Human embryo fragmentation in vitro and its implications for pregnancy and implantation.

OBJECTIVE: To evaluate the effects of fragmentation and fragment removal in day 3 human embryos on implantation and pregnancy. DESIGN: Retrospective analysis of ETs homogeneous with respect to embryo fragmentation. SETTING: A program of IVF-ET. PATIENT(S): The study population consisted of 2,410 patients. INTERVENTION(S): The degree and pattern of fragmentation were evaluated on days 2 and 3; microsurgical fragment removal was performed after assisted hatching on day 3. MAIN OUTCOME MEASURE(S): Clinical pregnancy and implantation rates. RESULT(S): The degree and pattern of fragmentation significantly impact pregnancy and implantation. With the application of microsurgical fragment removal before ET, embryos with 6%-35% fragmentation implant with similar frequency. The presence of large fragments (type IV) is detrimental to the developing embryo, whereas localized or small and scattered fragments do not significantly affect implantation. CONCLUSION(S): The potential of fragmented embryos for implantation is determined partly by the distribution of fragments. Adoption of an embryo classification system reflecting types of fragmentation is advisable. The use of microsurgical fragment removal significantly alters the course of development for some embryos and improves their implanting potential.

Cryopreservation of single human spermatozoa.

A procedure is described that allows cryopreservation and efficient post-thaw recovery of either a single or a small group of human spermatozoa. This is achieved by injecting them into cell-free human, mouse or hamster zonae pellucidae before the addition of cryoprotectant. The method involves a combination of physical micromanipulation procedures and glycerol-mediated cryoprotection. Zonae were tracked by positioning them in straws between two small air bubbles prior to freezing. Spermatozoa from poor specimens were cryopreserved and their fertilizing ability after thawing was compared with that of fresh spermatozoa from fertile men. Human eggs used for fertilization testing were either 1 day old or in-vitro matured. Only 2% of the frozen zonae were lost and >75% of spermatozoa cryopreserved in this manner were recovered and prepared for intracytoplasmic sperm injection. The feasibility of cryopreserving a single spermatozoon was assessed. Fifteen motile spermatozoa were frozen in 15 zonae, of which 14 were recovered after thawing. Ten were injected into spare eggs, of which eight became fertilized. Spermatozoa recovered mechanically from human zonae fertilized the same proportion of oocytes as fresh fertile control spermatozoa. The recovery and fertilization rates with spermatozoa frozen in animal zonae were 87 and 78% respectively. The fertilization rate was marginally higher (P < 0.05) than that for spermatozoa frozen in human zonae, perhaps because the latter may have acrosome reacted more frequently. The zona pellucida appears to be an ideally suited sterile vehicle for storage of single spermatozoa.

Embryo quality and pregnancy potential of fresh compared with frozen embryos--is freezing detrimental to high quality embryos?

To determine the effect of cryopreservation on embryo quality and the pregnancy potential of embryos, donated oocytes from the same donor (n = 24) were randomly allocated, with subsequent transfer to two or more different ovum recipients resulting in at least one fresh and one frozen embryo transfer cycle from the same cohort of oocytes. Endometrial receptivity was controlled in all ovum recipients, and male factor patients were excluded. The number of embryos transferred, mean embryo grade transferred, number of high quality embryos (grade < or = 2.5, grade 1 being best) transferred and embryo implantation and live birth rates are reported. Significantly more embryos (4.4 +/- 1.2 versus 3.3 +/- 1.2, P < 0.00003) of higher quality (1.9 +/- 0.5 versus 2.1 +/- 0.5, P < 0.013) and of a more advanced cell stage (3.0 +/- 0.6 versus 2.6 +/- 0.7, P < 0.019) were transferred fresh than after cryopreservation respectively. Implantation rates/embryo [19/151 (12.6%) and 9/111 (8.1%)] and live birth rates/transfer [11/42 (26.2%) and 6/45 (13.3%)], from fresh and frozen transfers respectively, were not significantly different despite the larger number of high quality embryos transferred fresh. Embryo cryopreservation adversely affects embryo quality, but does not have detrimental effects on the implantation or pregnancy potential of high quality embryos. Because of the loss of embryos during freeze-thawing during frozen embryo cycles, every effort should be made to attempt a fresh transfer.

Age-related decline in female fertility is not due to diminished capacity of the uterus to sustain embryo implantation.

OBJECTIVE: To evaluate the contribution of the uterus to age-related reproductive failure in women. PATIENTS: Thirty-eight ovum donors (30.2 +/- 4.9 years [mean +/- SD]) donating oocytes throughout 102 ovum donations. Fifty-one cycles were documented in "younger" recipients (35.8 +/- 3.1 years) and 51 in "older" recipients (44.0 +/- 3.1 years). The study was prospectively designed; same-cohort oocytes obtained from one young donor during a specific cycle were evenly distributed between "young" and "old" ovum recipients. Use of oocytes from a single source and a unique ovulatory cohort provides strict control over oocyte quality. Uterine age is varied by design, according to the age of the recipient at the time of ET. The role of the aging uterus in the decline of female fertility can be thus isolated and scrutinized. RESULTS: No significant (NS) difference in the number of ova received (7.9 +/- 3.4 versus 7.0 +/- 3.5), ova fertilized (4.4 +/- 1.5 versus 4.5 +/- 2.3), or embryos transferred (4.1 +/- 1.5 versus 4.1 +/- 1.6) was observed between the < 40 and > or = 40 recipient age groups. A total of 23 pregnancies occurred among the 102 ETs (22.6%). Eleven clinical pregnancies (21.6%) resulting in 10 deliveries were observed in the < 40 recipient age group, and 12 clinical pregnancies (23.5%) leading to 10 deliveries occurred in the > or = 40 recipient age group (NS). The pregnancy loss rates were 9.1% (1 of 11) and 16.7% (2 of 12) for the two recipient age groups, respectively, (NS). CONCLUSION: The capacity to conceive and to gestate a conception to term when oocyte quality is controlled appears to be independent of uterine aging through the fifth decade of life.

Premature luteinization in controlled ovarian hyperstimulation has no adverse effect on oocyte and embryo quality.

OBJECTIVE: To determine if premature luteinization has an adverse effect on oocyte and, hence, embryo quality. DESIGN: Retrospective evaluation of anonymous ovum donors/oocyte recipients. SETTING: A large oocyte donation program. PATIENTS, PARTICIPANTS: Sixty-eight women undergoing controlled ovarian hyperstimulation (COH) as ovum donors were matched to 68 women with ovarian failure as ovum recipients who had endometrial maturation exogenously controlled by an identical hormone replacement protocol. INTERVENTIONS: Serum was collected for E2 and P in donors and recipients. MAIN OUTCOME MEASURES: The incidence of premature luteinization was determined in donors. Cycle characteristics were compared between donors with and without premature luteinization, with emphasis on oocyte and embryo quality. Implantation rates per embryo and delivery rates per transfer were measured in recipients. RESULTS: Twenty-one (31%) of the donors demonstrated premature luteinization. Serum P was higher on day before hCG, day of hCG, and day after hCG in women demonstrating premature luteinization. However, there were no differences between donor cycles with or without premature luteinization as determined by donor age, ampules of gonadotropins used, day of hCG administration, peak E2, total number of oocytes, and number of mature oocytes retrieved. Ovum recipients were of similar age and had similar E2 exposure (area under the E2 curve) before P administration. Similar fertilization rates, incidence of polyspermia, number of embryos transferred of similar embryo grade, and similar implantation rates and deliveries per transfer were observed in women receiving oocytes from donors with and without premature luteinization, respectively. CONCLUSIONS: Similar oocyte quality, fertilization, and polyspermia rates, embryo quality, implantation, and delivery rates suggest that any negative impact of premature luteinization on pregnancy rates in COH cycles from young women is not due to an adverse effect of PL on oocyte and hence embryo quality, but rather on the endometrial environment.

Analysis of factors contributing to success in a program of micromanipulation-assisted fertilization.

OBJECTIVE: To determine factors important to clinical success in micromanipulation-assisted in vitro fertilization (IVF). DESIGN: Procedures invoked in two separate series of micromanipulation-assisted IVF cycles, one unsuccessful (series I) and the other successful (series II), were compared in an effort to identify changes that led to clinical success. SETTING: University-based IVF clinic. PATIENTS: In both IVF series involving micromanipulation, patients consisted of infertile couples who fit any of five categories of male-factor related infertility. The female patients underwent controlled hyperstimulation for oocyte retrieval and the oocytes were inseminated normally or were subjected either to partial zona dissection or subzonal sperm insertion to assist fertilization. Results in all groups were compared between the two patient series. RESULTS: In the successful series II, a noticeable improvement in fertilization rate and embryo quality was observed compared with series I. A significant increase in the percentage of patients reaching embryo transfer, the pregnancy rate per transfer, and the pregnancy rate per retrieval were noted in series II; a 25% ongoing pregnancy rate per retrieval was observed overall in this successful group, with "ongoing" defined as manifestation of at least a fetal sac on ultrasound with no detectable problems. Patients with a mixed transfer of embryos derived from manipulated and normally inseminated oocytes had a 75% rate of pregnancy per transfer in series II. Differences between the two series could not be attributed to patient selection or biases in selection of oocytes relegated to micromanipulation. However, oocyte handling, micromanipulation, and culture protocols differed significantly between the two series in that temperature and pH of oocytes was better controlled, and micromanipulation time was minimized in series II. CONCLUSION: Success in micromanipulation depends on maintenance of the oocyte in a stable and supportive environment throughout the micromanipulation procedure. It is also important to minimize trauma to the eggs by performing micromanipulation rapidly and with minimal distortion of the egg. Patients with a poor fertilization rate in standard IVF may experience a substantial increase in the likelihood of pregnancy when micromanipulation-assisted fertilization is performed on some eggs.

Cryopreservation of semen, oocytes, and embryos.

Human embryo cryopreservation is widely applied by programs of assisted reproductive technology. However, recent surveys have shown that relatively few in vitro fertilization programs in the United States substantially increase their per retrieval delivery rates through cryopreservation. While embryos may be successfully frozen and thawed at a range of developmental stages using several different cryoprotectants, the majority of in vitro fertilization programs use 1,2 propanediol to freeze early (pronuclear four-cell) embryos. Ultrarapid freezing techniques are under continued study; they have not yet been shown to offer any clinical advantage over slow freezing protocols. Although interest in oocyte cryopreservation remains high, experimental evidence shows that cryoinjury to human oocytes results in chromosome sorting defects, reduced fertilization, and compromised embryonic development. Micromanipulation of mammalian oocytes appears to have no deleterious effect on subsequent cryopreservation.

An intact zona pellucida is not necessary for successful mouse embryo cryopreservation.

OBJECTIVE: To determine the developmental potential of mouse embryos that underwent cryopreservation after micromanipulation of the zona pellucida. DESIGN: Gaps were produced in the zona pellucida of mouse oocytes or two-cell embryos by zona drilling with acid Tyrode's solution. Zona-drilled oocytes were fertilized in vitro and cultured to the two-cell stage. Two-cell embryos were frozen, thawed, and cultured to the expanded blastocyst stage. RESULTS: There was no difference in the rate of embryo survival post-thaw (248/318, 77% versus 288/345, 83.4%), or in the rate of development to the expanded blastocyst stage (91/248, 36.7% versus 88/288, 30.6%), between embryos that were zona drilled as oocytes and unmanipulated controls. Similarly, there was no difference in the rate of cryosurvival (206/217, 94.9% versus 168/187, 89.8%) or development to the blastocyst stage (154/206, 74.7% versus 132/168, 78.6%) between embryos that were fertilized in vivo and zona drilled at the two-cell stage and embryos that were unmanipulated. CONCLUSIONS: These findings indicate that small gaps in the zona pellucida, such as those that result from micromanipulation, do not significantly alter the ability of embryos to withstand cryopreservation.

The effect of female antisperm antibodies on in vitro fertilization, early embryonic development, and pregnancy outcome.

STUDY OBJECTIVE: To evaluate the extent to which human in vitro fertilization-embryo transfer (IVF-ET) alleviates immunological infertility. DESIGN: Retrospective. SETTING: In vitro fertilization program. PATIENTS: Thirty-three patients with positive antisperm antibodies undergoing 50 cycles of IVF-ET in which maternal serum was replaced by 5 mg/mL of bovine serum albumin (BSA) comprised the study group. Seventy-one patients with tubal infertility served as controls. In 50 of these, medium was supplemented with 7.5% maternal serum, and 21 were assigned to BSA substitution. RESULTS: Percentage of fertilization in the study group was significantly lower (41 +/- 31; mean +/- SD) than that of controls with maternal serum (77 +/- 15) and BSA (76 +/- 22). Early embryonic quality, as assessed by percentage of cleavage and morphological grading, was found to be inferior in patients with antisperm antibodies. The percentage of advanced embryos (greater than or equal to 4 blastomeres) at the time of transfer was 42 +/- 39 in the study group, compared with 65 +/- 23 and 75 +/- 35 for maternal serum and BSA controls, respectively. Percentage of morphologically favorable embryos (grades 1 and 2 in a 1 to 5 grading system) was 49 +/- 31 in the study group, compared with 78 +/- 35 and 74 +/- 23 for the controls. Percentage of clinical pregnancy was somewhat lower in the study group (12.5%) than in controls with either maternal serum (18%) or BSA (19%). CONCLUSIONS: Antisperm antibodies may have an adverse effect on fertilization and early embryonic development. Female immunological infertility may not be completely alleviated by IVF-ET.

Poor oocyte quality rather than implantation failure as a cause of age-related decline in female fertility.

Female fertility declines with advancing age. To establish whether this age-related reproductive failure results from diminished oocyte quality or uterine/endometrial inadequacy we investigated ovum donation in 35 infertile women, aged 40 years or older (mean 42.7 [SE 0.3]) who had failed at attempts at conception with their own (self) oocytes. Oocytes were donated by 29 young individuals (mean age 33.4 [0.7]) undergoing in-vitro fertilisation (IVF). 8 (5.3%) pregnancies were achieved in 150 cycles of ovulation induction with self-oocytes and 2 (3.3%) in 60 such cycles by in-vitro fertilisation (IVF), but none attained viability. By contrast in 50 cycles with donated oocytes 28 (56%) pregnancies and 15 (30%) deliveries were realised (p less than 0.005). The rate of implantation per embryo transferred was higher (14.7%) with donated oocytes than that with self-oocytes (3.3%) (p less than 0.01). To further elucidate the contribution of age to reproductive outcome, pregnancy results were compared between the young donors and older recipients. Both donors and recipients shared oocytes from the same induced cohort. Rates for clinical pregnancy and delivery did not differ between donors (33% and 23%) and recipients (40% and 30%). Our data suggest that the age-related decline in female fertility is attributable to oocyte quality and is correctable by ovum donation. The uterus can adequately sustain pregnancies even when reproductive potential is artificially prolonged into the late 40s.

An insight into early reproductive processes through the in vivo model of ovum donation.

To gain insight into early reproductive processes we have prospectively designed ovum donation protocols to elucidate several phenomena relating to embryo implantation and pregnancy sustenance. Artificial endometrial cycles with variable follicular phases were induced in 60 recipients by sequential estrogen and progesterone. A total of 964 oocytes were retrieved throughout 43 ovum donation attempts, for an average of 22.4 (range, 16-41) eggs/retrieval. The overall delivery rate per egg retrieval (donors and recipients combined) was 72.1% (31 of 43). The shortest estrogen stimulation (short follicular phase) resulting in ongoing pregnancies was 5 days in duration, while the longest (long follicular phase) was 35 days in duration before progesterone initiation. Utilization of variable length follicular phases, artificially extended the stage of endometrial receptivity to over 4 weeks. To assess the window of implantation, same age embryos were transferred onto endometrium of different maturational stages. Pregnancies were documented with embryo transfers between luteal day 1 (day 15) to luteal day 6 (day 20), extending the window of implantation in the human to at least 6 consecutive days. To evaluate the relative contribution of oocyte quality and endometrial receptivity to pregnancy outcome, common source ova were transferred onto endometrium with variable hormonal exposure. Despite the drastically different follicular phase estradiol levels and periods of exposure, similar delivery rates were attained in donor cycles (29.4%) and recipient cycles during short follicular phases (29.6%). Slightly higher delivery rates (39.4%) were observed with long follicular phases. The comparable pregnancy rates in donors and recipients are attributed to the common source oocytes regardless of endometrial stimulation.

Clinical evaluation of three approaches to micromanipulation-assisted fertilization.

Three different micromanipulation procedures were used to assist human fertilization in cases of severe male factor infertility. Zona drilling was performed either with acid Tyrode's solution, mechanically following zona softening with chymotrypsin, or by partial zona dissection. The fertilization rate was lowest in the zona drilling/acid Tyrode's group (7/40; 17.5%), although no differences between groups (zona drilling/chymotrypsin: 21/84, 25%; partial zona dissection: 31/143, 21.7%) were significant. The fertilization rate was significantly increased relative to untreated eggs from the same patients only in the partial zona dissection group (31/143, 21.7% versus 4/102, 3.9%). Oocyte damage occurred at a high rate as a result of zona drilling with acid Tyrode's solution (13/41, 37%). Embryonic development was compromised after zona drilling with chymotrypsin: only 7/12 (58.3%) of the fertilized oocytes cleaved, and the morphology of many of the cleaved embryos was abnormal. Although only 61% (16/26) of the diploid embryos resulting from partial zona dissection cleaved, the embryonic morphology of these embryos was comparable with controls. No pregnancies resulted from the transfer of manipulated embryos. We conclude that although zona manipulation increases the fertilization rate, losses due to oocyte trauma, low rates of diploid fertilization, low rates of cleavage, and a high frequency of abnormal cleavage reduce the number of embryos available for transfer.

The predictive value of zona-free hamster egg sperm penetration assay for failure of human in vitro fertilization and subsequent successful zona drilling.

The value of various sperm parameters and the zona-free hamster egg sperm penetration assay (SPA) in predicting human in vitro fertilization (IVF) failure and subsequent successful fertilization with zona drilling was assessed. In 19 couples, throughout 31 IVF cycles, a total of 153 oocytes failed to be fertilized. In subsequent 12 cycles with zona drilling, 33 of 131 (25%) were fertilized. The incidence of teratospermia and asthenospermia was significantly higher in the study group than in the control, 74% versus 32% and 42% versus 5%, respectively. Although the mean values for the performance of sperm in SPA and fertilization of human eggs after zona drilling were remarkably similar (28 +/- 6 versus 28 +/- 4), there was no correlation between individual parameters (r = 0.15). Thus, whereas male factor infertility is more likely to be associated with teratoasthenospermia, neither the SPA nor other sperm parameters have any predictive value for failure in IVF. In addition, no criterion of sperm function has yet been identified that would eliminate oligoteratoasthenozoospermic males from consideration of IVF with zona drilling.

Successful microsurgical removal of a pronucleus from tripronuclear human zygotes.

We have microsurgically extracted a pronucleus from 11 tripronuclear human zygotes under two different experimental conditions. One group of six zygotes was incubated in cytochalasin D for 30 minutes before manipulation. The remaining five zygotes were manipulated without preincubation in cytoskeletal relaxing agents. When cytoskeletal relaxants were used, all six embryos survived manipulation and cleaved. One embryo arrested spontaneously at the two-cell stage; all others cleaved to at least four cells, and one embryo reached the advanced morula stage. Of the five eggs manipulated without prior cytochalasin D and colcemid treatment, none survived for more than 30 minutes after manipulation. In none of the tripronuclear eggs was it possible to definitely identify the male pronuclei. Because development was interrupted for karyotyping, it is likely that these embryos would have otherwise continued to develop to more advanced stages. These results indicate that it is possible to microsurgically "epronucleate" tripronuclear human zygotes and obtain further development and that use of cytoskeletal relaxants facilities this manipulative procedure. However, because epronucleated embryos have significant developmental potential, chromosome studies are needed to confirm complete removal of a male pronucleus before such techniques can be applied clinically.

Fertilization of human oocytes by sperm from infertile males after zona pellucida drilling.

Infertile couples who had failed to achieve fertilization of oocytes in previous trials of in vitro fertilization (IVF) were treated by IVF with zona pellucida drilling. Zona drilling entails use of micromanipulation to introduce a gap in the zona pellucida either mechanically or by localized application of a zona solvent from a microneedle. Ten couples were treated, from whom 63 oocytes were recovered for manipulation. Sixteen eggs were denuded of the cumulus oophorus only, and the remaining 47 eggs were subjected to zona drilling. Of the 16 eggs denuded but not drilled, 4 (25%) were fertilized. Of the 47 oocytes drilled, 31 survived (67%) and 10 of the surviving eggs (32%) were fertilized. The polyspermy rate for drilled eggs that fertilized was high (5/10, 50%), and polyspermic eggs were often penetrated by more than two spermatozoa. The remaining five eggs fertilized after drilling were diploid fertilizations, and in three cases cleavage was followed by embryo transfer, although pregnancies were not obtained. These data indicate that zona drilling has the potential for establishing pregnancies in instances where treatment by standard IVF would fail. In addition, results indicate that the block to polyspermy in human eggs occurs at the level of the zona pellucida.