The Choice of the Most Appropriate Microfertilization Technique for Human Male Factor Infertility

Transcription

1 Reprod. Fertil. Dev., 1994, 6, The Choice of the Most Appropriate Microfertilization Technique for Human Male Factor Infertility Alan 0. Trounson Centre for Early Human Development, Institute of Reproduction and Development, Monash Universify, Monash Medical Centre, Clayton, Vic. 3168, Australia. Abstract: Comparisons were made among techniques used to treat male factor infertility. Patients with semen quality below that recognized by World Health Organization criteria as normal had a better success rate when treated by gamete intrafallopian transfer than by in vitro fertilization (25% v. 7% pregnancy rate per patient). When t2x lo6 motile sperm were recovered, the fertilization rate and embryo cleavage rate were higher for microdrop insemination than for conventional insemination. When motile sperm were recovered, microdrop insemination resulted in a higher fertilization rate (46%) and a higher incidence of pregnancies (23% of patients treated) than subzonal sperm microinjection (SUSM). However, for patients with motile sperm, the immediate transfer of SUSM oocytes to the Fallopian tube increased pregnancy rates for this technique to 24% of patients treated. Direct microinjection of epididymal sperm from azoospermic men into the cytoplasm of oocytes resulted in pronuclear formation in 27% of oocytes; in comparison, pronuclear formation occurred in 5% of SUSM oocytes. These data led to formulation of a logical treatment programme for male factor infertility. Extra keywords: fertilization, micromanipulation, sperm microinjection. Introduction Microfertilization techniques have undergone major developments over the past few years resulting in an array of choices for use with male factor infertility which requires detailed, controlled analysis to determine which method is best for the type of infertility problem presented by the couple. In vitro fertilization (IVF) enables sperm to be mixed with oocytes avoiding the bamers in vivo which selectively reduce the number of sperm reaching the site of fertilization in the ampulla. Normally very few sperm are in the vicinity of the oocyte in vivo and the number may be substantially increased by gamete intrafallopian transfer (GIFT), where both sperm and oocytes are transferred to the Fallopian tubes, even in cases of male factor infertility with reduced semen quality (Trounson 1993). Insemination of oocytes with sperm from male factor patients enables large numbers of sperm to interact with oocytes even though a small proportion (<lo%) are capacitated and capable of a physiological acrosome reaction (Holden and Trounson ; Tarin and Trounson 1993). Increasing the probability of collision of the few capacitated sperm with oocytes is important for low-quality sperm samples to achieve fertilization in vitro (Trounson and Osborn 1993). In this article, a range of microfertilization techniques are explored with respect to their use in male factor infertility in order to determine which are appropriate for use at the present stage of development. Data were provided by the Monash IVF infertility clinic, Melbourne. Materials and Methods GIFT v. IVF for Male Factor Infertilify One hundred couples with male factor infertility, according to the World Health Organization (1987) criteria, with at least one patent Fallopian tube in the female partner were allocated to a randomized controlled trial comparing GIFT and IVF; this involved transfer of 2-cell to 4-cell stage embryos laparoscopically to the Fallopian tubes (Calderon et al. 1992). Forty-three patients had GIFT and 47 patients had IVF. There was no difference in mean age of the patients, superovulation protocol used [short or long protocols of treatment with gonadotrophin-releasing hormone analogue and human menopausai gonadotrophin to induce muitipie foiiicuiar growth as described by Calderon and Healy (1993)], mean number of oocytes recovered or semen parameters between the two groups. Conventional Insemination v. Microdrop Insemination Fourteen couples with severe male factor infertility, for which only ~ lo6 motile sperm could be obtained from one or several ejaculates, and who had failed to become pregnant after conventional insemination, were treated by microdrop insemination during a subsequent cycle of IVF treatment. Sperm preparation included the addition of 2.3 mm pentoxifylline to the liquefied semen for 20 min to activate sperm motility and to act as an antioxidant to minimize superoxide damage to the sperm. Motile sperm were isolated on a 90% and 45% Percoll density gradient by centrifugation at 200g for 25 min. The lower portion of the 90% Percoll was retained, 3 mm 2-deoxyadenosine was added, and the sample was centrifuged at 600g for 5 min. All but the last 300 pl was discarded and the sperm were allowed to swim up into this small volume for 30 min at 37 C. The top 200 pl was diluted in human tuba1 fluid (HTF) culture medium (Quinn et al. 1985) and centrifuged at 600g for 5 min. The final sperm pellet (200 pl) was used for insemination.

2 38 A. 0. Trounson For conventional insemination, sperm were added to oocytes in 800,LL of HTF medium with 10% maternal serum at a concentration of x lo6 motile sperm m~-'. For microdrop insemination, sperm were added to oocytes in pl of culture medium under lightweight paraffin oil at a concentration of ~ lo6 motile sperm m~-'. Oocytes were removed from the insemination medium after h, the adhering cumulus cells were removed and the oocytes were then scored for fertilization by assessing the presence of pronuclei and the second polar body. Up to three 2-cell to 4-cell embryos were transferred to the uterus after a further h culture in vitro. Microdrop v. Subzonal Sperm Microinjectiot~ (SUSM) Twelve couples with oligoasthenoteratospermic infertility where only motile sperm were obtained from one or several ejaculates, and one couple in which a normospermic male had failed to fertilize his partner's oocytes on two occasions by conventional IVF insemination, were treated by SUSM. These patients had failed to achieve pregnancy and were treated in a subsequent cycle by microdrop insemination. Sperm were prepared as described earlier for microdrop insemination. Five to 12 sperm were microinjected into the perivitelline space using the techniques for micromanipulation described by Sakkas et al. (1992). Oocytes were examined for pronuclei h after insemination or microinjection, and 1-3 cleaving embryos were transferred to the uterus or Fallopian tubes via the uterine cavity under ultrasound guidance. The further clinical experience of SUSM for 37 patients where less than 5000 motile sperm were obtained from one or several ejaculates, and microdrop insemination for 34 patients where motile sperm were harvested, are also presented. Holding pipette Subzonal Sperm Injection t Injection pipette Transfer to Faiiopian Tubes Fig. 1. Microinjection and intrafallopian tube transfer (MIFT). Microinjection and Intrafallopian Tube Transfer (MIFT) The technique of MIFT has been developed at the Monash IVF infertility clinic (Calderon et al. 1993) and involves the laparoscopic transfer of microinjected oocytes to the Fallopian tube of patients within 1-3 h of SUSM. Normally, 1-5 sperm are microinjected into the perivitelline space after preparation of the sperm with pentoxifylline and 2-deoxyadenosine as described earlier for insemination and microinjection of male factor sperm samples. Up to four microinjected oocytes are placed in the ampulla of the Fallopian tube using GIJT transfer catheters (Fig. 1). The results of the initial 21 cases involving patients with male factor infertility, where motile sperm were recovered from one or more ejaculates, are presented. Intracytoplasmic Sperm Microinjection (ICI) In cases of azoospermia resulting from blockage of the corpus and cauda epididymidis, surgical recovery of spermatozoa can lead to fertilization and pregnancy (Temple-Smith et al. 1985). However, surgical recovery of sperm on each occasion required for insemination of oocytes is a major exercise for the patients and the clinic. The direct injection of epididymal sperm into the cytoplasm of oocytes reduces the number of sperm required and, furthermore, does not require the sperm to exhibit motility. As a result, we are cryopreserving epididymal sperm obtained by aspiration of epididymal ducts at surgery in dilute aliquots of culture medium and in cryoprotectant solutions. The thawed, immotile sperm are washed in culture medium and a single spermatozoon is drawn tail first into the end of a fine microinjection pipette with an outer diameter at the tip of about 7 pm. A sharp bevel is ground onto the tip of the pipette and the pipette is passed through the zona pellucida and into the cytoplasm at a site perpendicular to the polar body to avoid the metaphase plate. The sperm is gently expelled into the cytoplasm and the pipette is withdrawn. Fertilization is checked at 1&20 h and the embryos are cultured for a further h before transfer to the female. Using this technique, the female partner can have numerous cycles of treatment without the need to surgically recover sperm again from the azoospermic partner (Fig. 2). Results GIFT v, W F for Male Factor Infertility The meanf s.e.m. number of embryos transferred in the patients receiving IVF was 2.352~0.17 and in GIFT the meanks.e.m. of oocytes transferred was 3.1 f 0.1. In the IVF group, 17 patients (36%) had no fertilized oocytes and therefore had no embryos transferred. In the GET group, 11 patients had clinical pregnancies (25% pregnancy rate per patient) and in the IVF group there were 3 clinical pregnancies (7% pregnancy rate per patient). In this particular study there was a major benefit of GIFT over IVF for patients with male factor infertility. Conventional Insemination v. Microdrop Insemination A significantly higher fertilization rate was achieved in microdrop insemination (37%) than by conventional insemination (15%) and a higher proportion of oocytes cleaved following microdrop insemination (83%) than conventional insemination (71%) in these severe male factor patients (Table 1). A mean of 2.4 embryos were transferred to patients after microdrop insemination and a further 11 were cryopreserved, compared with a mean

3 Microfertilization Techniques Recovery of ep~didymal sperm qp Embryo transfer Egg recovery Aliquots of sperm cryopreserved Fig. 2. Embryo transfer after intracytoplasmic sperm microinjection (ICI) using sperm obtained by aspiration of epididymal ducts at surgery. Table 1. Success of microdrop insemination compared with conventional IVF Factor Conventional Microdrop IVF insemination No. of cycles (patients) 19 (14) 16 (14) Proportion of fertilized oocytes (2 PN)~ (15%)~ (37%)B Polypronuclear oocytes 2 (2%) 4 (3%), A *-- ucavcu UULY LC> i2 (7i%)" 45 (83%)" Mean no. of embryos transferred Mean embryo score (max. 10) No. of patients with transferred embryos No. of pregnancies 0 0 No. of embryos frozen (% of total) 0 11 (24%) A PN, pronuclei. x2-test, P < X2-test, P < Table 2. Success of microdrop insemination compared with sperm microinjection Factor Preceding Microdrop microinjection cycle insemination cycle No. of patients (cycles) Proportion of fertilized oocytes (2 PN)~ Polypronuclear oocytes Cleaved oocytes Mean no. of embryos transferred Mean embryo score (max. 10) No. of patients with transferred embryos No. of pregnancies (% per EF) No. of embryos frozen (% of total) A PN, pronuclei. x2-test, P < ET, embryo transfer. x2-test, P c: 0.01.

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5 Microfertilization Techniques Table 4. Preferred choice of treatment options for male factor infertility GIFT, gamete intrafallopian transfer; ICI, intracytoplasmic injection of sperm; MIFI: microinjection of oocytes and intrafallopian tube transfer; SUSM, subzonal sperm microinjection Reduced sperm quality Type of male factor infertility Repeated failure of IVF with normospermia Male factor: motile sperm recovered Severe male factor: t1500 motile sperm recovered Azoospermia where epididymal sperm are recovered Preferred treatment Several cycles of GIFT for patients with a patent tube if supernumerary oocytes fertilize in vitro SUSM or ICI Microdrop insemination MIFT or ICI ICI rates (Leeton et al. 1987), irrespective of whether the embryos are transferred to the tubes or uterus. After three cycles of GIFT, more than 50% of these patients deliver babies. Supernumerary oocytes are inseminated in vitro and if pregnancy is not established and supernumerary oocytes fail to fertilize, SUSM or MIFT is normally recommended for further treatment. Male factor patients whose partners have severe tubal pathology are treated by conventional IVF or microdrop insemination depending on the number of motile sperm that can be harvested. Insemination of oocytes in small volumes of medium under oil (200 pl; Cohen et al. 1985) and in straws ( pl; Hammitt et al. 1991) enables fertilization with relatively small numbers of sperm. Insemination of oocytes in pl droplets of medium under oil with relatively concentrated sperm, in a wide range of male factor indications (Trounson and Osborn 1993) yielded good results, although patients who had failed to fertilize oocytes in previous conventional insemination programmes were still a problem. In the present studies, microdrop insemination significantly increased fertilization rates without concentrating sperm and it is clearly the preferred option when motile sperm numbers are decreased. The carefi~l preparation af sperm zxl treatment with motility activators has improved the capacity to obtain progressively motile sperm from a large proportion of severe male factor patients. Providing that there are sufficient sperm for microdrop insemination, the present data indicate that microdrop insemination is preferable to SUSM. It is also apparent that fertilization rates are higher if the cumulus mass is retained around the oocytes rather than denuding them of cumulus cells prior to microdrop insemination (Trounson and Osborn 1993). Microdrop insemination effectively increases the interaction of gametes by reducing the volume occupied, and by minimizing the number of sperm required as well as their by-products from degradation and metabolism. It is apparent that MET increases the effectiveness of SUSM for the treatment of severe male infertility where insufficient sperm are available for microdrop insemination. The pregnancy rate of patients treated in this preliminary trial (25% of patients treated) was equivalent to that seen with GIFT for male factor patients with much less severe problems of sperm quality. Indeed, the clinical pregnancy rate was considerably higher than that determined from our own clinical data for SUSM (Trounson and Sathananthan 1993; Trounson et al. 1993) and than those of some other groups using the SUSM technique (Fishel et al. 1990; Cohen et al. 1991; Sakkas et al. 1992). A further comparison of SUSM and MET in a randomized controlled trial (McLachlan et al. 1993) has confirmed the increased MIFT success rate; MET was more than twice as successful as SUSM. These data suggest that the tubal environment may be beneficial for the induction of sperm-oocyte fusion because the pregnancy rate is higher than that which would be predicted from the fertilization rate (21%) observed after SUSM on the supernumerary oocytes. This observation deserves further exploration. Interestingly, Bongso et al. (1991) reported a significantly increased fertilization rate for conventional insemination of oocytes in coculture with cells from the human ampullary tube when the cells were freshly detached from passaged monolayers. The nature of the effect of human tubal cell secretions on sperm fertilizing ability is unknown, although we have shown that human ampullary cells secrete proteins that pdmg spe= rnotiliijj (C. A. Holden aiid A. Trounson, unpublished observations). Fertilization rates with SUSM tend to be low (12-30%) and vary according to the patients and sperm quality involved in the studies (Fishel et al. 1990; Cohen et al. 1991; Sakkas et al. 1992). Our own clinical results of SUSM in 1991 (Trounson and Sathananthan 1993) which involved 83 patients (1 11 cycles of treatment) resulted in a fertilization rate of 21% (1-10 sperm injected) and 8 pregnancies (10% pregnancy rate per patient), which is less than half the pregnancy rate of MET. As with most other studies, 40% of the SUSM treatment cycles did not result in embryo transfer, which limits the success of the SUSM procedure. The present preference for patients with very low quality sperm, or those for whom few motile sperm can be obtained, is MIFT. Rapid advances have been made with ICI of a single spermatozoon (Trounson and Sathananthan 1993) and our initial studies have involved azoospermic patients for whom surgery is required to obtain sperm from the

6 42 A. 0. Trounson epididymis. Although good results have been reported by Silber and colleagues for IVF involving patients with congenital absence of the vas deferens (Silber et al. 1988), repeated surgery to recover epididymal sperm for each IVF attempt and the disappointing results of our own studies have encouraged us to develop the ICI method for these patients. In our preliminary studies involving seven patients, pronuclear development in 27% of the oocytes compared with 5% for SUSM is very encouraging. We believe that the rate of embryo development will improve as our technical experience increases. The ICI technique is very attractive and, as demonstrated by Van Steirteghem and colleagues (Palermo et al. 1992), it is likely to be the technique of choice in the future for severe male infertility cases, including azoospermic patients who require surgical recovery of epididymal sperm. In conclusion, the preferred treatment options for male factor patients are summarized in Table 4. It is already apparent that male infertility can now be treated very effectively by a number of new microfertilization techniques. Although some of the procedures are still being optimized and experience in micromanipulation is still being obtained, the prospects for treating most types of male factor infertility are excellent. These are dramatic developments which have occurred in a very short time. However, there are many details yet to be elucidated on the specific types of sperm abnormalities and their prospects for resolution, including, for example, the puzzling normospermic husband who continually fails to fertilize in IVF and SUSM and therefore has a poor prognosis for resolution of infertility. References Balmaceda, J. P., Gastaldi, C., Remohi, J., Borrero, C., Ord, T., and Asch, R. H. (1988). Tuba1 embryo transfer as a treatment for infertility due to male factor. Fertil. Steril. 50, Bongso, A., Ng, S.-C., Fong, C.-Y., and Ratnam, S. (1991). Improved fertilization rates of human oocytes in coculture. J. In Vitro Fert. Embryo Transfer 8, Calderon, I., and Healy, D. (1993). Endocrinology of IVF. In 'Handbook of in Vitm Fertilization'. (Eds A. Trounson and D. K. Gardner.) pp (CRC Press: Boca Raton, FL.) Calderon, I., Kovacs, G., Cushnahan, L., and Osborn, J. (1991). GIFT vs. TEST for male infertility. Proceedings of 7th World Congress on in Vitro Fertilization and Assisted Procreations, Paris. p [Abstr.] Calderon, I., Fuscaldo, G., Yates, C., Azuma, K., Osborn, J., and Wood, C. (1992). GIFT versus TEST for male infertility. 48th Annual Meeting of the American Fertility Society, Fertil. Steril. Suppl., 223. [Abstr.] Calderon, I., McLachlan, R., Fuscaldo, G., and Wood, C. (1993). Sub-zonal microinjection and intrafallopian transfer (MIFT): a novel approach to severe male factor infertility. J. Assist. Reprod. Genet. (In press.) Cohen, J., Edwards, R. G., Fehilly, C., Fishel, S., Hewitt, J., Purely, J., Rowland, G., Steptoe, P., and Webster, J. (1985). In vitro fertilization: a treatment for male infertility. Fertil. Steril. 43, Cohen, J., Alikani, M., Malter, H. E., Adler, A., Talansky, B. E., and Rosenwaks, Z. (1991). Partial zona dissection or subzonal sperm insertion: microsurgical fertilization alternatives based on evaluation of sperm and embryo morphology. Fertil. Steril. 56, Fishel, S., Jackson, P., Antinori, S., Johnson, J., Grossi, S., and Versaci, C. (1990). Subzonal insemination for the alleviation of infertility. Fertil. Steril. 54, Hammitt, D. G., Walker, D. L., Syrop, C. H., Miller, T. M., and Bennett, M. R. (1991). Treatment of severe male-factor infertility with high concentrations of motile sperm by microinsemination in embryo cryopreservation straws. J. In Vitro Fert. Embryo Transfer 8, Holden, C. A., and Trounson, A. (1991). Staining of the inner acrosomal membrane of human spermatozoa with concanavalin A lectin as an indicator of potential egg penetration ability. Fertil. Steril. 56, Leeton, J., Rogers, P., Caro, C., Healy, D., and Yates, C. A. (1987). A controlled study between the use of gamete intrafallopian transfer (GIFT) and embryo transfer (ET) in the management of idiopathic and male infertility. Fertil. Steril. 48, McLachlan, R., Fuscaldo, G., Poulos, C., Rho, H., and Calderon, I. (1993). Subzonal sperm microinjection and intrafallopian transfer in severe male infertility. J. Assist. Reprod. Genet. 10, 71. [Abstr.] Palermo, G., Joris, H., Devroey, P., and Van Steirteghem, A. C. (1992). Pregnancies after intracytoplasmic injection of a single spermatozoon into an oocyte. Lancet 340, Quinn, P., Kerin, J. K., and Warnes, G. M. (1985). Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tuba1 fluid. Fertil. Steril. 44, Sakkas, D., Lacham, O., Gianarofi, L., and Trounson, A. (1992). Sub-zonal microinjection (SUSM) in cases of severe male factor infertility and repeated IVF failure. Fertil. Steril. 57, Silber, S. J., Balmaceda, J., Borrero, C., Ord, T., and Asch, R. (1988). Pregnancy with sperm aspiration from the proximal head of the epididymis: a new treatment for congenital absence of the vas deferens. Fertil. Steril. SO, Tarin, J. J., and Trounson, A. 0. (1993). Zona-free sperm penetration assays and inducers of the acrosome reaction: a model for sperm microinjection under the zona pellucida. Mol. Reprod. Dev. 35, Temple-Smith, P. D., Southwick, G. J., Yates, C. A., Trounson, A. O., and de Kretser, D. M. (1985). Human pregnancy by in vitro fertilization (IVF) using sperm aspirated from the epididymis. J. In Vitro Fert. Embryo Transfer 2, Trounson, A. 0. (1993). State of the art and future directions in human IVF, In 'In Vitro Fertilization and Embryo Transfer in Primates'. (Eds R. M. Brenner, D. P. Wolf and R. L. Stouffer.) pp (Serono Symposia: Boston.) Trounson, A., and Osborn, J. (1993). In vitro fertilization and embryo development. In 'Handbook of in Vitro Fertilization'. (Eds A. Trounson and D. K. Gardner.) pp (CRC Press: Boca Raton, FL.) Trounson, A., and Sathananthan, A. H. (1993). Fertilization using micromanipulation techniques. In 'Handbook of in Vitro Fertilization'. (Eds A. Trounson and D. K. Gardner.) pp (CRC Press: Boca Raton, FL.) Trounson, A., Yates, C., Temple-Smith, P., Southwick, G., and Laeham, 0. (1989). The value of IVF for male infertility management. In 'Perspectives in Andrology'. Vol. 53. (Ed. M. Serio.) pp (Raven Press: New York.) Trounson, A., Lacham-Kaplam, O., and Gianaroli, L. (1993). Subzonal and intracytoplasmic sperm microinjection for the

7 Microfertilization Techniques 43 treatment of severe male infertility. In 'Treating Male Infertility: New Possibilities'. (Eds G. M. Colpi and M. Balena.) (S. Karger: Basel.) (In press.) World Health Organization (1987). 'WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction'. (Cambridge University Press: Cambridge.) Yovich, J. L., Blackledge, D. G., Richardson, P. A., Matson, P. L., and Draper, R. (1987). Pregnancies following pronuclear stage tuba1 transfer. Fertil. Steril. 48, Manuscript received 17 November 1993 Lou Warnes (Adelaide): Open Discussion You raised the question of the effect of high concentrations of sperm around the oocyte. Is there any real evidence that there is a problem with the generation of toxic compounds, or do you think it is largely theoretical? Trounson: I guess it's rather empirical. I think most embryologists would agree that if you inseminate human eggs with one million sperm or more, you get fertilization but you get a lot of embryos that don't develop properly. So if you lower the number of sperm for insemination, you get better embryo quality. It seems to me that if you allow all the sperm to float down onto the egg in tubes you might have that situation more than if you did it in microdrops.