aberrations. The results show that chromosome aberrations in the man

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Introduction: In our unit, patients with teratozoospermia. (~20% normal forms) .... spermatocyte isolated from men with obstructive azoospermia. Abstracts of the ...

Abstracts of the 13th Annual Meeting of the ESHRE, Edinburgh 1997

aberrations. The results show that chromosome aberrations in the man and/or woman influence the success of ICSI. We support the argument for strict cytogenetic screening of male and female ICSI patients. 10.30-10.45 0-164. Effect of post-ovulatory age and omission of Ca2+ from the injection medium on male pronucleus formation and metaphase entry following injection of human spermatozoa into golden hamster oocytes . Goud A.P., Goud PT., Rybouchkin A., De Sutter P. and Dhont M.

Infertility Centre, Department of Obstetrics and Gynaecology, University Hospital, Ghent, Belgium Introduction: ICSI of human spermatozoa into hamster oocytes can provide important information on male pronucleus formation and the karyotype of poor quality human spermatozoa. Hamster oocytes are known to undergo both spontaneous activation with post-ovulatory ageing and injection-induced parthenogenetic activation when punctured in the presence of Ca2+. As we found recently, the mean percentage of male pronucleus formation after ICSI of hamster oocytes with human spermatozoa is inversely correlated with parthenogenetic activation. To study the relative contributions of spontaneous activation and injection-induced parthenogenetic activation to male pronucleus formation and metaphase entry, we observed the behaviour of hamster oocytes when retrieved at different post-ovulatory ages in response to sperm injection with or without Ca2+ in the injection medium. Materials and methods: Superovulated hamster oocytes were obtained at 13, 16 and 21 h post-HCG. Oocytes from each group were injected with spermatozoa prepared in a medium containing either 1.9 (regular) or 0 mM Ca (Ca-free). The spermatozoa were obtained from patients undergoing assisted reproduction treatment. The injected oocytes were checked at 8-12 h after injection for the number of pronuclei developed. They were fixed after pronucleus disappearance to analyse the behaviour of sperm chromatin. The media used for oocyte handling, sperm preparation and culture were HECM-3 with or without HEPES. Results: A progressive fall in the fertilization (2PN) rates was observed with increasing post-ovulatory age in both the Ca-free and regular groups. The mean fertilization (2PN) rate and the percentage reaching metaphase entry were higher in the Ca-free group at 16 and 21 h; difference was more marked in the latter. In the 13 h group both the Ca-free and regular groups had high and similar mean fertilization (2PN) rates. Effect of post-ovulatory age and omission of Ca2 + from the injection medium on male pronucleus formation and metaphase entry Oocyte age (h post-HCG)

13

Injection medium 1.9 mM Ca2 +

Ca2+ -free 16 21

1.9 ruM Ca2+

Ca2+ -free 1.9 mM Ca2+ Ca2 + -free

No. of oocytes Oocyte survival Fertilization injected

rate (%)

69 69 132 146 109 111

79.8 79.7 90.3 75.3 82.1 74.0

± ± ± ± ± ±

2.1 1.2 3.7' 5.4' 3.7' 3.1'

Metaphase

(2PN) rate (%) entry rate (%)a 662 ± 2.5 68.5 ± 7.0 36.6 ± 5.2b 51.0 ± 7.gb 11.1 ± 1.2' 22.2 ± 2.0'

53.7 58.9 26.9 36.3 5.2 13.3

± ± ± ± ± ±

aAll figures are means :t SEM. Differences in the groups were significant: a,b,dp < 0.04; cp < 0.02; ep < 0.001; fp < 0.01.

80

1.9 5.8 5.8' 6.5' 1.1' 2.2'

Conclusion: The injection of human spermatozoa into postovulatory young oocytes leads to a significant and high increase in the rate of male pronucleus formation. The beneficial effect of Ca omission on male pronucleus formation may be attributed to the differential sensitivity of hamster oocytes to injectioninduced parthenogenetic activation at later post-ovulatory stages. Our findings provide important information for the development of a new method of human sperm karyotyping following ICSI using hamster oocytes.

10.45-11.00 0-165. A prospective, controlled trial of ICSI versus IVF for the treatment of teratozoospermia Payne D., Flaherty S.P. and Matthews C.D. Reproductive Medicine Unit, Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital and Wakefield Clinic, Adelaide, Australia

Introduction: In our unit, patients with teratozoospermia (~20% normal forms) but adequate sperm recovery have a high risk of poor fertilization «35%) after routine IVF (Duncan et al., 1993). However, some will achieve normal fertilization rates. So committing all these couples to ICSI adds unnecessary cost for some, while requiring them all to have routine IVF first will result in failed fertilization for many. In this prospective study we used ICSI with control IVF oocytes in the same cycle to determine which method was appropriate for each couple. Materials and methods: Couples with eight or more mature oocytes collected and semen suitable for IVF (>500 000 motile spermatozoa after washing) but with ~20% normal morphology in the 'neat' semen were offered ICSI in their first treatment cycle. However, at least four of the oocytes were inseminated by routine IVF (control group). Oocytes were denuded prior to both procedures. Results: In all, 92 couples were admitted to the trial. Of these, 90 couples had a fresh embryo transfer resulting in 27 pregnancies (pregnancy rate 30%, implantation rate 13.5%). The transfers comprised ICSI embryos only (n = 81), IVF embryos only (n = 3) or a mixture of ICSI and IVF embryos (n = 6). The other two couples had embryos frozen because of ovarian hyperstimulation, and one pregnancy resulted from the transfer of thawed embryos. Overall the ICSI fertilization rate was 78% (636/814) while the routine IVF fertilization rate was only 26% (136/519). A total of 43 couples (47%) had failed fertilization by routine IVF, but they all had ICSI embryos transferred and achieved good pregnancy (35%) and implantation (16.6%) rates. In all, 19 couples (20%) achieved >50% ferti,lization by routine IVF. i/

Conclusions: About half of the couples with teratozoospermia (~20% normal forms) had complete fertilization failure by routine IVF, while 20% achieved satisfactory fertilization. Hence, when adequate numbers of mature oocytes are available in the first treatment cycle, the insemination of some oocytes by routine IVF and the remainder by ICSI identifies those couples who can anticipate normal fertilization in future routine

Abstracts of the 13th Annual Meeting of the ESHRE, Edinburgh 1997

IVF attempts and those who clearly require IeSL This approach does not compromise treatment in any way. Reference: Duncan et a!. (1993) Ferti!. Steril., 59, 1233-1238.

11.00-11.15 0-166. Deficiency in the oocyte-activating substance in spermatozoa: a cause of ICSI failure Sofikitis N.V., Kanakas N.!, Mantzavinos T., Antypas S., Loutradis D.2, Agapitos E.3 and Miyagawa 1. Reproductive Physiology and IVF Center, Department of Urology, Tottori University, Yonago, Japan, lA and 2B Departments of Obstetrics and Gynaecology and 3Department of Pathology, Athens University, Athens, Greece Introduction: The human sperm nucleus can undergo dec ondensation within inactivated oocytes under optimal conditions. However, oocyte activation is a prerequisite for the transformation of the male gamete nucleus into the pronucleus within the ooplasm. We attempted to investigate whether an assay evaluating oocyte-activating substance in spermatozoa (OASIS) activity can indicate the lack of oocyte activation due to subnormal sperm OASIS profiles as a cause of IeSI failure. Materials and methods: Human OASIS is known to activate hamster oocytes. One, two or three spermatozoa from fertile men (n = 11; group A), idiopathic infertile men (n = 13; group B), infertile men with two failed IeSI cycles resulting from a lack of oocyte activation (n = 4; group C) and participants of an IeSI programme (n = 15; group D) were injected into mature hamster oocytes using an electric pipettedriving microfertilization unit. Spermatozoa from men in group e failed to activate human donor oocytes. The X2 test (Yates' correction) was used for statistical analysis. Results:

No. of spermatozoa 2

A B C D

Conclusions: OASIS activity/content is significantly lower in infertile men than in fertile men. Furthermore, injections of one, two or three spermatozoa into ha!llster oocytes in two men from group B and two men from group e did not activate even a single oocyte. It appears that a subpopulation of infertile men has subnormal OASIS profiles. The significant differences in the percentage of hamster oocytes activated after injections of one or two spermatozoa between groups A and B and the absence of significant differences in the percentage of hamster oocytes activated after injections of three spermatozoa may suggest that spermatozoa from men with subnormal OASIS profiles can induce hamster oocyte activation if an additional amount of OASIS is provided. This hypothesis is supported further by the activation of three oocytes after injections of three spermatozoa in group e. Human secondary spermatocytes may have a lower OASIS content/activity than round spermatids, elongated spermatids, elongating spermatids and testicular spermatozoa. Injections of human spermatozoa into hamster oocytes (preferably two spermatozoa/oocyte) may reveal a subpopulation of men with a sperm OASIS content insufficient to activate human oocytes via IeSI procedures.

11.15-11.30 0-167. Treatment of male infertility caused by spermatogenic arrest at the primary spermatocyte stage with ooplasmic injections of round spermatids or secondary spermatocytes isolated from foci of early haploid male gametes Sofikitis N.V., Mantzavinos T.!, Loutradis D.2, Antypas S., Miyagawa 1. and Tarlatzis v. 3

Effects of a deficiency in OASIS Group

were injected into hamster oocytes. The mean percentage of hamster oocytes activated was similar after testicular spermatozoon, elongated spermatid, elongating spermatid or round spermatid injections. However, the percentage of hamster oocytes activated was significantly lower (P < 0.05) after injection of a secondary spermatocyte.

3

10

AO (%)

10

AO (%)

10

AO (%)

33 39 16

14 (42)a 6 (l5)b 0(0)

33 39 12 150

18 (54)a 8 (25)b 0(0) 35 (23)b

22 26 8

14 (63)a 12 (49)a 3 (37)

10 = injected hamster oocytes; AO a versus b: P < 0.05.

=

activated hamster oocytes.

Within group D, the percentage of human oocytes activated by IeSI was ~20%, while the percentage of hamster oocytes activated by two spermatozoa was ~1O%. Furthermore, no human oocytes were activated in cycles when the percentage of hamster oocytes activated equalled 0%. In additional experiments a testicular spermatozoon, an elongated spermatid, an elongating spermatid, a round spermatid or a secondary spermatocyte isolated from men with obstructive azoospermia

Reproductive Physiology and IVF Center, Department of Urology, Tottori University School of Medicine, Yonago, Japan, lA and 2B Departments of Obstetrics and Gynaecology, Athens University, Athens and 3Department of Obstetrics and Gynaecology, Aristoteleion University School of Medicine, Thessaloniki, Greece Introduction: Ooplasmic injections of round spermatids in the mouse, rabbit and human have resulted in pregnancies. The objective of this study was to use ooplasmic injections of spermatozoa, round spermatids or secondary spermatocytes for the treatment of non-obstructive azoospermia with spermatogenic arrest at the primary spermatocyte stage. Materials and methods: Testicular biopsy specimens from 36 men with a diagnosis of spermatogenic arrest at the primary spermatocyte stage were processed for the recovery of spermatogenic cells. Spermatozoa, round cells with acrosomal granulae (round spermatids of stage ~2) and round cells of 12-13 11m diameter without acrosomal granulae (secondary spermatocytes or stage 1 round spermatids) were found in 81

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