Transfer at the blastocyst stage of embryos derived from testicular round spermatid injection

Bulent Urman,1, Cengiz Alatas, Senai Aksoy, Ramazan Mercan, Alp Nuhoglu, Alper Mumcu, Aycan Isiklar and Basak Balaban

The Assisted Reproduction Unit, American Hospital of Istanbul


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Intracytoplasmic injection of testicular round spermatids has been suggested as a salvage treatment in couples when testicular sperm extraction does not yield any mature sperm. However, the success of the procedure is debatable, and controversy surrounds issues such as the presence and (if present) identification of spermatids in testicular tissue. Progression rate to the blastocyst stage of spermatid-derived embryos appears to be low. METHODS: In this study, we investigated the feasibility and outcome of blastocyst stage embryo transfer after round spermatid injection (ROSI). ROSI was undertaken in 58 couples who did not yield mature or elongated sperm to testicular sperm extraction. RESULTS: The incidence of blastocyst formation from two pronuclear oocytes was 7.6%. A total of 16 blastocysts were transferred in 12 patients (20.7%). None of the patients conceived. CONCLUSIONS: The results of this study indicate that the blastocyst stage is reached by only very few ROSI-derived embryos and these embryos do not implant.

Key words: blastocyst/spermatid/ICSI/IVF


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Round spermatid injection (ROSI) of oocytes and subsequent embryo transfer has been associated with very low pregnancy rates, with success reported by only select groups (Tesarik et al., 1996Go; Antinori et al., 1997Go). Results have unfortunately not been reproducible (Silber et al., 2000Go). The poor outcome with ROSI can be attributed to lower fertilization rates and suboptimal embryo quality. We have previously reported a lower and slower rate of blastocyst formation and poor blastocyst quality when round spermatids from azoospermic men with incomplete spermatogenic failure were utilized for intracytoplasmic injection (Balaban et al., 2000bGo). However, the blastocysts derived from round spermatids in this study were not transferred. The aim of the current study was to analyse the outcome of the transfer of blastocysts derived from ROSI.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ROSI, with the aim to transfer the embryos at the blastocyst stage, was undertaken in 58 cycles in 58 couples who did not have any mature sperm or elongated spermatids available. All men suffered from non-obstructive azoospermia, as determined by genital examination, transrectal ultrasound, serum FSH levels and testicular biopsy in some patients. All men were karyotypically normal. Microdeletions of the Y chromosome were not routinely investigated. All female partners were stimulated with a long luteal phase GnRH analogue combined with pure or recombinant FSH. Testicular sperm extraction was performed ~24 h prior to oocyte retrieval. This has been shown to be more convenient for the laboratory personnel. Furthermore, in-vitro culture of testicular sperm was shown to induce motility and increase pregnancy and implantation rates after testicular extraction (Balaban et al., 1999Go). Details of the procedure have been reported previously (Urman et al., 1998Go). As none of the patients described herein yielded mature sperm or elongated spermatids to the biopsy specimen, following informed consent and IRB approval, ROSI was offered to the couple.

Round spermatid identification
Identification, isolation, culture and injection of round spermatids have been described previously in detail (Balaban et al., 2000bGo). Although in stained preparations round spermatid identification is relatively easy, in wet preparations difficulties arise due to its resemblance to other cells in the extracted testicular tissue. Round spermatids were distinguished from other cells, i.e. spermatogonia, spermatocytes and polymorphonuclear leukocytes, according to their smaller size. The diameter of the round spermatid (6.5–8 µm) is similar to that of an erythrocyte and also to small lymphocytes (Tesarik et al., 1996Go; Angelopoulos et al., 1997Go; Vanderzwalmen et al., 1998Go; Verheyen et al., 1998Go).

Round spermatids may be observed at the Golgi, cap and acrosome phases (movement of the nucleus to a peripheral position). Golgi phase is the first phase after secondary spermatocyte division. The size of a Golgi phase spermatid (7–8 µm) is similar to that of a red blood cell and small lymphocyte and it contains a centrally located, rounded and thickened nucleus. Golgi phase round spermatids are very difficult to identify accurately, as they may be easily confused with haematopoetic cells. The round spermatids used in this study were in the acrosome phase (Sa1 spermatid). Sa spermatids, as recently described by Sousa et al., are round cells with a smooth outline, having a small rim of cytoplasm around the central nucleus and a bright spot corresponding to the round acrosomal vesicle that lies at the apical nuclear surface (Sousa et al., 1999Go). There are no visible flagella.

ROSI
Once a round spermatid was chosen as a potential candidate for injection, an attempt was made to aspirate it into the microinjection needle. The behaviour of the cell during this step was decisive for its final acceptance or rejection. Round spermatids were slightly larger than the internal diameter of the microinjection needle and were thus deformed as they entered the needle. The cells that were deformed but did not disintegrate upon aspiration were assumed to be viable spermatids and were deemed suitable for injection. These spermatids were transferred to a polyvinylpyrrolidone solution, and if they resumed their initial round forms during this step then they were thought to be alive. The spermatid injection technique has been previously and extensively described (Tesarik and Mendosa 1996Go; Vanderzwalmen et al., 1996Go, 1997Go). Following the aspiration of round spermatids into an injection pipette, the pipette was pushed through the zona pellucida and the oolemma at the equatorial level. Vigorous aspiration of the ooplasma was performed prior to injection. Oocyte activation was achieved mechanically by aspiration of the ooplasma during the ICSI procedure.

In-vitro culture of embryos to the blastocyst stage
In-vitro culture of spermatid-derived embryos was undertaken in a similar method to that of sperm-derived embryos (Gardner et al., 1998Go; Balaban et al., 1999Go). A sequential media system (S1 and S2 media; Scandinavian Science AB Products, Gotenborg, Sweden) designed for further embryonic development was used for blastocyst culturing. Embryos were cultured in groups in S1 medium until day 3 and in S2 medium until the blastocyst stage. Culture medium was refreshed every day. Cleavage stage embryos were graded as follows: grade 1 embryo, no fragmentation with equal sized homogeneous blastomeres; grade 2 embryo, <20% fragmentation with equal sized homogeneous blastomeres; grade 3 embryo, 20–50% fragmentation with unequal sized blastomeres; grade 4 embryo, >50% fragmentation with unequal sized blastomeres. Blastocyst grading was according to Dokras et al., (Dokras et al., 1993Go). Grade 1 blastocysts were characterized by early cavitation, resulting in the formation of an eccentric and then expanded cavity lined by a distinct inner cell mass region and trophectoderm layer. Grade 2 blastocysts exhibited a transitional phase where single or multiple vacuoles were seen, which over subsequent days developed into the typical blastocyst appearance of the grade 1 blastocysts. Grade 3 blastocysts were defined as blastocysts with several degenerative foci in the inner cell mass, with cells appearing dark and necrotic.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient characteristics and fertilization parameters are shown in Table IGo. The two pronuclear (PN) fertilization rate was <20%. Over half of the oocytes remained intact after injection. Of the 2PN embryos that cleaved, only 20.8% were grade 1 or 2 on day 3 (Table IIGo). The rate of blastocyst formation on day 5 was 7.6% for 2PN embryos and 6.0% for 1PN embryos. All blastocysts derived from 1PN embryos were grade 3. A total of 16 blastocysts were transferred in 12 patients; five of these blastocysts had originated from 1PN embryos. None of the patients conceived.


View this table:
[in this window]
[in a new window]
 
Table I. Patient and treatment cycle characteristics
 

View this table:
[in this window]
[in a new window]
 
Table II. Cleavage stage embryo quality and blastocyst quality
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Non-obstructive azoospermia is a relatively common cause of male sterility. The pathology may be traced to basically two conditions: germ cell aplasia or maturation arrest, both of which can be total or partial. Germ cell aplasia yields mature sperm to testicular sperm extraction procedures in ~50% of the subjects, irrespective of a previous testicular biopsy report. The use of spermatids is usually unnecessary. The diagnosis of maturation arrest is usually made in a man with normal testicular size and FSH levels, but a negative testicular biopsy. Maturation arrest can likewise be complete or partial. The critical issue is whether round spermatids can be used in cases of complete maturation arrest and whether this approach yields favourable results. There is a general consensus that when round spermatids are present, there should also be elongated ones and probably mature sperm (Silber et al., 2000Go). Elongated or elongating spermatids yield a much more favourable fertilization rate, and pregnancy rates are comparable with injection of testicular sperm (Vanderzwalmen et al., 1998Go). Thus, only when elongated spermatids are not numerous enough to inject all retrieved oocytes should one resort to ROSI. However, extremely poor results have been obtained with this approach. Levran et al., failed to achieve pregnancies with the injection of round spermatids from men with maturation arrest (Levran et al., 2000Go). In this study, ROSI was associated with lower fertilization (44.9 versus 69.0%) and higher cleavage arrest rates (40.8 versus 8.2%). The outcome with fresh and frozen spermatids was similar.

Sousa et al., studied the outcome of spermatid injection in relation to a spermatid developmental stage classification adapted to this purpose (Sousa et al., 1999Go). A complete block was detected at the earliest stage (Sa1). No pregnancies were achieved in ROSI cycles.

Conceptions after ROSI have been reported, albeit in only very few patients and by select groups (Tesarik et al., 1996Go; Antinori et al., 1997Go). Failure of ROSI may be due to several factors, including failure of oocyte activation, the use of apoptotic cells and development of genetically abnormal embryos (Tesarik et al., 1999aGo). However, pregnancies have been reported after in-vitro maturation of round spermatids (Tesarik et al., 1999bGo). Cells with a higher degree of maturity were observed at the end of the in-vitro culture period.

We recently reported a lower and slower rate of blastocyst formation after ROSI (Balaban et al., 2000bGo). As the azoospermic men who formed the basis of this report had incomplete spermatogenic failure, some of the oocytes were injected with elongated or mature sperm, and round spermatids were used when no mature sperm cells were available for the remaining oocytes. Embryos derived from round spermatids were not transferred and instead were observed in culture up to the blastocyst stage. All blastocysts resulting from ROSI were of poor quality (grade 3) and none showed spontaneous hatching. The pregnancy potential of these blastocysts was not assessed as none were transferred.

We have previously shown a clear association between blastocyst quality and implantation rates (Balaban et al., 2000aGo). Transfer of poor quality (grade 3) blastocysts was associated with very low implantation rates (7.1%); however, occasional pregnancies occurred yielding a clinical pregnancy rate per transfer of 15.3%. These results prompted us to reconsider transferring blastocysts derived from testicular sperm. We assumed that the few embryos reaching the blastocyst stage might have the potential to implant. Low chances of success were explained to the patient and, in patients who gave informed consent, round spermatid-derived blastocysts were transferred on day 5 or 6. Only 12 out of 58 couples (20.1%) progressed to the embryo transfer stage. Of the 16 blastocysts transferred in these patients, none implanted.

Several concerns may be voiced regarding our study. Accurate identification of the round spermatid is a difficult task. It may be argued that some of the cells injected into the oocytes were not round spermatids. This is unlikely in the case of 2PN embryos. For 1PN embryos, parthenogenetic activation cannot be ruled out. However, not all 1PN embryos can be discarded in that sense, as observation of two pronuclei may have been missed due to earlier occurrence of syngamy in ROSI oocytes. (Ogura and Yanagimachi, 1993Go; Sofikitis et al., 1994Go).

We can conclude that blastocyst transfer is not feasible for patients having round spermatid-derived embryos. Accumulated data in the literature and our own experiences suggest that ROSI should not be offered to azoospermic subjects, as there appears to be no realistic prospect for success.


    Notes
 
1 To whom correspondence should be addressed at: VKV American Hospital, Guzelbahce sok. No:20, Nisantasi 80200, Istanbul, Turkey. E-mail: burman{at}superonline.com Back

Submitted on February 16, 2001


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Angelopoulos, T., Krey, L., McCullough, A., Adler, A. and Grifo, J. (1997) A simple and objective approach to identifying human round spermatids. Hum. Reprod., 12, 2208–2216.[Abstract]

Antinori, S., Versaci, C., Antinori, M. and Selman, H.A. (1997) Successful fertilization and pregnancy after injection of frozen–thawed round spermatids into human oocytes. Hum. Reprod., 12, 554–556.[ISI][Medline]

Balaban, B., Urman, B., Sertac, A., Alatas, C., Aksoy, S., Mercan, R. and Nuhoglu, A. (1999) In-vitro culture of sperm induces motility and increases implantation and pregnancy rates after testicular sperm extraction and intracytoplasmic sperm injection. Hum. Reprod., 14, 2808–2811.[Abstract/Free Full Text]

Balaban, B., Urman, B., Sertac, A., Alatas, C., Aksoy, S. and Mercan, R. (2000a) Blastocyst quality affects the success of blastocyst stage embryo transfer. Fertil. Steril., 74, 282–287.[ISI][Medline]

Balaban, B., Urman, B., Isiklar, A., Alatas, C., Aksoy, S., Mercan, R. and Nuhoglu, A. (2000b) Progression to the blastocyst stage of embryos derived from testicular round spermatids. Hum. Reprod., 15, 1377–1382.[Abstract/Free Full Text]

Dokras, A., Sargent, I.L. and Barlow, D.H. (1993) Human blastocyst grading: an indicator of developmental potential. Hum. Reprod., 8, 2119–2127.[Abstract]

Gardner, D.K., Valla, P., Lane, M. et al., (1998) Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil. Steril., 69, 84–88.[ISI][Medline]

Levran, D., Nahum, H., Farhi, J. and Weisman, A. (2000) Poor outcome with round spermatid injection in azoospermic patients with maturation arrest. Fertil. Steril., 74, 443–449.[ISI][Medline]

Ogura, A. and Yanagimachi, R. (1993) Round spermatid nuclei injected into hamster oocytes form pronuclei and participate in syngamy. Biol. Reprod., 48, 219–225.[Abstract]

Silber, S.J., Johnson, L., Verheyen, G. and Van Steirteghem, A. (2000) Round spermatid injection. Fertil. Steril., 73, 897–900.[ISI][Medline]

Sofikitis, N., Miyagawa, I., Agapitos, E. et al., (1994) Reproductive capacity of the nucleus of the male gamete after completion of meiosis. J. Assist. Reprod. Genet., 11, 335–341.[ISI][Medline]

Sousa, M., Barros, A., Takahashi, K., Oliveria, C., Silva, J. and Tesarik, J. (1999) Clinical efficacy of spermatid conception: analysis using a new spermatid classification scheme. Hum. Reprod., 14, 1279–1286.[Abstract/Free Full Text]

Tesarik, J. and Mendosa, C. (1996) Spermatid injection into human oocytes. I. Laboratory techniques and special features of zygote development. Hum. Reprod., 11, 772–779.[Abstract]

Tesarik, J., Mendoza, C. and Greco, E. (1999a) In vitro culture facilitates the selection of healthy spermatids for assisted reproduction. Fertil. Steril., 72, 809–813.[ISI][Medline]

Tesarik, J., Rolet, F., Sedbon, J., Tibi, C. and Thebault, A. (1996) Spermatid injection into human oocytes. II. Clinical application in the treatment of infertility due to nonobstructive azoospermia. Hum. Reprod., 11, 780–783.[Abstract]

Tesarik, J., Bahceci, M., Ozcan, C., Greco, E. and Mendoza, C. (1999b) Restoration of fertility by in vitro spermatogenesis. Lancet, 353, 555–556.[ISI][Medline]

Urman, B., Aksoy, S., Alatas, C., Balaban, B., Sertac, A. and Nuhoglu, A. (1998) Early sperm retrieval and in vitro culture: preventing the injection of human chorionic gonadotropin to partners of azoospermic men. Ass. Reprod. Rev., 8, 76–78.

Vanderzwalmen, P., Bertin, G. and Geerts, L. (1996) Two essential steps for a successful ICSI; injection of immobilized spermatozoa after rupture of the oolemma. Hum. Reprod., 11, 540–547.[Abstract]

Vanderzwalmen, P., Zech, H., Birkenfield, A., Yemini, M., Bertin, G., Lejeune, B. et al., (1997) Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation. Hum. Reprod., 12, 1203–1213.[ISI][Medline]

Vanderzwalmen, P., Nijs, M., Stecher, A., Zech, H., Bertin, G., Lejeune, B. et al., (1998) Is there a future for spermatid injections? Hum. Reprod., 13 (Suppl. 4), 71–84.[Abstract]

Verheyen, G., Crabbe, E., Joris, H. and Van Steirteghem, A. (1998) Simple and reliable identification of the round spermatid by inverted phase-contrast microscopy. Hum. Reprod., 13, 1570–1577.[Abstract]

Submitted on February 16, 2001; accepted on October 29, 2001.