Extracorporeal maturation: Norfolk, 1984.

More than one-half of the laparoscopies undertaken for oocyte retrieval in a program of in vitro fertilization yielded immature (unripened) oocytes. Seventy-three percent of these still possessed an intact germinal vesicle at aspiration. During the period from September 1981 to March 1984, in vitro maturation techniques were applied to the handling of these immature oocytes, with a resulting extracorporeal maturational success reaching 82-85%. The monitoring of germinal vesicle breakdown was essential to determining the fate of the oocytes. No oocytes were successfully fertilized that had normal dipronuclear development unless first polar body extrusion was observed prior to insemination. However, once matured in vitro, these oocytes were fertilized at rates nearly equal to that of mature or preovulatory oocytes. When interval periods of 29 hours between follicular aspiration and oocyte insemination were constant, the incidence of polyspermic fertilization was higher in oocytes that did not possess a germinal vesicle at aspiration compared to that in oocytes with a germinal vesicle. The method of follicular stimulation for ovulation induction was examined to determine whether differences in developmental potential existed between cycles stimulated with hMG alone, FSH alone, or a combination of FSH/hMG. Although fertilization rates were somewhat lower in the combination group, this may have been due to the small number of oocytes involved in that sample. A 50% increase in the number of immature oocytes harvested from FSH cycles was noted. The increasingly higher number of immature oocytes collected for in vitro fertilization underscores the importance of developing reliable in vitro maturation techniques.

ICSI and its outcome.

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

New method of evaluating sperm morphology with predictive value for human in vitro fertilization.

A prospective study was planned to evaluate sperm morphology as a parameter to predict the fertilization outcome in an in vitro fertilization program. Couples applying to in vitro fertilization were admitted into this project when the sperm concentration was greater than 20 million per mL and motility greater than 30 per cent. Based on new strict criteria for evaluating normal sperm morphology, patients were divided prospectively into 2 groups. In group I (25 patients) normal sperm morphology was less than 14 per cent, and in group II (71 patients) normal sperm morphology was greater than 14 per cent, using a threshold established previously. Multiple regression analysis was used to evaluate different parameters: concentration, motility, and morphology against the dependent variables, fertilization, and cleavage. The only factor which was significantly correlated with fertilization and cleavage was normal sperm morphology (P less than 0.0001). The fertilization rate (per oocyte) and the cleavage rate were 49.4 per cent and 47.6 per cent in group I and 88.3 per cent and 87 per cent in group II (P less than 0.0001). The ongoing pregnancy rate per laparoscopy and per embryo transfer was 4 per cent and 5.5 per cent, respectively, in group I and 18.3 per cent and 18.5 per cent, respectively, in group II (no significant difference). This study demonstrates the value of analyzing sperm morphology using the criteria recommended in terms of predicting fertilization and perhaps pregnancy outcome. Patients can be better counseled and the probability of fertilization or no fertilization can be more accurately established. Furthermore a trend is shown in the pregnancy rate that may indicate the importance of the male genome in establishing a pregnancy.

Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia.

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.

Correlation of follicular fluid volume with oocyte morphology from follicles stimulated by human menopausal gonadotropin.

The maturity of human oocytes was correlated with corresponding follicular fluid volumes in 547 cycles stimulated with human menopausal gonadotropin, with or without the supplementation of follicle-stimulating hormone. Mature oocytes were found to be associated with larger follicles (average volume, 2.7 ml). Immature or degenerating oocytes were found to be associated with smaller follicles (average volume, 1.0 and 0.8 ml, respectively). Follicles without oocytes were generally quite small (average volume, 1.0 ml). We studied follicular fluid volumes associated with mature oocytes that were responsible for the establishment of pregnancy after single conceptus transfer. Analysis of these data demonstrated that the rate of spontaneous abortion was very high with conceptuses derived from smaller follicles, suggesting that oocytes from larger follicles may be of better quality.

Basal follicle-stimulating hormone level and age affect the chance for and outcome of pre-embryo cryopreservation.

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.

Vital initiation of pregnancy (VIP) using human menopausal gonadotropin and human chorionic gonadotropin ovulation induction: Phase I--1981.

Laparoscopies for oocyte aspiration in 31 cycles were performed on 25 patients receiving human menopausal gonadotropin and human chorionic gonadotropin. Sixty oocytes were aspirated, of which 48 were considered preovulatory. Ninety-seven percent (58 of 60) of the oocytes were found in the original aspirate, and the remaining oocytes were found in either the first or second follicle wash. The fertilization rate per preovulatory oocyte was 33% (16 of 48), whereas on a per cycle basis it was 39% (12 of 31). A total of 15 conceptuses (2-cell = 5; 3-cell = 3; 4-cell = 7) were transferred to 12 patients, and two pregnancies were established. These pregnancies were established by transfers of 3-cell and 4-cell conceptuses at approximately 47 hours after insemination. Both pregnancies resulted in term deliveries of normal infants.

Delayed fertilization during in vitro fertilization and embryo transfer cycles: analysis of causes and impact on overall results.

This study evaluated possible causes of delayed fertilization during in vitro fertilization (IVF) cycles, its repetitiveness, and its influence on IVF results in 23 patients (27 cycles) with delayed fertilization of greater than or equal to 1 preovulatory oocyte(s). In 15 cycles, reinsemination with husband's semen was performed at 18 hours. Possible causes of delayed fertilization were oocyte defects (10 cycles, 37.0%), sperm defects (4 cycles, 14.8%), oocyte and sperm defects (4 cycles, 14.8%), and no detectable gamete defects (9 cycles, 33.3%). Overall fertilization rate was 47.9%. No pregnancies were observed in 10 patients with one embryo transferred. Recurrence rate of delayed fertilization per patient was 17.3%; overall ongoing pregnancy rate/cycle was 10.3%. Although repetitiveness of delayed fertilization is low, it seems to impact negatively on IVF results.

A preclinical evaluation of pronuclear formation by microinjection of human spermatozoa into human oocytes.

In vitro fertilization (IVF) is recognized as an accepted treatment for male infertility. However, the fertilization rate is significantly lower than the fertilization rate of other IVF patient groups. Some male factor infertility patients still have a basic semen quality too poor for treatment by IVF. Microinjection of a spermatozoon directly into ooplasm has been recommended to assist fertilization in this subfertile population. This study found that oocytes from 5 of 11 patients microinjected with human spermatozoa demonstrated successful pronuclear formation and correlated with the incidence of pregnancy in these patients transferred with same-source oocytes inseminated by standard protocols. This initial evidence promotes the supposition of clinical feasibility of assisted fertilization by sperm microinjection.

Cryopreserved/thawed semen for in vitro fertilization: results from fertile donors and infertile patients.

We evaluated the in vitro fertilization (IVF) outcome in 54 cycles using cryopreserved/thawed semen from fertile donors. Controls were other IVF patients matched by time frame, female age, stimulation protocol, number of pre-embryos transferred, and absence of a male factor using freshly ejaculated normal semen samples. In the study group and controls, respectively, post-thaw swim-up motility was 83.1% and 89.5%; fertilization rate of preovulatory oocytes (91.8%, 95.7%) and ongoing pregnancy rate (PR) per transfer (21.1%, 25.0%) were similar. The excellent fertilization rate with frozen/thawed semen was achieved through high-concentration insemination (0.5 x 10(6) motile sperm/mL). With use of frozen/thawed samples from infertile men (normal and subfertile samples), PR was similar but fertilization rate was lower. Cryopreserved semen is a valuable option for infertile couples in IVF therapy.

Vital initiation of pregnancy (VIP) using human menopausal gonadotropin and human chorionic gonadotropin ovulation induction: phase II--1981.

In a program for in vitro fertilization, laparoscopies for oocyte aspiration were performed on 24 patients receiving human menopausal gonadotropin and human chorionic gonadotropin. Of the 40 preovulatory oocytes that were recovered from these patients, 33 (83%) were fertilized and 30 (75%) cleaved and were transferred. Ten immature oocytes were collected, and attempts were made to mature these in vitro prior to insemination. All ten oocytes (100%) did fertilize, and seven (70%) cleaved and were transferred. Morphologic variation was noted between cleaving conceptuses, even in those conceptuses responsible for establishing pregnancies. Five pregnancies resulted from 19 embryo transfers (26%).

Characteristics of consecutive in vitro fertilization cycles among patients treated with follicle-stimulating hormone (FSH) and human menopausal gonadotropin versus FSH alone.

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.

Predictive value of pregnancy during original in vitro fertilization cycle on implantation and pregnancy in subsequent cryothaw cycles.

OBJECTIVE: To understand the homogeneity of oocyte quality within a cohort. DESIGN: All women (n = 367) that had cryopreserved pre-embryos subsequently thawed were studied. Pregnancy and implantation rates in the in vitro fertilization (IVF) and cryothaw transfers were examined. SETTING: Tertiary care academic center. RESULTS: Pregnancy in original IVF cycle predicted higher implantation rates at subsequent cryothaw transfers. Similarly, pregnancy in cryothaw cycles was associated with higher implantation and ongoing pregnancy rates at the initial IVF cycle. CONCLUSIONS: Within a cohort of oocytes, pregnancy with some of the pre-embryos in a cohort predicts pregnancy with the remaining oocytes in the cohort. Thus, oocytes within a cohort have similar pregnancy potential.

The window of embryo transfer and the efficiency of human conception in vitro.

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

Embryo quality and pregnancy rates in patients attempting pregnancy through in vitro fertilization.

Patients at low risk for producing abnormal gametes produce a wide range of embryo qualities that correlate well with PRs. These data may be useful in counseling patients and in reviewing treatment and laboratory protocols.

Transfer of cryopreserved-thawed pre-embryos in a natural cycle or a programmed cycle with exogenous hormonal replacement yields similar pregnancy results.

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.

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

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

Low-dose glucocorticoids after in vitro fertilization and embryo transfer have no significant effect on pregnancy rate.

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.

Messenger ribonucleic acid kinetics in human oocytes--effects of in vitro culture and nuclear maturational status.

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.

High-dose follicle-stimulating hormone stimulation at the onset of the menstrual cycle does not improve the in vitro fertilization outcome in low-responder patients.

In an attempt to improve their outcome with in vitro fertilization (IVF), 34 low-responder patients were stimulated with six ampules of follicle-stimulating hormone (FSH) daily starting on day 1 (n = 17) or day 2 (n = 17) of their menstrual cycles. The stimulated cycles showed a mean peak estradiol of 443 +/- 173 pg/mL, mean days of human chorionic gonadotropin of 7.6 +/- 1.4, 2.67 +/- 1.5 preovulatory oocytes per retrieval, and 2.56 +/- 1.3 oocytes per transfer. Three clinical pregnancies resulted after 25 embryo transfer cycles (12%). With paired analysis, we compared 8 patient cycles with prior six ampules of FSH stimulation starting on day 3; all parameters examined showed no significant differences. In a comparison of 22 patient cycles with prior 4 ampules of FSH stimulation on cycle day 3, no significant differences in any parameters were observed except in the higher number of ampules used in the present study. We conclude that high-dose FSH stimulation at the onset of the menstrual cycle does not improve the IVF outcome in low-responder patients.