of urofollitropin; (2) to evaluate the output of estradiol, testosterone, progesterone, and androstenedione by the cultured oocyte-cumulus complex; and (3) to ascertain if steroid hormone production of the oocyte-cumulus complex correlates with final oocyte maturation stage. Methods:Fifty-eight immature oocytes were obtained from 11 regularly cycling women undergoing oophorectomy. The oocyte-cumulus complexes were randomly assigned to control medium (Ham’s F-10 supplemented with 7.5% fetal bovine serum) or test medium (control medium supplemented with 75 mIU/ml of urofollitropin). Results:(1) The addition of urofollitropin to oocyte culture medium does not significantly increase the ability of the oocyte to achieve the metaphase II stage; (2) the addition of urofollitropin significantly increases the production of progesterone, testosterone, and androstenedione by the oocyte-cumulus complex; and (3) there is no difference in the production of estradiol, progesterone, testosterone, and androstenedione by the oocyte-cumulus complex at the germinal vesicle, metaphase I or metaphase II stage of oocyte maturation. Conclusions:This information is of importance in the use of oophorectomy specimens for patients who must undergo an oophorectomy but desire to attempt pregnancy using their oocytes, in the use of oophorectomy specimens for donor oocytes, or for patients undergoing in vitro fertilization using immature oocyte collection.

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.

Spermatozoa mature during epididymal transit, acquiring the abilities to swim progressively, fertilize oocytes, and produce viable offspring. In this study, we investigate the capacity of spermatozoa retrieved from the midcorpus and distal cauda regions of the epididymis of the cynomolgus monkey to penetrate homologous zona pellucida. Successful in vitro fertilization by ejaculated macaque sperm is dependent upon the addition of caffeine and dbcAMP. Therefore, the effect of these cyclic nucleotide mediators was also examined in this study. Results of sperm motion analysis indicate no difference in baseline values (without stimulators) for any motion parameter. With the addition of caffeine and dbcAMP, curvilinear velocity significantly increased only for the distal cauda sperm (P = 0.05). Amplitude of the lateral head displacement was significantly increased for distal cauda sperm (P < 0.01); although elevated above baseline, the increase observed after activation by corpus sperm was significantly lower than that achieved by cauda sperm (P < 0.05). The addition of caffeine and dbcAMP was an absolute requirement for zona penetration by both midcorpus and distal cauda sperm. With activation, zona penetration was significantly decreased for corpus sperm compared to cauda sperm (P < 0.001). These results suggest that cynomolgus monkey sperm reaching the midcorpus region of the epididymis have not completed all of the maturational changes requisite for successful fertilization; this immaturity is evidenced by decreased sperm motion and by impedance at the level of zona penetration.

Objective To determine if the developmental potential of embryos resulting from in vivo-and in vitro-matured monkey oocytes could be increased through the use of a coculture system. Design Randomized prospective comparison of embryos resulting from either in vitro- or in vivo-matured oocytes cocultured with Vero cells or cultured in medium alone (control). Setting Basic research laboratory. Main Outcome Measures In vitro embryo development to the blastocyst stage and blastocyst hatching. Results No significant difference in development was noted between coculture and control groups with embryos resulting from in vivo-matured oocytes. However, coculture was found to improve significantly the development of monkey embryos resulting from in vitro-matured oocytes. Conclusions These results demonstrate that primate embryos resulting from in vitro-matured and in vitro-fertilized oocytes differ in their culture requirement when compared with embryos resulting from in vivo-matured oocytes.

In mice, expression of the transcription factor Oct-3 and the proto-oncogene c-mos is limited to germ cells, suggesting a specific role for these factors in gamete physiology and early embryonic development. We have studied the expression pattern of Oct-3 and c-mos in various reproductive as well as control tissues in the cynomolgus monkey, using reverse transcriptase polymerase chain reaction (RT-PCR) and Northern analysis. Analogously with the data from the mouse model, strong expression of Oct-3 and c-mos could be detected in monkey ovary and oocytes. Unexpectedly, strong expression of c-mos was demonstrable in the pituitary gland and the amount of mRNA expression in the pituitary was roughly equal to that found in the ovary. Of the tissues examined, the testicular expression of c-mos was the most intense. Weak signal for c-mos mRNA was also seen in hypothalamus and brain; however, all other tissue types examined were negative for c-mos expression. In addition to the oocytes, expression of Oct-3 mRNA was detected in the ovarian granulosa cells, fallopian tube, myometrium, cervix, breast, liver, adrenal gland, pituitary, hypothalamus, brain cortex, prostate, and in testis. Thus, in the cynomolgus monkey, Oct-3 is predominantly, but not specifically, expressed in reproductive tissues. In the female monkey reproductive organs, the expression of c-mos seems to be germ cell specific. Therefore, further characterization of c-mos and Oct-3 functions in primate reproductive physiology, especially in gametogenesis and early embryonic development, is highly warranted.

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.

Mammalian fertilization, whether it takes place within the female reproductive tract or within a laboratory dish, is comprised of many processes which must follow a specific sequence. The spermatozoon must bind to and pass through the zona pellucida, fuse with the oolemma and become incorporated into the cytoplasm of the oocyte. Fusion of the two gametes triggers oocyte activation, resulting in exocytosis of the cortical granules and completion of the second meiotic division of the oocyte. A block in one or more of these processes, due either to abnormalities in the spermatozoon or oocyte, may result in fertilization failure.

Little is known about the molecular mechanisms governing the development of human oocyte and pre-embryo. We characterized the expression pattern of c-mos, cyclin B1 and beta-actin mRNA in oocytes and granulosa cells from human and monkey, and in human early embryos, using both qualitative and semi-quantitative reverse transcriptase polymerase chain reaction. The proto-oncogene c-mos was expressed in an oocyte-specific manner and no mRNA for c-mos could be detected in the granulosa cells. Similarly, strong expression of cyclin-B1 was seen in the oocytes. In human pre-embryos, the expression of cyclin-B1 and beta-actin increased from the 6-cell stage onwards, indicating active transcription and thus activation of embryonic genome either at or before the 6-cell stage. The expression of c-mos was transient and very little c-mos mRNA could be detected in the human embryos beyond the 6-cell stage. Thus, both its time-specific and site-specific expression suggest meiosis-specific functions for the proto-oncogene c-mos in human oocytes. As judged by the disappearance of c-mos, the maternal pool of mRNA seems to be degraded towards the 6- to 8-cell stage. The transient expression of c-mos and high levels of cyclin-B1 mRNA suggest that mechanisms similar to those found in lower organisms govern the growth and development of the human oocyte and preimplantation embryo.

Little is known about the molecular mechanisms governing the development of human oocyte and pre-embryo. We characterized the expression pattern of c-mos, cyclin B1 and beta-actin mRNA in oocytes and granulosa cells from human and monkey, and in human early embryos, using both qualitative and semi-quantitative reverse transcriptase polymerase chain reaction. The proto-oncogene c-mos was expressed in an oocyte-specific manner and no mRNA for c-mos could be detected in the granulosa cells. Similarly, strong expression of cyclin-B1 was seen in the oocytes. In human pre-embryos, the expression of cyclin-B1 and beta-actin increased from the 6-cell stage onwards, indicating active transcription and thus activation of embryonic genome either at or before the 6-cell stage. The expression of c-mos was transient and very little c-mos mRNA could be detected in the human embryos beyond the 6-cell stage. Thus, both its time-specific and site-specific expression suggest meiosis-specific functions for the proto-oncogene c-mos in human oocytes. As judged by the disappearance of c-mos, the maternal pool of mRNA seems to be degraded towards the 6- to 8-cell stage. The transient expression of c-mos and high levels of cyclin-B1 mRNA suggest that mechanisms similar to those found in lower organisms govern the growth and development of the human oocyte and preimplantation embryo.

The objective of these studies was to evaluate the modulatory effect(s) of progesterone on sperm functions crucial to fertilization in infertile men with abnormal sperm parameters. A prospective, controlled study applying a sequential diagnostic analysis capable of identifying specific dysfunctions of the male gamete was performed. Patients (n = 14) were allocated to the study group if they had a history of infertility of > 1 year duration and after semen evaluation showed teratozoospermia (< 14% normal sperm forms as diagnosed by strict criteria) or terato-asthenozoospermia (< 50% progressive motility). After swim-up separation of the motile sperm fraction, the following functions were assessed with and without previous exposure to progesterone (1.0 micrograms/ml): acrosome reaction (using Pisum sativum agglutinin), hyperactivated motility (using a computerized semen analyser), sperm-zona pellucida binding (in the hemizona assay), sperm-zona pellucida penetration (in a sperm-zona penetration assay), and sperm-oocyte penetration (using the hamster zona-free oocyte/sperm penetration assay). Progesterone did not affect the percentage of acrosome-reacted spermatozoa after 1 or 3 h of incubation. Hyperactivated motility was significantly enhanced by progesterone after 1 h (12 +/- 4 versus 6 +/- 2% in controls; P < 0.02). Although progesterone did not affect sperm-zona binding, it significantly enhanced both sperm-zona pellucida penetration (27 versus 12% in controls; P = 0.03) and sperm-oocyte penetration (15 versus 8% in controls; P < 0.05). Because those sperm functions enhanced by progesterone are crucial to fertilization, the steroid may have value in the treatment of some male-factor patients undergoing assisted reproductive therapy.

Our purpose was to utilize current reproductive technologies to treat an infertile female lowland gorilla.Following pituitary down-regulation with a gonadotropin releasing hormone agonist and follicular stimulation with human menopausal gonadotropins, transrectal ultrasound-guided aspiration of ovarian follicles yielded nine oocytes. Following failed fertilization with both epididymal and electroejaculated sperm, zona dissection and sperm injection were performed and produced one embryo, which was cryopreserved. Immature oocytes were obtained from ovarian sections and were either cultured in vitro or cryopreserved.This report demonstrates that in vitro fertilization techniques, including pituitary suppression, ultrasound-guided aspiration, micromanipulation, and cryopreservation, can be used to assist reproduction in the lowland gorilla.

The hemizona assay is a diagnostic test used to evaluate the binding potential of spermatozoa to zonae pellucida and has been used to predict fertilization potential in the human. In this study, frozen-thawed gorilla spermatozoa were coincubated with human hemizonae to evaluate tight binding and to assess the use of human zonae in evaluating sperm fertility. Matching hemizonae were incubated with human sperm to serve as a control. For evaluation of binding studies in a homologous system, matching halves of gorilla hemizonae were coincubated with both gorilla and human sperm. Whole, intact zonae of both human and gorilla oocytes were also coincubated with heterologous sperm to determine if penetration into the perivitelline space could occur. This study found that gorilla sperm bound well to both gorilla and human hemizonae, with a mean of 112.5 and 81.0 tightly bound sperm, respectively. Human sperm also bound to gorilla (mean 229.5) and human (mean 236.5) hemizonae. Following incubation with intact gorilla zonae, motile human sperm were found within the perivitelline space. However, gorilla sperm were not visible within the perivitelline space of nonviable human oocytes. These findings demonstrate that the zonae of nonviable human oocytes can be used to assess sperm binding of gorilla sperm. Studies continue for optimizing assay condition and correlation of findings with the fertility potential of gorilla sperm.

The purpose of this study was to evaluate the in vitro fertilizabilily of rhesus monkey oocytes and the developmental capacity of the resulting embryos as they relate to oocyte maturation at the time of follicular aspiration. Animals were hyperstimulated with human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH), with follicular aspiration performed 27 Ii after administration of an ovulatory stimulus (1000 IU human chorionic gonadotropin (hCG] or 3 x 100 p.g gonadotropin-releasing hormone (GnRHJ). In 7 animals exhibiting a continuously rising pattern of serum estradiol through Day 10 of hyperstiniulation, 45 germinal vesicle-intact (GV), 106 metapha.ce I (MI), and 24 metaphase ii (MU) oocytes were collected and cultured in vitro. Upon reaching Ml!, oocytes were inseminated with 5 x 10� motile spermlml. Twenty-four percent of GV oocytes cultured in vitro matured to MI! with 11 inseminated and none fertilized. Seventy-three percent of MI oocytes matured toM!! in vitro with 50% inseminated and 32% fertilized. Oocytes collected at MU stage and inseminated underwent fertilization at a high rate of efficiency (93%). Pronuclear to 8-cell stage embryos were frozen and, upon thawing, 67% (10/15) survived with all blastomeres intact. Frozen-thawed embryos (2- to 6-cell) were transferred to the oviducts of 4 recipients (2 embryos/recipient) during the early lutealphase(1 - 3 days postLH surge) of natural menstrual cycles. Three twin pregnancies resulted. Thus, a positive correlation exists between the degree of nuclear maturation of rhesus monkey oocytes at collection and their potential for fertilization in vitro. In addition, those oocytes that fertilize are capable of early development in vitro, before and after cryopreservation, with continued embryogenesis, efficient implantation, and successful gestation in surrogate mothers.

Objective To determine if the size of human oocytes at collection from unstimulated ovaries is related to their ability to resume meiosis and undergo maturation in vitro. Design A comparative study of oocyte diameter at collection. Setting Department of Obstetrics and Gynecology, Northwestern University Medical School. Patients Women age 25 to 39 years of age undergoing gynecological procedures yielding oophorectomy specimens. Intervention Oocytes obtained from ovarian tissue were cultured in Ham's F-10 and fetal bovine serum for 72 hours and observed two times per day. Main Outcome Measure The oocytes ability to resume meiosis and complete maturation based on their diameter at collection. Results Chi-squared analysis revealed a significant difference in oocytes measuring 86 to 105 μ m versus those measuring 106 to 125 μ m. Conclusion The unstimulated human oocyte appears to have a size-dependent ability to resume meiosis and complete maturation.

Telomerase, a ribonucleoprotein, has been described as an essential component of highly proliferative cells as it stabilizes the telomeres and avoids cellular senescence. The objective of this study was to modify the polymerase chain reaction-based telomeric repeat amplification protocol to detect telomerase activity in the single cell and to characterize the activity expressed in the human oocyte through to the blastocyst stage embryo. A comparative evaluation of telomerase activity and developmental stage was conducted using discarded or donated human oocytes and embryos. Telomerase activity was detected in all developmental stages evaluated from immature oocytes through to blastocyst stage embryos. Immature oocytes and blastocysts had similar levels of telomerase activity; however, both groups had significantly (P < 0.05) higher activity than zygote through to pre-morula stage embryos. Seventy-five thawed zygotes were cultured to day 3, biopsied by removing 1-2 cells, and the biopsied embryos were cultured to blastocyst stage. There was no difference (P < 0.05) in telomerase activity between cells biopsied from embryos that reached the blastocyst stage and cells from those that arrested in growth. This study has shown that human oocytes through to blastocyst stage embryos express telomerase activity, but that the level of telomerase activity in biopsied blastomeres, of the day 3 cleavage stage embryo, is not predictive of embryonic growth potential.

Objective: The aims of this study were to compare preimplantation embryo quality in intracytoplasmic sperm injection (ICSI) with standard IVF and to examine the impact of age and number and quality of embryos transferred on implantation and pregnancy. Design: Retrospective, controlled clinical study. Setting: Academic tertiary center. Patient(s): We examined 211 consecutive couples undergoing ICSI who were matched with 211 couples undergoing IVF therapy during the same time frame. Intervention(s): In vitro embryo culture. Main Outcome Measure(s): Day 3 embryo quality as judged by the number of blastomeres and morphology scoring. Result(s): Patients undergoing ICSI had a significantly reduced number of embryos with good morphology and cleavage compared with IVF cases. Nevertheless, pregnancy and abortion rates were similar when adjusted by age and number of embryos transferred. Average cleavage status and age were significant predictors of implantation. Women of advanced age had significantly lower embryo cleavage and implantation rates. Conclusion(s): [1] The cleaving status of day 3 embryos is a valuable, although limited, indicator of implantation outcome. [2] In vitro fertilization-derived embryos had better cleavage rates and morphology scores than ICSI-derived embryos; however, the implantation potential was similar for both groups. [3] The age-related decline in implantation rate was associated with impaired embryo growth rates.

Objective: To determine if frozen-thawed donor oocytes could be used to provide cytoplasm for transfer into patients' oocytes to improve subsequent embryonic development. Design: Prospective evaluation of the procedure in consenting IVF patients. Setting: Assisted reproductive technology program. Patient(s): The study was open to consenting IVF patients (of any age) with a history of poor embryo quality or those couples in which the wife's age was ≥40 years. Intervention(s): Transfer of donor egg cytoplasm from frozen-thawed oocytes into the oocytes of infertile recipients. Main Outcome Measure(s): Donor oocyte survival following cryopreservation, fertilization following cytoplasmic transfer into recipient oocytes, embryo quality, and pregnancy outcome. Result(s): Oocytes collected from four donors were cryopreserved and 61% (28/46) survived the thaw procedure. Cytoplasmic transfer was performed on the eggs of four patients, with fertilization occurring in 70.3% (26/37). Twin pregnancy was established in one patient (35 years of age) with a history of poor embryo quality. Conclusion(s): Cryopreserved donor oocytes may provide a source of cytoplasm for transfer into recipient oocytes, eliminating the need for cycle synchronization between donor and infertile patient.

In the human, intracytoplasmic sperm injection is typically performed using "viable" sperm which has been mechanically rendered nonmotile. The purpose of the present study was to determine the ability of nonviable sperm to fertilize human oocytes and the early developmental normalcy of the resulting embryos.In this study, immature, prophase I oocytes from a total of 27 consenting patients were matured in vitro and then randomized into two groups: injection with a viable human sperm or injection with a sperm rendered nonviable by freeze-thawing in liquid nitrogen. The rates of fertilization and cleavage were compared between the two groups.The results demonstrated a significantly higher two-pronuclear fertilization rate when oocytes were injected with viable sperm (62.2%) compared to when oocytes were injected with nonviable sperm (16.2%). Oocytes injected with viable sperm also demonstrated a higher cleavage rate (91 vs 33%).These findings suggest that while the intracytoplasmic injection of nonviable human sperm can result in normal fertilization, it does so at a much reduced rate compared to viable sperm and may not result in normally cleaving embryos.

Purpose: The purpose was to evaluate methods of DNA preparation in a single cell to determine the ability to amplify and correctly diagnose a targeted gene. Methods: One- or two-cell lymphoblasts (n=100/group), heterozygous for the normal and 4-base pair insertion on exon 11 of the β-hexosaminidase A gene, were collected and prepared under the following conditions: (1) freeze-thaw liquid nitrogen, then boiling (LN2); (2) potassium hydroxide/dithiothreitol, heated to 65°C, followed by acid neutralization (KOH); (3) boiling only (BI); and (4) water only (H2O). Cells were analyzed by polymerase chain reaction using nested primers. Results: The total number of cells amplifying [in brackets] and the cells with amplification for both alleles (heterozygous), the normal allele, or the mutant allele were as follows, respectively: LN2 [38], 11, 16, 11; KOH [97], 91, 5, 1; BI [41], 17, 13, 11; and H2O [85], 41, 16, 28. With two cells per reaction tube the results were as follows: LN2 [85], 53, 14, 18; and KOH [97], 96, 1, 0. Conclusions: KOH lysis was significantly greater than with all other methods (P

Objective To determine whether intracytoplasmic sperm injection has an adverse effect on the meiotic spindle of oocytes after injection of a sperm into the ooplasm. Design Hamster oocytes were injected with human sperm (Test group) and evaluated for meiotic spindle and chromosome morphology using immunofluorescent staining. Results were compared with control uninjected oocytes exposed to the microscope environment (CS group) and untreated oocytes remaining in the incubator (CI group). Setting Basic research center at a medical school. Results No significant differences were noted in spindle appearance and chromosome alignment between Test (13 abnormal/68 normal) and both control groups (CS 9/73 and CI 12/71; P = 0.602). Conclusion Our results demonstrate that injection of human sperm into the cytoplasm of hamster oocytes may not result in a significant increase in damage to the meiotic spindle provided care is taken to orient the polar body away from the site of injection.

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.

OBJECTIVES: Our purpose was to identify and evaluate practical methods within a preimplantation genetic diagnosis program that will increase the percentage of embryos for which a genetic diagnosis can be obtained, including clinical responses after failure of deoxyribonucleic acid implantation has occurred. STUDY DESIGN: Known human lymphoblast cell lines and human embryo blastomeres were evaluated in a single-cell, nested primer polymerase chain reaction system with primer sequences for the specific locus surrounding the four base pair insertion mutation on exon 11 of β-hexoaminidase A—Tay-Sachs disease, the ΔF508 mutation of cystic fibrosis, and the sex-determining region on the Y chromosome. Reamplification polymerase chain reaction with standard polymerase chain reaction and primer extension preamplification was performed in deoxyribonucleic acid preparations after previous polymerase chain reaction amplification attempts had resulted in failure of amplification. RESULTS: The amplification efficiency of Tay-Sache disease, 51% (97187), was significantly lower than that for cystic fibrosis, 85% (87107), and for the sex-determining region on the Y chromosome, 85% (7790). Tay-Sache disease polymerase chain reaction amplification occurred in 51% of one-cell lymphoblasts, 89% of two-cell lymphoblasts, and 94% of samples when more than two cells were processed together. When previous amplification failure had occurred, standard Tay-Sachs disease polymerase chain reaction resulted in an amplification efficiency of 16% (three of 19), whereas primer extension preamplification polymerase chain reaction for Tay-Sachs disease resulted in amplification of 52% (3159) lymphoblasts and 54% (1324) of polyspermic human blastomers. Four of six human blastomeres in which amplification failure occurred in a Tay-Sachs disease preimplantation genetic diagnosis cycle amplified by primer extension preamplification polymerase chain reaction, which increased the diagnostic information obtained from four to six of the seven embryos on which biopsy was performed. CONCLUSIONS: We suggest that practical approaches for consideration within a clinical preimplantation genetic diagnosis program to limit the net effect of amplification failure (i.e., reduced embryo transfer number) include increasing the deoxyribonucleic acid content in the polymerase chain reaction tube by using more than one blastomere and by using primer extension preamplification when the initial attempt at amplification fails.

Objective To determine the ability to apply preimplantation genetic diagnostic techniques to screen for and prevent Tay-Sachs disease (TSD). Design A couple, both carriers for the 4 base pair (bp) insertion in exon 11 of the β-hexosaminidase A gene, which results in TSD, underwent IVF, pre-embryo biopsy, polymerase chain reaction (PCR) DNA amplification of the biopsied blastomeres, and pre-embryo transfer. One to two blastomeres were aspirated using a biopsy pipette that was inserted through an opening in the zona formed with acidified phosphate buffer. Polymerase chain reaction was performed on the individual blastomeres for 20 cycles followed by an additional 30 cycles using nested primers. This yielded amplified DNA products of 272 and 276 bp for the normal and mutant gene, respectively. Heteroduplex formation was used for identification of normal, homozygous affected, and heterozygous pre-embryos. Results Seven of 13 oocytes fertilized normally and were biopsied at the four- to eight-cell stages. Deoxyribonucleic acid amplification occurred in four of seven pre-embryos (one homozygous affected and three homozygous normal pre-embryos). The three normal pre-embryos that continued to cleave after biopsy were transferred on the evening of day 3 after retrieval. Subsequently, a single gestational sac was observed and the genetic diagnosis was confirmed at amniocentesis. Conclusion A successful pregnancy and birth were accomplished after preimplantation genetic diagnostic screening for the prevention of TSD.

The predictive value of 2 tests for ovarian response to controlled ovarian hyperstimulation in the cynomolgus monkey model was evaluated. The tests utilized were: 1) the cycle Day 3 (Day 1 = onset of menses) FSH value and 2) the acute estradiol (E(2)) response to a GnRH agonists (GnRHa) administered on Day 3. Both tests were performed during the cycle preceding control ovarian hyperstimulation. Subsequently, monkeys (n = 26) were stimulated with Metrodin(T) (Days 2-6, 25IU/d) and Pergonal(T) (Day 7 to hCG administration, 25IU/d). Laparoscopic oocyte retrieval was performed 32 to 34 after hCG administration. Analysis of the data revealed that Day 3 FSH values could not predict whether an animal would respond well to control ovarian hyperstimulation in a subsequent cycle (P = 0.77). However, the E(2) change 24 h post-GnRHa administration was significantly greater for animals responding well to control ovarian hyperstimulation compared with the animals deleted after 6 d of stimulation (P = 0.042). The mean change in E(2) levels in animals taken to aspiration was 97.8 pg/ml compared with only 21.6 pg/ml for the deleted animals. This differential response of E(2) production after GnRHa treatment was used to correctly identify (by discriminant analysis) 78% of the animals subsequently deleted for poor response. Thus, the increase in serum E(2) level after GnRHa, but not the basal FSH level, was found to be predictive of ovarian response to stimulation in the cynomolgus monkey.

Objective o 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.

PROBLEM: It has been reported that massive amounts of immunoreactive endothelins (ETS) exist in human seminal plasma. However, the physiological role of ETS in seminal plasma remains to be determined. We speculated that ETS might affect sperm function. METHOD: The present study was designed to investigate the effect of endothelium-1 (ET-1) on: (a) sperm motion parameters, (b) hyperactivated motility, (c) sperm-zona pellucida-binding capacity, (d) sperm-oocyte penetration capacity, (e) acrosome reaction and its prerequisite, an increase in intracellular Ca2+ concentration ([Ca2+]i), and to examine (f) the presence of binding sites for ET-1 in human sperm. Forty-six semen ejaculates from 14 fertile men were assessed under capacitating conditions after separation of the motile sperm fraction by wash and swim-up. RESULTS: ET-1 (1 (μm) exhibited significant stimulatory effects on sperm velocity at 30 min (P = 0.01), amplitude of lateral head displacement (ALH)max (P = 0.05), and ALHmean at 60 min (P = 0.04) in some samples (n = 10). However, these effects were not observed in experiments using a larger number of samples (n = 39). ET-1 had no effect on hyperactivated motility of sperm at 30 min to 24 h. Neither ET-3 nor IRL 1620, a selective ETB receptor agonist, affected sperm motion parameters or hyperactivated motility. ET-1 did not affect sperm-zona-binding capacity, sperm-oocyte penetration capacity, acrosome reaction, or [Ca2+]i of sperm. Specific binding sites for ET-1 were not detected on the cell surface of human sperm. CONCLUSIONS: Although ET-1 is present in massive amounts in human seminal plasma and may have the capacity to alter the quality of motile sperm in some samples, a physiological role of ET-1 in the modulation of the function of mature, ejaculated sperm still remains unknown.

To evaluate, using the hemizona assay (HZA), whether egg yolk treatment of human sperm enhances binding to the human zona pellucida in vitro and to determine whether such a treatment is as efficient as the standard swim-up procedure for promoting sperm binding ability.Ejaculates were divided into aliquots and half incubated at 37 degrees C for 21 hours in standard culture medium or combined with buffered medium containing chicken egg yolk and stored at 4 degrees C for 21 hours. A second set of ejaculates were split and processed by a standard 1-hour rise alone or treated with egg yolk medium.University teaching hospital.Two healthy sperm donors.The number of sperm tightly bound to the hemizona were measured and compared between the groups.A significant increase (P less than 0.0001) in hemizona binding (n = 46) for egg yolk treatment (90.1 +/- 9.8; range 7 to 258) as compared with standard culture medium (53.0 +/- 8.8; range 0 to 228) was observed. Similarly, a significant increase (P less than 0.0001) in binding (n = 37) for egg yolk treatment (74.9 +/- 8.2; range 7 to 219) as compared with samples obtained with a sperm rise (37.1 +/- 5.7; range 2 to 122).Treatment of human spermatozoa with an egg yolk medium at 4 degrees C overnight significantly increases sperm binding in the HZA.

This study was performed to evaluate the effects of mechanical stimulation and sucrose treatment on the oocyte activation process. Fresh and aged human oocytes were exposed to sucrose and zonae were dissected with microneedles before fixation and quantitative analysis of cortical granules by transmission electron microscopy. Examination of the mean number of cortical granules/analyzed segment revealed no significant differences between control oocytes or oocytes treated with sucrose or sucrose treatment followed by zona dissection. A significant decline in the number of cortical granules/segment was observed for oocytes undergoing prolonged culture after dissection (P less than 0.05). Thus, zona dissection and sucrose exposure of freshly aspirated mature human oocytes do not result in classical oocyte activation.

Clinical IVF-ET was initiated with the birth of baby Louise Brown in 1978 following several years of pioneering effort by Patrick Steptoe and Robert Edwards. In the ensuing 12 years, human IVFET has evolved into standard medical practice for the treatment of many types of infertility, and the service is now offered by some 700 clinics in 48 countries. The application, confined originally to the hopelessly infertile patient with irreparably damaged or absent fallopian tubes, has expanded to include idiopathic, male, endometriosis, cervical, and immunologic etiologies. About 15% of egg retrieval cycles end in pregnancy and live birth resulting from the transfer of, on average, three embryos. Over 15,000 babies have been born by IVF-ET, which means that the experience base in man exceeds that in all other mammalian species with the possible exception of the mouse. A summary of the IVF experience in the United States for 1987 has appeared (Medical Research International et al., 1989). Several peer-reviewed journals now devote major attention to the clinical and applied aspects of IVF—Fertility and Sterility, Human Reproduction, and Journal of In Vitro Fertilization and Embryo Transfer.