Human embryo: a biological definition.

This paper defines a human embryo from a biological standpoint that takes into account emerging technologies in reproductive science. The paper does not consider legal, moral, religious or social views. As the definition of a human embryo must reflect the multifactorial processes of development, an approach has been adopted which combines recognition of observed events with potential for further development. This acknowledges that fertilization and development are not static processes, and as such embryo status can only be defined by observation of specific markers. The following biological definition of 'human embryo' is proposed. A human embryo is a discrete entity that has arisen from either: the first mitotic division when fertilization of a human oocyte by a human sperm is complete or any other process that initiates organized development of a biological entity with a human nuclear genome or altered human nuclear genome that has the potential to develop up to, or beyond, the stage at which the primitive streak appears, and has not yet reached 8 weeks of development since the first mitotic division.

Cytogenetic analysis of embryos generated from in vitro matured mouse oocytes reveals an increase in micronuclei due to chromosome fragmentation.

PURPOSE: (i) To determine the prevalence of micronuclei in the cytoplasm of embryos generated from in vitro matured oocytes. (ii) Assess whether micronuclei presence are the result of chromosome fragmentation or the loss of whole chromosomes. METHODS: In vitro fertilization was performed on mature oocytes generated from superovulated mice (control) and in vitro matured mouse oocytes. Fertilized oocytes were cultured to the two-cell stage and fixed to slides. Micronuclei assessment was performed after staining with Giemsa. Centromere assessment was made using immunofluorescent staining (CREST) of the centromeric kinetochores. RESULTS: Micronuclei were observed in 2% (4/197) of control two-cell embryos and 36.2% (46/127) of two-cell embryos generated from in vitro matured oocytes (P < 0.02). Centromeres were not detected in micronuclei from either group. CONCLUSIONS: A significant increase in micronuclei was observed in embryos generated from in vitro matured oocytes. The lack of accompanyingcentromeres would suggest the micronuclei are the result of chromosome fragmentation.

Cytogenetic analysis of unfertilized oocytes following intracytoplasmic sperm injection using spermatozoa from a globozoospermic man.

A man with globozoospermia was treated in our in-vitro fertilization-intracytoplasmic sperm injection (ICSI) programme. In the treatment cycle, 24 oocytes were collected from his wife. All the oocytes were at metaphase II stage. The semen sample produced on the day had a normal sperm count, good motility, but with 100% globozoospermia. All oocytes were injected with randomly selected spermatozoa and of these, two oocytes showed two pronuclei and another contained a single pronucleus. The remainder were unfertilized. The normally fertilized oocytes (two pronuclear) cleaved to the four-cell stage and were transferred to the patient. At 48 h after ICSI, the 21 unfertilized oocytes were processed for cytogenetic analysis. All oocytes contained a haploid chromosome set. The only abnormality seen was a chromosome fragment in one metaphase. Eighteen oocytes contained decondensed sperm nuclei and of these, 14 nuclei were beginning to show signs of premature chromatin condensation (PCC) and the other four showed strong signs of PCC. Thus it appears that in some forms of globozoospermia, arrest of nuclear decondensation and/or PCC are another cause of fertilization failure. The most likely cause for this is the absence or down-regulation of spermatozoa associated activating factor in round-headed spermatozoa.

Cryopreservation of oocytes and embryos: use of a mouse model to investigate effects upon zona hardness and formulate treatment strategies in an in-vitro fertilization programme.

Mouse oocytes and embryos were obtained following ovulation induction of (C57B16 x CBA) F1 animals. Zonae pellucidae were exposed to alpha-chymotrypsin in phosphate-buffered medium (PB1) supplemented with 3 mg/ml bovine serum albumin upon a heated stage (37 degrees C) and were observed constantly through an inverted microscope. The endpoint of the bioassay was the limits of the zona no longer being seen clearly at x 200 magnification, and the time taken for each zona to dissolve was recorded. A dose-dependent response in dissolution time was clearly seen, with 1% alpha-chymotrypsin being chosen as the routine working solution. Cryopreservation of 2-cell mouse embryos using propanediol did not cause zona hardening but induced a small and significant softening, as gauged by the time taken for zona dissolution (2181 +/- 167 versus 1864 +/- 82 s). Zona hardening was not suspected to occur after the freezing of human embryos as there was no difference in implantation rates per embryo for in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles between fresh [IVF: 63/644 (9.7%); ICSI: 51/330 (15.5%)] and frozen embryos [IVF: 36/458 (7.9%); ICSI: 18/112 (16.1%)]. Conversely, significant hardening of the zonae of mature oocytes was seen following cryopreservation (747 +/- 393 s) compared with freshly ovulated oocytes (151 +/- 68 s). It is concluded that (i) the freezing of murine oocytes with propanediol results in zona hardening, implying a possible benefit of ICSI after the cryopreservation of human oocytes, and (ii) the cryopreservation of embryos is not associated with zona hardening or reduced implantation, making microdissection of the zona in such cases generally unwarranted.

Birth from cryopreserved embryos following in-vitro maturation of oocytes and intracytoplasmic sperm injection.

This case report describes the birth of a baby following the transfer of cryopreserved embryos generated from intracytoplasmic sperm injection (ICSI) carried out on the second day after oocyte pick-up of in-vitro-matured metaphase I and germinal vesicle stage oocytes. The couple had a history of three failed intrauterine insemination attempts and reduced fertilization rates in two previous in-vitro fertilization (IVF) cycles. In the IVF-ICSI treatment cycle, 6/11 mature oocytes became fertilized following ICSI on the first day. However, the patient failed to conceive following the transfer of three embryos. Five oocytes were immature (two at metaphase I stage and three with a germinal vesicle) and these were cultured overnight. All had extruded a polar body by the following day and ICSI was therefore performed; four oocytes became fertilized, and were cryopreserved at the pronulear stage in propanediol. In the next treatment cycle, transfer of frozen embryos was planned. The pronuclear zygotes were thawed and cultured for 24 h prior to the transfer of two embryos in a cycle stimulated with low doses of follicle stimulating hormone. This resulted in a pregnancy and the delivery of a healthy baby boy. In-vitro maturation of metaphase I and germinal vesicle oocytes which are routinely collected in IVF-ICSI cycles, followed by second day ICSI fertilization, may provide a valuable source of embryos for infertile couples.

Changes in motility patterns during in-vitro culture of fresh and frozen/thawed testicular and epididymal spermatozoa: implications for planning treatment by intracytoplasmic sperm injection.

The present report describes the motility changes in vitro (percentage motile and progressively motile) of freshly collected testicular and epididymal spermatozoa and following freeze/thaw of the same spermatozoa from a man with obstructive azoospermia. Washed spermatozoa were cultured in micro droplets under paraffin oil or in test tubes using HEPES-buffered or bicarbonate-buffered medium containing 10% human serum. In fresh testicular sperm cultures 60-65% of the sperm cells became motile within 2 days of culture; the motility was maintained for a further 4-5 days before a decline was observed. The progressive motility improved markedly on the third day of culture and it peaked around day 5. Only a small number of frozen/thawed testicular spermatozoa became motile during in-vitro culture (15-20%) and the motility was maintained for only 2-3 days before it declined. Furthermore, only 10-12% of the spermatozoa showed progressive motility. Spermatozoa recovered from micro-epididymal sperm aspiration (MESA) showed a gradual decrease in progressive motility and in 5 days all sperm cells were found to be immotile in both freshly collected and frozen/thawed spermatozoa. All culture systems supported sperm motility. It is clear that testicular spermatozoa, particularly from men with obstructive azoospermia, can be collected and maintained in vitro for up to 1 week before the oocyte retrieval but when frozen testicular or epididymal spermatozoa are used it is more reliable to thaw these spermatozoa on the day of intracytoplasmic sperm injection.

An argument for the past and continued use of pentoxifylline in assisted reproductive technology.

Pentoxifylline was first used within an in-vitro fertilization (IVF) programme before the advent of alternative treatment strategies such as oocyte micromanipulation. Over the years, it has continued to be useful in aiding fertilization in selected IVF cases, with a beneficial effect also being seen in certain cases treated by intrauterine insemination. In both instances, the acrosome reaction to ionophore challenge test appears to have been invaluable in identifying suitable patients. The stimulation of spermatozoa by pentoxifylline should remain a therapeutic option in the treatment of couples with a male factor present. As an adjunct to IVF, it has the advantage of being simpler and less costly to perform compared with micromanipulation. However, its use should be restricted to selected cases, and the merits over and above those of invasive procedures such as intracytoplasmic sperm injection should be discussed with the individual patients. The pretreatment of spermatozoa prior to intrauterine insemination in selected cases gives an alternative therapeutic strategy to those patients not wishing or unable to undertake IVF.

The incidence and influence upon fertility of antisperm antibodies in seminal fluid following vasectomy reversal.

Seminal plasma samples from men undergoing vasovasostomy were analysed for antisperm antibodies using the indirect immunobead test. A pre-operative assessment showed antisperm antibodies of either IgA or IgG class to be present in 9/27 (33.3%) men. A significant increase (P less than 0.05) in the post-operative incidence of the antibodies was seen in the men who achieved patency (27/45, 60%) but not in those men for whom no sperm were seen in the ejaculate (4/10, 40%). After follow-up for a minimum of 1 year, conception rates for couples in which the male partner had achieved patency were similar in the groups with no antibodies detected post-operatively (12/18, 66.7%) or with IgA alone (2/3, 66.7%), but was reduced significantly in the presence of IgG (1/9, 11.1%; P less than 0.05) or IgA + IgG (3/15, 20.0%; P less than 0.01).

Effect of antispermatozoal antibodies in seminal plasma upon spermatozoal function.

The indirect immunobead test for antispermatozoal antibodies of the class IgA, IgG and IgM was applied to the seminal plasma of male partners of infertile couples. The presence of both IgA and IgG was associated with a decreased incidence of good post-coital test results and a reduced rate of fertilization of human oocytes. No significant differences were found for men with IgA or IgG alone when compared to men with no detectable antispermatozoal antibodies.

The fertilization of human oocytes by spermatozoa from men with antispermatozoal antibodies in semen.

Seventy-two couples, including 15 with antispermatozoal antibodies in the male partner's semen, were studied in a program of in vitro fertilization and embryo transfer. Cases were further subclassified as normospermic or oligospermic and antispermatozoal antibodies were assessed with categorization into the respective human immunoglobulin classes as determined using the indirect immunobead test. The study reveals that fertilization is significantly reduced (P less than 0.001) only if both IgA and IgG antibodies are present in semen but there is no reduction if either class is present alone. The fertilization rate of oocytes is significantly reduced (P less than 0.001) by sperm from oligospermic samples, and there is a further reduction in those cases with combined IgA/IgG antispermatozoal antibodies.

Use of immunobeads to detect human antispermatozoal antibodies.

Immunobeads for IgA, IgG and IgM were used in an indirect test (immunobead test: IBT) to detect human antispermatozoal antibodies, with positive results for at least one class of antibody being found in the serum of 13/169 (7.7%) men tested and 12/172 (6.9%) women. Of those men with antibodies present in serum, 100% had IgG, 62% had IgA and none had IgM, whilst the proportion for women was 75%, 100% and 33% respectively for each class of antibody. Antispermatozoal antibodies in men do not always appear both in semen and serum, but may be present in only one of the fluids tested for IgA (7/13 men; 53%) and IgG (6/14 men; 42.9%). The incidence of antibodies in the serum of oligospermic men was not significantly different from that of normospermic men (chi 2 = 0.06). A total of 481 serum and semen specimens were assayed by both the IBT and tray agglutination tests, and agreement between the two assays occurred in 97.3% (468/481) samples (P less than 0.001).