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Background: Assisted reproductive technologies (ART) to treat infertility have been available for nearly three decades. There have been a number of systematic comparisons of the health and development of ART-conceived with spontaneously-conceived (SC) children. Data are equivocal, some finding no differences and others that there are more health and developmental problems in the ART group. It is agreed that perinatal mortality and morbidity are worse after assisted than spontaneous conception and the impact of the hormonally altered intrauterine environment on puberty and later fertility of offspring are unknown. To date however, there has been no investigation of the health and development of ART-conceived young adults, including from the world's few prospective cohorts of ART conceived children. Obtaining these data requires contact to be made with people at least twenty years after discharge from the treating service. Given the ethical difficulties of approaching families to participate in research up to two decades after cessation of treatment, the aim of this exploratory qualitative investigation was to assess the feasibility and acceptability of approaching mothers treated for infertility prior to 1988, and their recall of the health and development of their ART-conceived young adult children., Methods: Mothers treated for infertility at the Royal Women's Hospital Reproductive Biology Unit in Melbourne, Australia prior to 1988 were approached by a senior clinician and invited to participate in individual semi-structured interviews which could include their partners and/or young adult children if they wished. Recruitment continued until theoretic saturation had been reached., Results: Ten mothers, two of their husbands and five young adults participated in interviews, and the health and development of 15 ART-conceived young adults were described. The experience of conception, pregnancy, birth and the health and development of the children were recalled vividly and in detail. Families were pleased to have been approached and supported the need for systematic data collection. Mode of conception had been disclosed from childhood to all the offspring., Conclusion: With careful and sensitive recruitment strategies it is feasible and acceptable to contact women treated for infertility at least two decades ago and their families, to assess the health and development of ART-conceived young adults.

Background: Data show that differences exist in the birthweight of singletons after frozen embryo transfer (FET) compared with fresh transfer or gamete intra-Fallopian transfer (GIFT). Factors associated with low birthweight (LBW) after assisted reproduction technology (ART) were studied., Methods: Birthweight, distribution of birthweight, z-score, LBW (<2500 g), gestation and percentage preterm (<37 weeks) for singleton births >19 weeks gestation, conceived by ART or non-ART treatments (ovulation induction and artificial insemination) between 1978 and 2005 were analysed for one large Australian clinic., Results: For first births, the mean birthweight was significantly (P < 0.005) lower, and LBW and preterm birth more frequent for GIFT (mean = 3133 g, SD = 549, n = 109, LBW = 10.9% and preterm = 10.0%), IVF (3166, 676, 1615, 11.7, 12.5) and ICSI (3206, 697, 1472, 11.5, 11.9) than for FET (3352, 615, 2383, 6.5, 9.2) and non-ART conceptions (3341, 634, 940, 7.1, 8.6). Regression modelling showed ART treatment before 1993 and fresh embryo transfer were negatively related to birthweight after including other covariates: gestation, male sex, parity, birth defects, Caesarean section, perinatal death and socio-economic status., Conclusions: Birthweights were lower and LBW rates higher after GIFT or fresh embryo transfer than after FET. Results for FET were similar to those for non-ART conceptions. This suggests IVF and ICSI laboratory procedures affecting the embryos are not causal but other factors operating in the woman, perhaps associated with oocyte collection itself, which affect endometrial receptivity, implantation or early pregnancy, may be responsible for LBW with ART.

Background: Although ovarian tissue cryopreservation for women at risk of losing ovarian function is offered by many clinics, there is a lack of evidence relating to the developmental potential of the stored tissue and, therefore, its clinical potential. This study was designed to use xenografting of cryopreserved tissue from multiple patients to assess the reproducibility of preservating developmental potential, the variation in developing follicle profile and the relationship between pre-freeze histology and post-thaw development., Methods: Using previously published methods, cryopreserved ovarian cortex from nine patients was thawed and grafted under the kidney capsules of immunodeficient mice. Development of follicles was assessed after 26 weeks and compared to histology prior to freezing., Results: Multiple growing follicles including antral stages were observed in multiple grafts of tissue from all patients. Metaphase II oocytes (n=9) were observed in follicles in grafts from five patients. There was no relationship between pre-freeze histology and developing follicle profile in xenografts., Conclusions: The propanediol freezing method used in this study is capable of reproducibly preserving the developmental potential of human ovarian follicles. The developing follicle profile after cryopreservation cannot be accurately predicted from pre-freeze histology. Xenografting provides a powerful tool for assessing the potential of human cryopreserved ovarian tissue.

Background: Previous studies have demonstrated development of antral follicles in cryopreserved human ovarian tissue after autografting and xenografting, thus indicating successful preservation of follicular function. The study aim was to assess whether these follicles could also undergo periovulatory changes in response to hCG., Methods: Ovarian tissue from three patients were dehydrated in propanediol (PROH)/sucrose and cryopreserved using the slow cooling/rapid thaw procedure. Thawed tissue was placed under the kidney capsule in immunodeficient mice. Following growth (>20 weeks) in the presence of gonadotrophin, hCG was administered and ovarian tissue examined histologically., Results: Thirty-two antral follicles (diameter range 0.6 to 5 mm) were examined. Histological evidence of a response to hCG was evident in all follicles. Disruption of the concentric layers of mural granulosa and theca cells was apparent in all antral cavities. In 17 (53%) follicles the exterior follicular wall had reduced to a few cells thick, and in eight (25%) the wall had ruptured. Mucified oocyte-cumulus cell complexes were present in 32 follicles, 17 of which had begun to detach from the pedicle. Resumption of meiosis had occurred in over half the oocytes (five metaphase II and seven metaphase I oocytes, eight germinal vesicle breakdown). Two corpora lutea were also detected., Conclusions: Follicles cryopreserved within human ovarian tissue using the PROH procedure, can develop to the antral stage and undergo periovulatory changes following xenografting and exposure to a luteinizing stimulus.

This study reports the first gross morphological and histological evidence of antral follicle development in human ovarian tissue following cryopreservation. Human ovarian tissue was cryopreserved using propanediol and sucrose and grafted under the renal capsule of bilaterally oophorectomized severe combined immunodeficient (SCID) mice. Follicles at all stages of development were observed in the grafted tissue whereas, prior to grafting, only primary and primordial follicles were present. Antral follicles were rarely observed on grafts examined <20 weeks after grafting either non-frozen tissue (fresh, 1/7) or cryopreserved tissue (0/11). In contrast, development of at least one antral follicle was evident on the majority of sites > or = 20 weeks after grafting (fresh 7/9, cryopreserved 18/24). Antral follicle diameters ranged from 0.1 to 5.0 mm. Histological examination of these antral follicles indicated normal follicular morphology, i.e. antral cavities encapsulated by concentric layers of theca and granulosa cells. Pedicles containing germinal vesicle oocytes were observed protruding from the granulosa cell layers. The development and morphology of the cryopreserved and fresh tissue following grafting was similar.

Purpose: To determine whether the relatively low implantation rate of cryopreserved Day 2 embryos with only 2 blastomeres can be increased as a consequence of increasing their blastomere content by extending the prefreeze culture time., Methods: Of a total of 3480 Day 2 embryos studied, 1921 (55.2%) had reached the 4-cell stage by 40 h postinsemination (FAST) and were transferred or cryopreserved. The remaining embryos that underwent subsequent cell division by 46 h (INTERMEDIATE; 18.3% of total) or 66 h (SLOW; 20.3% of total) were also cryopreserved whereas the 6.2% that remained arrested at 66 h were discarded. Thawed embryos from each category were assessed for survival, post-thaw cleavage, and implantation., Results: The proportion of thawed embryos that survived, the proportion of surviving embryos that underwent post-thaw cleavage, and the implantation rate of transferred embryos were all reduced in the slower growing cryopreserved embryos., Conclusions: The growth rate, and not the number of blastomeres per se, is a critical factor in predicting the developmental potential of cryopreserved embryos.

Using rigorously matched non-frozen controls we have shown that cryopreservation does not alter the implantation potential of early cleavage stage (day 2) human embryos if no blastomere loss occurs. Thawed intact 4-cell embryos have a significantly higher implantation (fetal heart) rate (16.9%) than similar 2-cell embryos (7.2%). This difference is not due to blastomere number per se since increasing the cell number in frozen embryos by allowing an extended period in culture prior to freezing does not alter their intrinsic developmental potential. Blastomere loss, which occurred in almost half of all thawed embryos, is directly related to a reduction in developmental potential. We estimate that approximately 30% of the expected fresh embryo implantations are lost as a consequence of cryopreservation. Both preimplantation and peri-implantation losses may contribute to this outcome.

The impact of cryopreservation on the implantation potential of early cleavage stage (day 2) embryos was assessed by analysing the outcome from > 5000 thawed embryos in relation to the outcome from a similar number of fresh embryos. Analysis of procedures in which all transferred embryos fulfilled equivalent defined criteria revealed no significant difference in the implantation rates (fetal hearts/100 embryos transferred) of fresh 4-cell embryos (16.6%) and fully intact thawed 4-cell embryos (16.9%). Although 2-cell embryos implanted at significantly lower rates, there was again no significant difference between fresh (6.5%) and fully intact thawed (7.2%) embryos. Similar analysis of all embryos (irrespective of cell number on day 2) demonstrated that the implantation potential of partially intact thawed embryos was related to the extent of blastomere loss with the implantation rate of embryos with 50% cell survival (5.4%) being approximately half the rate of fully intact embryos (11.3%). Combining the values obtained from 'pure' data for the implantation rates of embryos with defined levels of survival with their relative prevalence in the total population of thawed embryos gave a predicted number of implantations (441) which was similar to the observed outcome (463). This number was approximately 30% less than the number expected had the same embryos been transferred fresh (635). The results suggest that intact thawed embryos have the same implantation potential as equivalent fresh embryos and that the impact of cryopreservation is limited to blastomere loss which is directly related to loss of implantation potential. The observed frequency of blastomere loss results in a reduction of approximately 30% in the implantation potential of a population of embryos following cryopreservation.

Donor insemination (DI) using cryopreserved semen commenced at The Royal Women's Hospital in 1976. Over the next 15 years we performed 5953 treatment cycles to achieve 816 pregnancies (13.7% per cycle) and 706 live births. In-vitro fertilization (IVF) using donor spermatozoa commenced in 1986. Over the next 5 years we performed 303 treatment cycles for 185 couples. Including subsequent transfer of cryopreserved embryos, a total of 33% of couples achieved a successful pregnancy by IVF. Statistical analysis indicated that, for DI pregnancies, the most important semen variable was the percentage post-thaw motility, whilst for normal fertilization in IVF it was the pre-freeze motility. These results may be explained by the compensatory effects of post-thaw processing of spermatozoa for IVF, but not for DI in our clinic.

Six tubal ectopic pregnancies occurred in a series of 193 pregnancies following ovulation induced with human pituitary gonadotropin (hPG) and human chorionic gonadotrophin (hCG). The ectopic pregnancy rate of 3.1 per cent is higher than quoted incidences in the general population and occurred in the absence of predisposing factors. There was an association between ectopic pregnancy and elevated urinary oestrogen excretion in the peri-ovulatory phases of the induced ovulatory cycles. A urinary oestrogen excretion of greater than 200 microgram/24 hours on day 0 (the day after hCG was given) was associated with a 10 per cent chance of ectopic pregnancy (P less than 0.05).

Preovulatory eggs were aspirated from the ovaries of women 36-38 h after hCG and incubated with washed sperm suspensions for 3 or 6 h; 5 out of 9 eggs were fertilized. Sperm penetration into the ooplasm and decondensation of the male chromatin was observed by 3 h, and an early pronucleate egg was found by 6 h, suggesting that human spermatozoa may become capacitated in vitro in 3-4 h.

Serum levels of the growth factor non-suppressible insulin-like activity (NSILA-S) have been measured in eleven pregnant women with diabetes mellitus during pregnancy and immediately (2 days) after birth. A significant increase in mean levels was observed with increasing gestation, reaching a peak at the time of delivery and subsequently returning to non-pregnant levels within 2 days. The NSILA-S levels in diabetic pregnancies were not different from those in non-diabetic pregnancies and there were no significant correlations between NSILA-S and birth weight, maternal diabetic control, age or duration of diabetes or the treatment required. The physiological role of high NSILA-S levels during pregnancy still requires elucidation.

Thirty anovulatory patients, previously unresponsive to clomiphene citrate in standard dosage, were treated with a new incremental method of clomiphene therapy. Clomiphene was given continuously with dosage increments every 5 days, up to a maximum dose of 3750 mg, and ovarian response was monitored with urinary estrogen estimation and with ultrasound. Twenty-one patients (70%) ovulated on this regimen and 8 (27%) conceived; 6 patients who ovulated but did not conceive had other infertility factors. Clomiphene response was associated with a progressive rise in follicle-stimulating hormone, luteinizing hormone, and estrogen excretion; these parameters were unchanged in nonresponders. Side effects of treatment were minimal and only 1 case of hyperstimulation occurred. Preliminary testing with estrogen-amplified gonadotropin-releasing hormone did not differentiate responders from nonresponders. Incremental clomiphene treatment appears to be a less complicated alternative for patients who would otherwise require exogenous gonadotropin therapy.

A case of non-immunological hydrops fetalis with gross ascites is reported. Following definitive ultrasonic diagnosis, a fetal abdominal paracentesis was performed under ultrasonic control with a view to minimising fetal trauma during delivery.

To determine whether the absence of one ovary would influence adversely the outcome of in vitro fertilization, the results of 60 laparoscopic oocyte retrieval procedures in 34 single-ovary patients were compared with the outcome for all laparoscopic retrieval cycles during the same period (January 1984-August 1986) in patients with tubal infertility and two accessible ovaries (559 cycles in 335 patients). The median age was younger in the one-ovary group (31 vs 32) (P less than 0.05). The group with two accessible ovaries had significantly more follicles aspirated (median, 7 vs 5; P less than 0.001) and more oocytes obtained (median, 5 vs 4; P less than 0.001) per retrieval compared with the single-ovary group. However, the single-ovary group had a higher percentage fertilization (69 vs 62%; P less than 0.05) so that the number of embryos transferred per transfer patient (median, 3 vs 3) and pregnancy rate per cycle (9.7 vs 15.6%) did not differ significantly between groups. We conclude that patients with a single ovary have a compensatory increase in the ability to produce oocytes which may have greater potential for fertilization and subsequent development.

In vitro fertilization (IVF) and embryo transfer results were analyzed for three women with sperm antibodies in their plasma and follicular fluid. These preliminary results suggest that substitution of the patients' serum by replacement serum in the fertilization and embryo growth media may prove to be an effective means of improving IVF treatment for women with sperm immunity.

The gestation sac size in pregnancies resulting from in-vitro fertilization (IVF) and embryo transfer have been compared with those in spontaneous pregnancies. Small-for-dates gestational sac sizes were found in 36% of the IVF pregnancies. This proportion held for both singleton and multiple pregnancies. With increasing gestation beyond 8 weeks the gestation sac volume increasingly approached normal. In contrast to spontaneous conceptions, IVF pregnancies had a low rate of pregnancy loss once fetal heart movements were demonstrated, when the gestation sac size was small-for-dates. Small sac size in an IVF pregnancy may lead to the misdiagnosis of a failed pregnancy.

The dynamics of 17-alpha-hydroxy-progesterone (17-OHP) production during the onset of the luteinizing hormone (LH) surge were evaluated at 8-hour intervals and correlated with plasma estradiol (E2), LH, and progesterone (P) in 22 women superovulated for in vitro fertilization. Plasma 17-OHP levels rose 8 to 16 hours before the earliest rise in LH and P in 18% of patients, but had risen significantly in 82% of patients by the onset of the LH surge. In the late follicular phase, 17-OHP levels correlated significantly with E2 and LH levels, and the number of follicles greater than 10 mm. Because rising 17-OHP levels preceded the LH surge in only a few patients, it has no clinical value as a marker for human chorionic gonadotropin administration for timed oocyte recovery. Plasma 17-OHP levels, however, may complement LH levels better than P levels in determining the most appropriate stage for surge-timed oocyte retrieval.

Three hundred seventy-two patients underwent laparoscopy for in vitro fertilization and embryo transfer. Of these, 156 were treated with clomiphene citrate alone, 203 with clomiphene citrate and hMG, and 13 with hMG alone. Two-hundred seventy-two of these patients underwent embryo transfer, and 55 pregnancies resulted. Of these, 30 were ongoing, 14 biochemical, 6 ectopic, and 5 aborted. Where four embryos were transferred, the pregnancy rate was found to be significantly higher than when a lesser number were transferred. When the embryos transferred were analyzed, however, it was found that fewer than 10% of all transferred embryos implanted and it was largely a function of multiple embryo transfer per patient that led to success. When the type of hyperstimulation was compared, clomiphene citrate cycles were found to be less successful in that they produced fewer oocytes and embryos than did cycles in which hyperstimulation was achieved by supplementary hMG. It is conceded that this is an overall analysis and that individuals, especially those producing twins, had more than one good embryo transferred into a receptive uterus. The data suggest that unsuccessful cycles had somewhat poorer quality embryos, nonreceptive endometria, or a combination of these factors when compared with those of the successful embryo transfer cycles. The only predictive factor as to outcome relates to the multiplicity of embryos transferred. The fact that some IVF cycles produce multiple pregnancies indicates that a predictor of embryo health is urgently required. However, until such a reliable predictor is obtained, multiple embryo transfer remains a major advance in the success of IVF and ET.

A retrospective analysis was carried out to assess the outcome of ovarian stimulation on in vitro fertilization when human chorionic gonadotropin (hCG) was administered after 5, 6, or 7 days of continuously rising plasma estradiol (E2). There was no significant difference in the number and size of large follicles in each group although the number of small follicles (less than 15 mm in diameter) decreased significantly after 7 days of E2 rise. After hCG injection in the 7-day group, the E2 level fell below the previous day's value in 40% of patients, whereas a similar fall was observed in only 16% of patients in the 5- and 6-day groups. In those cycles where a luteinizing hormone surge occurred, most surges were detected during the seventh day of E2 rise. The pregnancy rate was 31% when hCG was given after 6 days of rising E2, 21% after 5 days, and 14% after 7 days. In patients achieving pregnancy in the 6-day group, 53% of embryos were derived from leading follicles. In the 7-day group, only 15% of embryos associated with pregnancies were derived from leading follicles. These results strongly suggest that in stimulated cycles, hCG should be administered after 6 days of continuously rising E2. It is therefore postulated that 6 days of rising E2 represents a mean optimal period for follicular growth and oocyte maturation in stimulated cycles.

A case, in which pregnancy (unfortunately ectopic) followed oocyte collection by an ultrasound technique, is reported. Nevertheless, the procedure is less invasive than laparoscopic follicular aspiration, does not require general or major regional anaesthesia, and is suitable for patients in whom access to the ovaries by means of laparoscopy is either not possible or severely restricted.

In 161 consecutive gamete intrafallopian transfer (GIFT) cycles in which supernumerary oocytes were inseminated, a failure to fertilize any of these oocytes was no more predictive of an unsuccessful outcome than the simple overall pregnancy rate in this group. This is possibly related to the significantly reduced proportion of oocytes graded as good in the supernumerary group.

Positive sperm immobilization tests were found in the sera of 6.3% of 491 infertile men. There was a higher frequency of family histories of autoimmune diseases than for other subfertile men. Prognosis for fertility was related to results of sperm-mucus penetration tests: progressively motile sperm indicated a fair outlook--8 of 15 couples conceived within 7 months either spontaneously or with artificial insemination with husband's semen. Absence of progressively motile sperm indicated severe infertility - only 6 of 72 couples produced pregnancies. Treatment of the latter group by temporary suppression of spermatogenesis with testosterone or with Artificial Insemination by Husband (AIH) appeared to be ineffective. However, sperm immobilization titres fell and sperm concentrations, motility and mucus penetration increased during treatment of 14 of the men with prednisolone (0.5-0.75 mg/kg per day) for 4-7 months and there were three pregnancies. It is concluded that sperm autoimmunity in men causing failure of sperm to penetrate cervical mucus produces severe but not absolute infertility. Immunosuppression with continuous high dose glucocorticoid reduces antibody levels and improves semen quality. A controlled trial is needed to determine if this treatment is a practical method for improving fertility in men with sperm autoimmunity.

In order to determine whether infertility of unexplained aetiology would respond to therapy with bromocriptine, 50 regularly ovulating infertile patients were studied in a double-blind placebo controlled trial. Following an observation cycle in which midluteal urinary oestrogen and pregnanediol excretion and late luteal serum PRL and hCG levels were determined, patients were randomly allocated to treatment with either bromocriptine or placebo for three cycles. Patients who did not conceive were then treated for three cycles with the alternative therapy. Eighteen women (36%) conceived, five during the observation cycle, four during treatment with bromocriptine, four whilst receiving placebo and five during the 12 month follow-up period. The pregnancy rate achieved in patients treated with bromocriptine was thus no better than that for the group as a whole.

The effect of sperm antibodies on the human fertilization process was evaluated by analyzing the Royal Women's Hospital in vitro fertilization (IVF) data. The results suggest that sperm autoantibodies, particularly those of IgA immunoglobulin class (determined by immunobead test (IBT] can interfere with IVF. Thus, in a group (n = 8) of couples where the male partner had 80% or more of his motile spermatozoa coated with IgG and IgA class sperm antibodies, an overall fertilization rate of only 27% (18/66 ova) was obtained. In contrast, in a group (n = 9) with positive IBT results, but with less than 80% of motile spermatozoa coated with IgA class antibodies, a normal fertilization rate of 72% (47/65 ova) was obtained. Three of these patients had 90% or more of their motile spermatozoa coated with IgG and less than 70% coated with IgA class antibodies. In this subgroup a good fertilization rate of 16/21 (76%) was obtained. Another observation derived from this investigation is that the oocytes that do fertilize in the presence of sperm antibodies can subsequently proceed with normal cleavage, implantation, and pregnancy. This provides a rationale for attempting to treat these patients by reducing the proportion of antibody-coated sperm in vitro for future IVF cycles.

A simple procedure for detection of antisperm antibodies of IgA class in human cervical mucus is described and the results of its application to samples from 102 patients are presented. The results suggest that the IgA immunobead test (IgA-IBT) is a specific and clinically useful test for sperm antibodies. There was a strong correlation between the IgA-IBT and the presence of complement-dependent sperm immobilization in serum (Spearman's, r = 0.92, p less than 0.001). Positive IgA-IBT results occurred only in mucus samples that showed poor penetration by normal sperm. An added advantage of the IgA-IBT is that both the immunoglobulin class and the site of binding to the sperm surface can be determined simultaneously.

When investigations fail to reveal a cause for infertility, treatment must then be based on possible, but unproven, causes, and since there is a high spontaneous pregnancy rate in unexplained infertility the effect of any treatment is difficult to assess. Such treatment has included correction of anatomical variants such as uterine retroversion and the use of hormonal manipulation during the follicular and luteal phases of the menstrual cycle. Ovum entrapment, occult spontaneous abortion and faults in sperm fertilizing capacity have all been implicated, and it is likely that immunological factors play a substantial role in unexplained infertility. Evidence does not support the use of bromocriptine in the absence of hyperprolactinaemia. Successful treatment by intrauterine insemination is unlikely if there are circulating anti-sperm antibodies in the partner. Improving cervical mucus by treatment with oestrogens and clearing infections with antibiotics may have a modest place but it is very difficult to show that these treatments have more than a placebo effect. Endometriosis is often missed and the possibility of it having developed after initial investigation warrants repeat laparoscopy after 2 years. Three approaches are currently acceptable in the management of the couple with unexplained infertility: await spontaneous pregnancy, the empirical use of clomiphene and in-vitro fertilization.

Serum and prolactin levels and urinary oestrogen and pregnanediol excretion were measured during early pregnancy in 12 hyperprolactinaemic subjects who became pregnant during treatment with bromocriptine. Prolactin levels were suppressed below normal whilst taking the bromocriptine, but rose significantly when this drug was stopped and by eight weeks of gestation reached values significantly higher than those observed in normal pregnancy. Further increases in prolactin levels were, however, not observed. Urinary oestrogen and pregnanediol excretion was initially normal but, following cessation of bromocriptine, urinary pregnanediol excretion decreased and was significantly less than normal at 11 to 14 weeks gestation. These results suggest that elevated prolactin levels affect corpus luteum function of early pregnancy. Because the effect is maintained until at least 14 weeks gestation, prolactin may also affect progesterone production by the placenta.

The effects of maternal preovulatory serum and human fetal cord serum supplement in culture medium in human in vitro fertilization and embryo development were compared in 208 cases over an 8-month period when there were no significant changes in other variables. A significantly higher pregnancy rate was observed in the fetal cord serum group despite no significant difference being found in fertilization and embryo cleavage rates. This suggests a difference in the health of the embryo cultured in different serum supplements.

Preovulatory eggs in cumulus were inseminated in vitro with washed spermatozoa which had been preincubated for 1.5 hours. After 3 hours, three eggs were processed for electron microscopy and each was sectioned serially from pole to pole. In the two eggs which had been fertilized, the expanded chromatin of the fertilizing sperm head and the chromatin of the ovum were almost completely surrounded by a developing pronuclear envelope. In one of the penetrated eggs the developing male pronucleus and associated midpiece and sperm tail were located within an incorporation cone. The surface of the cone was free of microvilli but contained a zone of microfilaments immediately beneath the plasma membrane. A similar zone of microfilaments was present beneath the advancing surface of the extruding second polar body (PB2) which was connected to the ovum by an interbody and microtubules of the meiotic spindle. Cortical granules were completely absent from the fertilized eggs but were present in the unfertilized egg. PB2 contained a nucleus at a stage of development similar to that of the early pronuclei.