The transfer of two embryos instead of three to reduce the risk of multiple pregnancy: a retrospective analysis.

PURPOSE: Our purpose was to investigate whether a reduction in the number of embryos transferred from three to two would help reduce the incidence of multiple pregnancies and yet leave the pregnancy rate unaffected. METHODS: Women were treated in a routine clinical in vitro fertilization program and the results analyzed retrospectively. RESULTS: There was no reduction in the pregnancy rate when two embryos were transferred compared with three. Indeed, there was actually an increase in pregnancy rate after the transfer of two embryos in those cases with one or more embryos remaining after the transfer. CONCLUSIONS: The transfer of only two embryos compared to three in women younger than 40 years of age does not compromise the chance of pregnancy. Triplets were not seen in the limited series of patients when only two embryos were transferred, but the incidence of twins remained the same. Further consideration should be given to strategies that enable the transfer of single embryos without compromising the pregnancy rate.

Internal quality control and external quality assurance in the IVF laboratory.

The existence of internal quality control programmes and external quality assurance schemes is important in enabling the maintenance of good service to patients. All aspects of our work in laboratories involved in the diagnosis and treatment of human infertility can benefit from such programmes and schemes, moving the work from being a subjective art form to an objective science. Equally, many clinical procedures are amenable to such scrutiny. Acceptance and introduction of such schemes and programmes will rely initially on the self-motivation of the laboratories themselves and then pressure brought to bear by accrediting authorities.

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.

A prospective evaluation of cryopreservation strategies in a two-embryo transfer programme.

A total of 364 consecutive patients requesting in-vitro fertilization (IVF) treatment were divided randomly into two groups. In the first group, two embryos in the original IVF cycle were allowed to divide prior to transfer, with any remaining embryos being cryopreserved at the pronucleate (PN) stage. In the second group, all the embryos were allowed to divide to the early cleavage (EC) stage, and the best two replaced; any suitable remaining embryos were frozen at the 2- to 4-cell stage. A total of 134 cycles (36.8%) fulfilled the study criteria for a fresh embryo replacement and supernumerary embryos cryopreserved. In the PN group, 72 out of 182 (39.6%) patients had a fresh embryo replacement accompanied by embryo cryopreservation, which was not significantly different from the EC group (62/182; 34.1%). The livebirth rate per fresh embryo transfer in the EC group (17/62; 27.4%) was significantly higher than that for the PN group (8/72; 11.1%; P < 0.05). Embryo survival following thawing was similar for the PN (96/129; 74.4%) and EC (79/102; 77.4%) stages. Although not significant, the livebirth rate following the transfer of thawed embryos was higher in the PN group (11/44; 25.0%) than in the EC group (4/38; 10.5%). Following one fresh and two freeze-thaw embryo replacements, the observed cumulative viable pregnancy rates were comparable for patients in both the PN (40.2%) and EC (41.1%) groups.

Clinical value of tests for assessing male infertility.

The laboratory assessment of the male partner of an infertile couple is an important aspect of the overall investigation of that couple. The laboratory tests are designed essentially to determine whether (a) the semen samples contain adequate numbers of normal motile sperm, and the sperm are able (b) to migrate to the site of fertilization and (c) to fertilize oocytes. Within this framework, tests can be viewed as being either descriptive, in terms of describing the ejaculate and sperm, or assessing functional qualities of the sperm. Irrespective of the nature of the test, it must satisfy simple criteria, namely being reproducible and able to discriminate between the fertile and infertile populations reliably. External quality assurance programmes now exist for semen analysis and allied techniques to help laboratories to standardize their reporting and to identify the source of possible errors.

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 quarantine of fertilized donated oocytes.

The fertilization and subsequent cryopreservation of donated oocytes have enabled the resulting embryos to be quarantined for a minimum of 6 months, and to be thawed and replaced only after the donor has had a second negative human immunodeficiency virus (HIV) test. In the study described here, a total of 39 women had 56 embryo transfers, and 12 pregnancies (21% per transfer) were achieved. The logic of the protocol for minimizing the risk of infection of the recipient, which is in line with that of semen donation, is presented, together with an argument for the feasibility of such an approach.

Use of buserelin and low-dose human menopausal gonadotropin for in vitro fertilization in women at risk of ovarian hyperstimulation syndrome.

PURPOSE: The purpose of the present study was (i) to assess the value of using a low dose of hMG (75 IU/day) to achieve ovarian stimulation in women who have previously shown an exaggerated response to a standard dose of 150 IU human menopausal gonadotropin/day in a desensitization (group I) or flare-up (group II) protocol and (ii) to determine whether the choice of GnRH-a regimen in a subsequent cycle, namely, a desensitization or flare-up protocol, influenced the effectiveness of the low dose of hMG. RESULTS: In group I, 75% (12/16) and 57% (8/14) of the subsequent desensitization and flare-up protocols, respectively, were cancelled because of inadequate ovarian response. Similarly, the cancellation rates in group II were 10 of 10 and 7 of 11 (64%), respectively. The total cancellation rate (groups I and II together) with the desensitization protocol was higher than that using the flare-up protocol (P < 0.05). CONCLUSION: The simple use of a reduced dose of hMG (75 IU/day) for subsequent in vitro fertilization in women to minimize the risk of the development of ovarian hyperstimulation is of limited benefit since a large proportion then shows an inadequate response. This is particularly pronounced with a subsequent desensitization protocol which does not utilize endogenous gonadotropins to initiate follicular development.

External quality assessment for semen analysis and sperm antibody detection: results of a pilot scheme.

The need for quality assurance in the seminology laboratory is clear, as the techniques of semen analysis and sperm antibody detection are just as susceptible to variation as any other routine pathology test. Semen samples were distributed to 20 laboratories on six occasions, four samples per distribution, for sperm concentration and morphology assessment under routine conditions, together with an equal number of serum samples for sperm antibody detection. The semen analysis results showed a wide range of values for any given sample, which did not seem to be related to the methodology used. However, this variation appears to be related to the presence of persistent errors, as most laboratories showed reasonable between-assay and within-assay variation. Detection of sperm antibodies by the tray agglutination, gelatin agglutination or indirect immunobead test showed a consistent discrimination between the intended positive and intended negative samples. However, the use of fluorescent microscopy was unable to do this. This study has shown the feasibility of operating external quality assessment schemes for semen analysis and sperm antibody detection. These schemes provide the opportunity for individual laboratories to fully evaluate their own methods against those of others and to determine the stages at which any errors occur. An increased number of participants will ultimately enable a systematic comparison of different methods.

The routine assessment of sperm motility at room temperature and 37 degrees C.

The World Health Organization (WHO) laboratory manual (1992) states that assessment of sperm motility can be performed at either 37 degrees C or room temperature (20-24 degrees C). The motility of spermatozoa in 44 semen samples (22 fresh samples and 22 frozen-thawed samples) was assessed at both of these temperatures and a significant difference in the motility profiles was noted, specifically an increase at 37 degrees C in the percentage (expressed here as median and ranges) of spermatozoa with excellent progressive motility and an overall increase in the percentage with total progressive motility. With fresh samples the excellent progressive motility increased from 41 (19-53) to 54 (30-66) and the overall motility from 58.5 (39-74) to 65.0 (40-79). With the frozen-thawed samples the excellent motility increased from 14 (1-33) to 25 (6-45) and the overall motility from 30.5 (14-51) to 33.0 (16-52). As the WHO laboratory manual was published 'In response to a growing need for the standardisation of procedures for the examination of human spermatozoa' it is proposed that only one temperature for routine analysis should be used, namely 37 degrees C, which may have more physiological relevance and eliminate effects of fluctuations in ambient laboratory temperature.

The screening for cytomegalovirus antibody in semen donors and recipients within a donor insemination programme.

A total of 67 semen donors (62 Caucasian and five non-Caucasian) were tested for the presence of immunoglobulin G antibodies to cytomegalovirus (CMV). Ten (16%) of the Caucasian donors tested positive for CMV, while four (80%) of the non-Caucasian donors were positive. A total of 131 women receiving donor insemination (114 Caucasian and 17 non-Caucasian) were tested. Of these, 43 (38%) of the Caucasian recipients tested positive for CMV, while 16 (94%) of the non-Caucasian recipients were positive. There was a significant association between the incidence of positive tests and the age of the Caucasian recipients.

The acrosome reaction to ionophore challenge test: assay reproducibility, effect of sexual abstinence and results for fertile men.

Since the human acrosome reaction is considered a prerequisite for normal fertilization and the spontaneous acrosome reaction rate is low, laboratory tests using calcium ionophores to induce the acrosome reaction have been devised and applied to the investigation of patients. The introduction of any new laboratory test into routine clinical practice is usually accompanied by the determination of intra- and inter-subject variability within the normal population, and the derivation of reference values to distinguish between affected and unaffected populations. The acrosome reaction to ionophore challenge (ARIC) test was evaluated and found to have (i) intra- and inter-assay coefficients of variation of 10.8 and 18.8% respectively, (ii) a high degree of intra-subject variability for three subjects studied over a 10 week period, (iii) a high degree of inter-subject variability when aliquots of 20 ejaculates of donor semen of proven fertility were tested, and (iv) no effect of length of sexual abstinence on ARIC values. The results of this study suggest that the use of fresh semen samples from subjects of proven fertility for quality control purposes in the ARIC test may be inadequate due to the high degree of intra-subject variability, and that this problem may be overcome by utilizing a frozen quality control sample. The results also suggest that an isolated negative ARIC test is not necessarily indicative of functionally incompetent spermatozoa, and highlight the importance of examination of the normal population prior to the clinical application of such a test.

The UK Human Fertilisation and Embryology Act 1990--how well is it functioning?

The Human Fertilisation and Embryology Act became law in the UK in 1990, and a statutory body, the Human Fertilisation and Embryology Authority, was established to administer the Act. The opinions of those persons responsible for licensed activity under the Act were canvassed anonymously to assess the initial effect of the Act on their activities, and the administration of the Act by the 'Authority'. The views expressed reflect the opinions of 80% of the 'responsible persons' and are thus likely to be of value to those responsible for administration of the Act and also those planning legislation in this field of human endeavour.