Fertilization of human oocytes in capillary tubes with very small numbers of spermatozoa

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1 Human Reproduction vol no. pp.-6, 99 Fertilization of human oocytes in capillary tubes with very small numbers of spermatozoa Hans H.van der Ven, K.Hoebbel, S.AI-Hasani, K.Diedrich and D.Krebs University of Bonn, Department of Obstetrics and Gynaecology, Sigmund-Freud-Strasse, D-OO Bonn, FRG Human oocytes can be fertilized with high rates of success under in-vitro conditions even if only low numbers of spermatozoa are used. A culture system has been developed in which fertilization is performed in haematocrit capillary tubes (length mm; i.d mm). Oocytes were fertilized in -0 /d of different sperm s containing a total of 00, 000, 000 and 000 spermatozoa per oocyte (0.-0. x 0* spermatozoa/ml). Oocytes were obtained from 0 patients participating in an in-vitro fertilization programme; of these, oocytes were fertilized in capillary tubes and oocytes were cultured using standard methods ( ml culture medium in tissue culture tubes; x 0* spermatozoa/ml). The overall fertilization rate of oocytes cultured in tissue culture tubes was % (/) and the fertilization rates in capillary tubes using 000, 000, 000 or 00 spermatozoa per oocyte were % (/), 6% (6/), 60% (6/0) and 0% (/), respectively. The fertilization rate of mature oocytes was higher compared with immature oocytes when fertilization was performed in culture tubes ( and 6%) or in capillary tubes ( and %). Fertilization in capillary tubes using a 0 /tl of oocyte and spermatozoa compared to ^ seemed to provide better culture conditions, resulting in higher fertilization and cleavage rates. These preliminary results indicate that fertilization of human oocytes under in-vitro conditions can be achieved even with very low numbers of spermatozoa. Key words: IVF/spermatozoa/volume Introduction According to investigations of Ahlgren (9), only a few hundred spermatozoa reach the ampulla of the oviduct (the natural site of fertilization) in humans. Accordingly, this relatively small number of spermatozoa appears to constitute a selection of spermatozoa with high fertilization capacity, which ensure a high degree of successful fertilization in the favourable medium of the oviduct. In contrast to the situation in the oviduct, conventional in-vitro fertilization systems need relatively large numbers of spermatozoa in order to attain high rates of fertilization. Various sperm concentrations are used for in-vitro fertilization in humans. In most cases, 0 (XX) mobile spermatozoa are added per oocyte. However, there are individual variations from million spermatozoa per oocyte between different laboratories, culture systems and semen qualities (e.g. Lopata et al., 90; Veeck et al., 9; Mahadevan and Trounson, 9; Cohen et al., 9; Diamond et al., 9). In this study, a culture system is described which enables successful fertilization of human oocytes and normal subsequent embryonic development using very small quantities of spermatozoa. Materials and methods Ovarian stimulation and harvesting of oocytes In this study, the patients exclusively received HMG (human menopausal gonadotrophin) to stimulate the ovary. The HMG treatment consisted of daily administration of two ampoules of HMG ( IU FSH and IU LH per ampoule) from day to day 6 of the menstrual cycle followed by three ampoules of HMG per day up to the day before administration of IU human chorionic gonadotrophin (HCG). In cases of insufficient ovarian response, the HMG dose was raised individually. Follicular development was monitored by daily ultrasound investigation and by determination of serum oestradiol (E) and luteinizing hormone (LH). When the leading follicle attained a diameter of 0 mm and the serum oestradiol value reached a level of pg/ml for each follicle over 6 mm diameter, IU HCG was administered and follicular puncture was carried out 6 h later. The maturity of the oocytes was classified in accordance with morphological criteria (Veeck et al., 9). The major proportion of the cumulus oophorus was then mechanically removed by means of fine needles. Each oocyte was preincubated for 6 h in ml culture medium in ml culture tubes (Falcon no. 0) before spermatozoa were added or the oocytes were introduced into the capillary tubes. Culture conditions For oocyte preincubation, fertilization and embryo culture, modified Ham's F0 medium was used as culture medium (Lopata etal., 90). In addition, 0% heat-inactivated umbilical cord serum was included in the medium. For better maintenance of the culture medium within the culture and capillary tubes, these culture vessels were placed in a desiccator which was in turn located in a CO^-incubator (Forma scientific, model 6). The floor of the desiccator was filled with distilled water and a gas mixture (% CO, % O, 90% N ) was continuously released into the desiccator through the water. This incubator-desiccator IRL Press

2 Human oocyte IVF with low spermatozoa levels system ensures constancy in the culture conditions (temperature, osmolarity, ph) which may otherwise be altered slightly by opening and closing the incubator (Kruger et al., 9; Abramczuk and Lopata, 96). Semen processing After complete liquefaction, a standard semen analysis was performed on the semen specimens. Semen specimens were classified as normospermic when they fulfilled the following criteria: >0 X 0 6 spermatozoa/ml, >0% progressively motile spermatozoa, >0% normally formed spermatozoa, > ml volume. Normozoospermic semen samples were mixed with culture medium (:) and separated from semen plasma by washing twice (00 g, min). The amount of ejaculate used was determined by the sperm concentration in the ejaculate. Finally, the sedimented spermatozoa were carefully overlayered with fresh culture medium (0..0 ml). The culture tube was then incubated for 0-60 min (% CO, C) in order to enable the spermatozoa to swim up into the supernatant. The upper half of the supernatant, normally with >% motile spermatozoa, was removed and the semen concentration and motility determined three times in the Makler counting chamber. Oocytes in culture tubes were inseminated with 0. or 0. X 0 6 spermatozoa (see below). For fertilization in capillary tubes, various semen s (0., 0. or 0. x 0 6 spermatozoa/ ml) were prepared in ml equilibrated culture medium (% CO, % O, 90% N, C for 6 h) and incubated until use in the desiccator-incubator system. Semenoocyte incubation In patients with normozoospermic semen samples, two methods of sperm-oocyte incubation were performed. Some oocytes from each patient were incubated in ml culture tubes (Falcon no. 0) with X 0 6 spermatozoa in ml medium in accordance with the standard method of this laboratory. The remaining oocytes of each respective patient were cultivated in sterile, nonheparinized haematocrit capillary tubes (R.Brand, 690 Wertheim, no. 9-0; length, mm; i.d., mm). In the capillary tube, each oocyte was incubated with -0 jtl medium containing spermatozoa. The capillary tube was filled as follows. Equilibrated culture medium, equilibrated paraffin oil, sperm and the oocytes in culture medium were placed into single 'centre well' Petri dishes (Falcon no. 0) and incubated until use in the incubator desiccator system. Sterile capillary tubes in sterilization bags were placed on a hotplate ( C) for several minutes. Afterwards, one capillary, each with an adapter, (Unopette, no., Becton and Dickinson) was mounted on a tuberculin syringe (Rekord mounting) (Figures and ). The capillaries were then flushed a few times with fresh culture medium in order to remove loose glass particles and to warm the capillary. The capillary was then filled in accordance with the description in Figure. The semen-oocyte ( -0 /il) was surrounded by a small amount of sterile, equilibrated paraffin oil on both sides in the capillary and was thus protected from rapid alterations of the culture medium. Instead of the paraffin oil, the semen-oocyte could also be protected by culture medium separated Fig.. Tuberculin syringe, adapter and haematocrit capillary tube used in the apparatus. Oil Oil Sperm - oocyte - Fig.. Filling of the capillary tube with the of spermatozoa and oocytes. Adapter by small columns of air ( /tl). A volume of 0 jil of medium fills the capillaries over a length of ~ mm. To take up the spermoocyte, the oocytes can either be added to the semen beforehand and spermatozoa and oocytes can be drawn up together afterwards or spermatozoa and oocytes can be drawn up consecutively. Here, care must be taken that, if possible, the oocyte lies in the middle of the semen column within the capillary and that no bubbles of air are aspirated between the oocyte and the semen. The sticky cumulus complex can be mechanically reduced in size beforehand (by preparation with fine needles) for easier aspiration into the capillary. After some practice, the capillary can be filled in a few seconds. For protection of the sterile ends of the capillaries, the adapter can be left on one end and a further adapter can be mounted on the free end (Figure ). The adapter can also be used to mark the capillaries. For better positioning of the capillaries in the incubator, the capillary tubes can be inserted into test tube racks (e.g. Figure ). Fertilization and embryo culture The sperm-oocyte was incubated in culture or capillary tubes for -6 h. Afterwards, the cumulus cells still adhering were mechanically removed from the oocytes and the oocytes were investigated for the presence of pronculei. The oocytes could be readily flushed out of the capillary tubes. For this purpose, the adapters were carefully removed from the capillaries. A syringe filled with culture medium was introduced via a fine needle (Gauge ) and the oocyte was flushed out. After observation of two or more pronuclei as signs of fertilization, all embryos were cultivated singly in -ml culture tubes in ml medium for a further -0 h up to embryo transfer. The embryo quality was appraised according to morphological criteria (symmetry of the blastomeres, presence of fragmentation and granulation) and classified as poor, good and very good. Results In 0 patients from the in-vitro fertilization programme, a total of 6 oocytes was incubated with normospenmic semen from the

3 H.H.van der Ven et al. Table I. Fertilization of human oocytes in tissue culture tubes and in capillary tubes Motile sperm per oocyte , Volume of sperm-oocyte Sperm concentration per ml Patients (n) Test tube.0 ml x 0* 0 i.t\ /tl. 0 M>. 0 Ml 0. x x 0" 0., 0. x x 06 Culture vessel Fertilized oocytes Oocytes Total Mature Immature (%) 0 (%) (6%) 6 (%) 6(6%) 6 (60%) (0%) total mature 0 0 immature Table n. Fertilization of human oocytes in capillary tubes: effect of volume of sperm-oocyte on fertilization rate and early embryo development Volume of spermoocyte Test tube sperms sperms 00 sperms.0 ml 0 n\ Ml /«! Oocytes Cleavage rate Cleavage stage - - cell embryos >6 Poor Good Very good Embryo quality Total Fertilized (%) / (%) 9 6 (%) (6.%) (0%) / (9%) / (60%) / (0%) Oocytes in capillary tubes and tissue culture tubes were separated from spermatozoa after -6 h and then cultured in tissue culture tubes in ml of medium. See Material and methods for detail. husbands; oocytes were incubated in culture tubes in accordance with standard culture conditions and oocytes were cultured with different amounts of spermatozoa in capillary tubes. As shown in Table I, the rate of fertilization for oocytes in culture tubes was % (/ oocytes). The fertilization rate of mature oocytes (0/) was in excess of that of immature oocytes (/). Fertilization could also be achieved in capillary tubes. At comparable semen concentrations ( or 0. x 06 spermatozoa/ml), however, a total of only spermato zoa per oocyte was present in the small volume of 0 /tl in capillary tubes. The rate of fertilization of oocytes in capillaries with spermatozoa did not show any significant difference to the rate of fertilization in culture tubes. Although the total number of oocytes in the study is small, the rate of fertilization in capillaries appears to be reduced with only spermatozoa per oocyte (four out of eight or 6 out of 0 oocytes fertilized) compared to higher sperm numbers. However, this may also be due in part to the small volume of Fig.. An illustration of the apparatus.

4 Human oocyte FVF with low spermatozoa levels only y\ semen-oocyte. The rates of fertilization in capillaries containing and 0 /tl medium are listed for comparison in Table II. If semen counts of per oocyte are compared, a volume of 0 fd appears to provide better culture conditions compared to /il leading to higher rates of fertilization and cleavage. A distinct difference between the different culture systems in the embryo quality determined at the time of the embryo transfer could not be observed after cleavage. Discussion The results of this investigation show that human oocytes can be successfully fertilized under in-vitro conditions, even if only very low numbers of spermatozoa are present. Under in-vitro conditions, various prerequisites must be fulfilled for successful fertilization. A spermatozoon capable of fertilization must make contact with a fertile, mature oocyte within a suitable medium. For this purpose, the sperm concentration near the oocyte must be sufficient in order to enable an encounter of spermatozoon and oocyte to be highly probable. The dependence of the rate of fertilization on the concentration of mobile spermatozoa can be shown in numerous animal species (Bavister, 99; Siddiquey and Cohen, 9; Corselli and Talbot, 96) and also in the heterologous system of hamster oocytes and human spermatozoa (Martin and Taylor, 9; Van der Ven et al., 96). It is interesting to note that human spermatozoa show an as yet unclarified impairment of their quality (Mann, 96) with a high dilution of semen concentration. For in-vitro fertilization in humans, usually spermatozoa per oocyte are used in a volume of ml. Higher sperm concentrations appear to lead to a reduction in the rates of fertilization (Mahadevan and Trounson, 9) and to an increase in the rate of polyploidy (Van der Ven et al., 9) in normozoospermic specimens. On the other hand, for pathological semen specimens, an increase in the sperm concentration (up to 0. x 0 6 per oocyte) appears to bring about an improvement in the rate of fertilization (Mahadevan and Trounson, 9; Wolf et al., 9; Diamond et al., 9). The lowest sperm numbers with which successful in-vitro fertilizations of human oocytes can be attained so far are reported by Mahadevan and Trounson (9) with spermatozoa and by Cohen et al. (9) with ~ spermatozoa. Mahadevan and Trounson (9) used ml test tubes filled with ml culture medium as culture systems. Cohen et al. (9) used microdroplets of 0. ml which were overlayered with paraffin oil (Purdy, 9). By reduction of the volume of the sperm-oocyte and also by modification of the culture medium, successful fertilizations can be carried out in hamsters and in mice, even with very low sperm counts (Bavister, 99; Siddiquey and Cohen, 9; Corselli and Talbot, 96). Various culture sysems have been used successfully for human in-vitro fertilization, e.g. test tubes, Petri dishes, multi-well dishes, centre-well dishes. The volume of the semenoocyte was ml in most cases. For better maintenance of the culture medium (osmolarity, ph, temperature), especially in use of low volumes of culture medium, it has been recommended that the culture medium be overlayered with paraffin oil (Purdy, 9). The capillary system described in this study uses only very low medium volumes (-0 yx). For this reason, it might later be susceptible to alterations in the culture conditions (especially outside the incubator). The results of fertilization in the capillary tubes are very satisfactory, however, and contradict the specified theoretical disadvantages of this culture system. This might possibly be due to the rapid and simple handling of the capillary-pipette system, the low surface/volume ratio of the culture medium within the capillaries as well as the stable culture conditions within the incubator-desiccator system. The capillary system described here is able to maintain culture conditions which enable successful fertilization of human oocytes and thus might also be used for investigations of the process of human fertilization with very low sperm numbers. Although spermoocyte of -0 /tl, a sperm concentration of X lofyml, a total sperm count of and a sperm-oocyte incubation of -6 h were used for normozoospermic semen samples in this study, the most favourable culture conditions must still be determined. The results available so far indicate that the use of 0 ^ compared to /tl for the sperm-oocyte incubation as well as higher sperm numbers ( 000 per oocyte) appears to be advantageous. The incubation time of spermatozoa with oocytes may possibly be shortened, since the investigations of Plachot et al. (96) were unable to show any difference in rates of fertilization after - h compared with -0 h. In addition, a shorter incubation time, especially in the use of small voumes, might reduce negative effects on the culture medium by metabolic activity of spermatozoa and cumulus cells. On the other hand, although it is not essential for successful fertilization (Mahadevan and Trounson, 9), the cumulus oophorus nevertheless appears to promote successful fertilization and increase sperm motility, capacitation and acrosome reactions (Rogers, 9; Bradley and Garbers, 9). Investigations in this laboratory were able to show an increase in sperm binding and oocyte penetration in the heterologous hamster oocyte system in the presence of human cumulus cells (unpublished results). Besides various substances (e.g. steroids, proteins), cumulus cells may secrete a glycoprotein which binds to spermatozoa and is possibly significant in capacitation and fertilization (Tesarik et al., 9). The effect of certain cumulus constituents on fertilization could also be demonstrated in the hamster (Bavister, 9). In the event that secretion products of cumulus cells are significant in the process of human fertilization, these might possibly be present at a higher concentration in the small volume of the capillary system, compared to conventional culture systems. In this case, fertilization in capillary tubes might indeed afford an advantage compared to conventional culture systems. Relatively small volumes and sperm numbers are also used in the culture system described by Cohen et al. (9): 0. ml microdroplets with spermatozoa per oocyte. On the other hand, even smaller volumes (up to ~ /xl) can be taken up simply and very quickly in the capillary system. The observation that mature oocytes show a higher rate of fertilization than immature oocytes is consistent with the investigations of Diamond et al. (9) and Trounson et al. (9). But Veeck et al. (9) were unable to detect any reduction in the rate of fertilization, compared to pre-ovulatory oocytes after

5 H.H.van der Ven et al. preincubation of immature oocytes for h. However, the number of oocytes which underwent normal cell division after fertilization (and thus the proportion of transferred embryos) was reduced in the group of immature oocytes (Veeck et al., 9). In this connection, it is interesting to consider the investigations of Vanderhyden et al. (96), who were able to show that the in-vitro fertilization of rat oocytes can lead to defects in embryos with a delay in early embryonic development and a high proportion of disorders in implantation. For successful fertilization with low sperm numbers, it is important to maximize the probability of encounter of spermatozoa with oocytes. Under in-vitro conditions, this can be attained by the reduction in volume of the sperm-oocyte and possibly by constant transport of spermatozoa from the lower genital tract with the relatively large dimension of the cumulusoocyte complex within the small lumen of the Fallopian tube under in-vivo conditions. The results of in-vitro fertilization with low quantities of spermatozoa in capillary tubes demonstrate the effectiveness of sperm preparation techniques and of culture medium and conditions. They question the existence of a very effective, specific 'fertilization-promoting factor' in the oviduct fluid. In summary, it can be observed on the basis of these preliminary results that successful fertilization of human oocytes can be attained with only very low sperm numbers even under in-vitro conditions. The capillary method hence opens up the possibility of investigating the process of fertilization with 'physiological' sperm numbers. In addition, the capillary method might possibly be used in the treatment of male infertility, especially in the presence of low sperm counts, e.g. in oligozoospermia, or spermatozoa collected from an alloplastic spermatocele and before considering extreme therapies such as heterologous insemination or the micro-injection of spermatozoa into oocytes. However, the capillary method can only increase the possibility of contact between spermatozoa and oocytes. A direct enhancement of the fertilization capacity of spermatozoa after they have penetrated the cumulus oophorus cannot be attained with this procedure. References Abramczuk.J.W. and Lopata,A. (96) Incubator performance in the clinical in vitro fertilization program: importance of temperature conditions for the fertilization and cleavage of human oocytes. Fertil. Steril., 6, -. Ahlgren,M. (9) Sperm transport to and survival in the human fallopian tube. Gynaecol. Invest., 6, 06-. Bavister.B.D. (99) Fertilization of hamster eggs in vitro at sperm: egg ratios close to unity. J. Exp. Zool., 0, 9-6. Bavister.B.D. (9) Evidence for a role of postovulatory cumulus components in supporting fertilizing ability of hamster spermatozoa. J. Androl.,, 6-. Bradley.M.P. and Garbers.D.L. (9) The stimulation of bovine caudal epididymal sperm forward motility by bovine cumulusegg complexes in vitro. Biochem. Biophys. Res. Commun.,,. Cohen,J., Edwards,R., Fehilly.C, Fishel.S., Hewitt.J., Purdy,J., Rowland.G., Steptoe.P. and Webster,J. (9) In vitro fertilization: a treatment for male infertility. Fertil. Steril.,, -. Corselli.J. and Talbot.P. (96) An in vitro technique to study penetration of hamster oocyte-cumulus complexes by using physiological 6 numbers of sperm. Gamete Res.,, 9-0. Diamond,M.P., Rogers,B., Vaughn.K. and Wentz,A.C. (9) Effect of the number of inseminating sperm and the follicular stimulation protocol on in vitro fertilizauon of human oocytes in male factor and non-male factor couples. Fertil. Steril.,, Kruger.T.F., Lopata,A., Rosichme,R.N., De Villiers.J.N., Stander,F.S.H., Van der Merwe.J.P., Smith,K., Menkveld.R. and van Zyl.J.A. (9) Comparative analysis of in-vitro fertilization methods for establishing successful embryo transfer clinics. Ada Eur. Fertil., 6, -0. Lopata.A., Johnston.I.W.H., Hoult.I.J. and Speirs,A.I. (90) Pregnancy following intrauterine implantation of an embryo obtained by in-vitro fertilizauon of a preovulatory egg. Fertil. Steril.,,. Mahadevan,M. and Trounson.O. (9) The influence of seminal characteristics on the success rate of human in-vitro fertilization. FertiL Steril.,, Mahadevan,M. and Trounson.O. (9) Removal of the cumulus oophorus from the human oocyte for in-vitro fertilization Fertil. SteriL,, 6-6. Mann,T. 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Tesarik.J., Kopecny.V. and Dvorak,M. (9) Selective binding of human cumulus cell-secreted glycoproteins to human spermatozoa during capacitation in vitro. Fertil. Steril.,, Trounson.A.O., Mohr,L.R., Wood,C. and Leeton.J.F. (9) Effect of delayed insemination on in-vitro fertilization, culture and transfer of human embryos. J. Reprod. Fertil., 6,. Vanderhyden,B., Rouleau.A., Walton.E.A. and Amstrong,D.T. (96) Increased mortality during early embryonic development after in-vitro fertilization of rat oocytes. J. Reprod. Fertil.,, 0. Van der Ven,H., Al-Hasani,S., Diedrich.K., Hamerich,U., Lehmann,F. and Krebs.D. (9) Polyspermy in in-vitro fertilization of human oocytes: frequency and possible causes. In Sepalla,M. and Edwards,R.G. (eds), In Vitro Fertilization. New York Academy of Science, p.. Van der Ven,H., Jeyendran.R.S., Perez-Pelaez,M. and Zaneveld.L.J.D. (96) Does sperm motility reflect the potential fertilizing ability? Int. J. Fertil.,, -6. Veeck,L., Wortham.J.W., Witmeyer,J., Sandow,A., Acosta.A., Garcia,J., Jones,S. and Jones,W. (9) Maturation and fertilization of morphologically immature human oocytes in a program of in-vitro fertilization. Fertil. Steril., 9, Wolf.D.P., Byrd,W., Dandekar,P. and Quigley.M.M. (9) Sperm concentration and the fertilization of human eggs in vitro. Biol. Reprod.,,. Received on March, 9; accepted on July II, 9