Progression to the blastocyst stage of embryos derived from testicular round spermatids

Basak Balaban, Bulent Urman1, Aycan Isiklar, Cengiz Alatas, Senai Aksoy, Ramazan Mercan and Alp Nuhoglu

Assisted Reproduction Unit, American Hospital of Istanbul, Turkey


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Progression to the blastocyst stage of embryos derived from testicular round spermatids in men with non-obstructive azoospermia was studied. A total of 56 men were studied in whom partial spermatogenesis failure had occurred where only very few spermatozoa (fewer than the number of oocytes retrieved) were extracted from multiple testicular biopsy specimens. Oocytes remaining after intracytoplasmic injection of testicular spermatozoa (group 1) were injected with round spermatids (ROSI, group 2). Only embryos derived from group 1 were transferred. Remaining embryos were observed under culture for 8 days and their progression to the blastocyst stage was recorded. Of the 546 oocytes injected with testicular spermatozoa, 404 (73.9%) showed evidence of 2-pronuclear (2PN) fertilization. Injection of testicular round spermatids resulted in 2PN fertilization rate of 50% (P < 0.05). Using a four-point grading system, 53% of the good quality embryos (grade 1 or 2) in group 1 reached the blastocyst stage compared with 25% in group 2 (P < 0.05). The rate of progression to the blastocyst stage of grade 3 and grade 4 embryos was 46 and 8.5% in the two groups respectively (P < 0.05). Using a different three-point grading system for the blastocysts, 75.3% of the blastocysts in group 1 were either grade 1 or grade 2 and 24.7% were grade 3. However, in group 2 all blastocysts were grade 3. All embryos observed in group 1 reached the blastocyst stage by day 5 or 6 compared with 25% of the embryos reaching the blastocyst stage by this time in group 2. While 31.2% of the blastocysts in group 1 showed evidence of spontaneous hatching in vitro, none of the blastocysts in group 2 hatched. In conclusion, progression to the blastocyst stage occurred at a much lower and slower rate in embryos derived from testicular round spermatids. Furthermore, all blastocysts resulting from ROSI were of poor quality and none showed spontaneous hatching. These results may explain the dismal outcome associated with ROSI.

Key words: azoospermia/blastocyst/spermatid/spermatozoa/testicular sperm extraction


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Intracytoplasmic injection of round spermatids from the ejaculate or testicular tissue (ROSI) has been proposed as a salvage treatment in men with non-obstructive azoospermia where the testicular sperm extraction (TESE) procedure does not yield spermatozoa. In males with so-called non-obstructive or secretory azoospermia, the chances of finding a mature spermatozoon for use in intracytoplasmic sperm injection (ICSI) is at best 50% (Tournaye et al., 1997Go). In situations where there are no or not enough mature spermatozoa in the testicular biopsy specimen, spermatids have been utilized for ICSI. Spermatids are the end result of a completed second meiotic division and contain a haploid set of chromosomes. Spermatid-derived mammalian embryos showed the potential to form male pronuclei, participate in syngamy, to develop beyond the 2-cell stage and to produce live offspring (Ogura and Yanagimachi 1993Go, 1995Go; Ogura et al., 1994Go). Subsequent pregnancies and live births reported using this technique in humans led to enthusiasm in the scientific community and spermatid injection enjoyed widespread application (Tesarik et al., 1996Go; Vanderzwalmen et al., 1997Go). However, not all assisted reproductive technology programmes report similar success with spermatid injections. Fertilization rates are low especially when round spermatids are utilized for ICSI and embryos obtained implant at a much lower rate, yielding only occasional pregnancies (Aslam et al., 1998Go; Fishel et al., 1996Go, 1997Go; Tesarik et al., 1998aGo). Poor results have been ascribed to variations in centrosome function, oocyte activation, abnormal nuclear protein maturation and cell cycle asynchrony (Vanderzwalmen et al., 1998Go). Furthermore, accurate morphological identification of the spermatid remains a perplexing issue, rendering interpretation of results from different studies even more complicated. It is therefore vital to define as exactly as possible the different types of spermatids, to avoid confusion regarding the success of microinjection of their different subtypes. Light and electron microscopic studies established a classification that makes an accurate distinction between the start of spermiogenesis, followed by nuclear changes and development of the tail (Clermont, 1963Go; Holstein and Roosen-Runge, 1981Go). However, in the embryology laboratory, using light microscopy and under the Hoffman modulation contrast system, it is difficult to make a strict distinction between the various phases of spermatid development. In the IVF laboratory, four categories of spermatids can be distinguished according to the cell shape, amount of cytoplasm and the size of the tail. These are round spermatids, elongating spermatids, elongated spermatids, and mature spermatids.

An overview of the literature suggests a better ICSI outcome when elongating or elongated spermatids are used for ICSI (Fishel et al., 1997Go). However, extremely poor results have been reported with ROSI. Vanderzwalmen et al. reported a 4% ongoing pregnancy and a 2% live birth rate after the transfer of ROSI-derived embryos (Vanderswalmen et al., 1998). Similarly, our experience with ROSI has been very disappointing; only one biochemical pregnancy was achieved in 33 cleavage stage embryo transfers (unpublished data). The reason for the poor results obtained with ROSI may be the diminished or absent potential of ROSI derived embryos to progress to advanced stages of development. Goto et al. studied blastocyst formation following ICSI of spermatids in the bovine model (Goto et al., 1996Go). There were no significant differences in embryo quality of blastocysts obtained by injection of spermatids derived from spermatocytes, spermatids or spermatozoa. They concluded that various types of spermatogenic cells could be used for ICSI to produce viable embryos. However, the implantation potential of these embryos was not assessed. More recently it was shown that round spermatids obtained from hybrid sterile mice can result in normal embryos which progress to the blastocyst stage (Sasagawa et al., 1998Go). To our knowledge, no study in the literature has examined the progression of human ROSI embryos to the blastocyst stage. Therefore we aimed to evaluate the progression of human embryos obtained by ROSI to the blastocyst stage. We compared the rate of progression of ROSI embryos to the blastocyst stage with that of sibling embryos injected with testicular spermatozoa.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
In a total of 56 men with non-obstructive azoospermia, ROSI was undertaken due to the presence of very few mature spermatozoa in the TESE specimen. In 1997 following institutional review board (IRB) approval, we performed 33 spermatid injection cycles in azospermic men who did not yield spermatozoa to TESE. As only one biochemical pregnancy was obtained, we decided to stop further attempts. Subsequently another application to the IRB was made to use spermatids in azoospermic men when TESE did not yield enough spermatozoa to inject all retrieved oocytes. This application was granted provided the embryos obtained from spermatid injections were not transferred or cryopreserved. We thus proposed to observe these embryos under in-vitro culture and to document their progression to the blastocyst stage. Written consent was obtained from all couples who agreed to participate in this study. Due to the experimental nature of intracytoplasmic spermatid injection and lack of long-term follow-up data, only mature spermatozoa are currently used for ICSI in our clinic. We do not utilize elongating spermatozoa and classify elongated spermatozoa attached to the Sertoli cell as mature spermatozoa. We thus injected all excess oocytes with round spermatids. The patients were notified that the embryos obtained from spermatid injections would be discarded after an 8 day observation period.

Transfer was performed on day 3 only of embryos derived from ICSI of mature spermatozoa. Remaining embryos (immature spermatozoa, elongated spermatids and round spermatid derived) were observed under in-vitro culture in sequential defined media. Excess blastocysts from mature sperm injections were frozen at the blastocyst stage, taking into account their morphological quality.

All men were karyotypically normal. Microdeletions of the Y chromosome were not routinely investigated. Of the 56 men, 21 had undergone a prior diagnostic testis biopsy that showed hypospermatogenesis in four, Sertoli cell-only syndrome in 15 and maturation arrest in two subjects. Ovarian stimulation, oocyte retrieval, embryo transfer and luteal phase support were undertaken as described previously (Balaban et al., 1998Go). Briefly, gonadotrophin-releasing hormone analogue (GnRHa) was initiated in the luteal phase and was combined with FSH after down-regulation was achieved. In some cycles a flare GnRHa protocol was used. Transvaginal follicle aspiration and oocyte retrieval was performed following the injection of human chorionic gonadotrophin (HCG) at a leading follicle diameter of ~19–20 mm.

TESE and processing of testicular tissue
TESE was performed as previously described at ~24 h prior to ICSI (Urman et al., 1998Go). Briefly, testicular tissue samples obtained by open biopsy were placed into a Falcon 2001 tube containing 2 ml of IVF-50 medium (Scandinavian Science, AB, Gothenburg, Sweden). The tissue was washed several times with IVF-50 and then placed in a Petri dish (Falcon 3004). The cellular contents were extracted by gentle crushing between two needles and sterile glass slides. The suspension thus obtained was transferred into a Falcon 2001 tube and agitated for 60 s on a vortex to separate the different cell types. The vortexed suspension was then placed into a Falcon 3004 tube for observation under an inverted microscope equipped with Hoffman modulation contrast system. Magnifications of x200 (for the presence of spermatozoa) and x400 (for the identification of round spermatids) were used. The suspension was then centrifuged (Vanderzwalmen et al., 1991Go) at 600 g for 20 min through a three-layer (50, 70, 90%) discontinuous Percoll gradient (Percoll, Sigma P-1644, Sigma- Aldrich, Chemie GmbH, Stenheim, Germany). Each layer was washed with IVF-50 medium and centrifuged for 5 min at 1000 g. The spermatids were most often found in the 50 and 70% fractions whereas spermatozoa were collected from the 90% fraction. The pellet of each gradient was kept at 25–30°C until the ICSI procedure (Tesarik et al., 1998cGo). The pellets were prepared in a Falcon 3001 Petri dish as a swim-out droplet covered with mineral oil (Sigma- M-8410 Sigma-Aldrich Co. Ltd., St Louis, MO, USA) for spermatozoa or spermatid collection.

The injection of testicular spermatozoa was similar to that of conventional ICSI of mature ejaculated spermatozoa (Vanderzwalmen et al., 1996Go). However, in view of the different size and form of spermatids some modifications had to be adopted to the instrumentation and technique.

Spermatid identification and classification
The major concern about round spermatid injection is the identification of the cell. There are many other cells in the extracted testicular tissue that need to be distinguished from round spermatids (Figure 1Go). Although in stained preparations round spermatid identification is relatively easy, in wet preparations difficulties arise due to the resemblance to other cells in the extracted testicular tissue. Round spermatids at the Golgi cap phase and acrosome phase were observed and identified under an Olympus IX70 microscope equipped with a Hoffman modulation contrast system. Magnifications of x200 and x400 were used. Round spermatids were distinguished from other cells, e.g. spermatogonia, spermatocytes and polymorphonuclear leukocytes, by their smaller size (Figure 1Go). The diameter of the round spermatid (6.5–8 µm) is similar to that of an erythrocyte and to a small lymphocyte (Tesarik et al., 1996Go; Angelopoulos et al., 1997Go; Vanderzwalmen et al., 1998Go; Verheyen et al., 1998Go). Round spermatids may be observed at the Golgi phase, cap phase and the acrosome phase (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) (Figure 2Go). These were the Sa spermatids described previously (de Kretser and Kerr, 1969Go). Sa spermatids were recently described (Sousa et al., 1999Go) as 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. There were no visible flagella.



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Figure 1. Fresh testicular tissue sample. An acrosome phase (Sa1) round spermatid (large arrow) is shown together with erythrocytes (small arrow) and spermatogonia/primary spermatocytes (star). Scale bar = 10 µm.

 


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Figure 2. Acrosome phase (Sa1) spermatid. Scale bar = 10 µm.

 
Spermatid injection
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 the polyvinylpyrrolidone (PVP) solution and during this step if they resumed their initial round forms they were thought to be alive. Spermatid injection technique has been previously and extensively described (Tesarik and Mendosa, 1996Go; Vanderzwalmen et al., 1996Go, 1997Go). Needles used for spermatid injection had a less sharply bevelled tip than those used for standard ICSI. This was particularly important in view of the slightly larger size of round spermatids. Round spermatids that do not enter the needle as easily may spin at the needle tip because of turbulence caused by aspiration of the medium. Thus the needles used had a bevel angle of 35° and inner and outer diameters of 7 and 9 µm respectively. Following the aspiration of round spermatids into the injection pipette, the pipette was pushed through the zona pellucida and the oolemma at the equatorial level. Vigorous aspiration of the ooplasm was performed prior to injection (Tesarik and Mendoza, 1996). Oocyte activation was achieved mechanically by aspiration of the ooplasm during the ICSI procedure. Enzymatic oocyte activation was not performed, as this was not deemed necessary in microinjection procedures using round spermatids from men with incomplete spermatogenesis failure (Yamanaka et al., 1997Go).

In-vitro culture of embryos to the blastocyst stage
In-vitro culture of spermatid-derived embryos was similar to that of sperm-derived embryos (Balaban et al., 1998Go; Gardner et al., 1998Go). A sequential media system (S1 and S2 media from Scandinavian Science AB Products) 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 (G1): no fragmentation with equal sized homogeneous blastomeres; grade 2 (G2): <20% fragmentation with equal sized homogeneous blastomeres; grade 3 (G3): 20–50% fragmentation with unequal sized blastomeres; grade 4 (G4): >50% fragmentation with unequal sized blastomeres. Blastocyst grading was according to a previous method (Dokras et al., 1993Go): grade 1 (B1) characterized by early cavitation, resulting in the formation of an eccentric and then expanded cavity lined by a distinct inner cell mass region and trophoectoderm layer; grade 2 (B2) 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 (G3) with several degenerative foci in the inner cell mass with cells appearing dark and necrotic.

Embryo transfer
Embryos derived from intracytoplasmic injection of testicular spermatozoa were transferred 3 days after oocyte retrieval and supernumerary embryos were cryopreserved at the blastocyst stage. Embryos derived from ROSI, however, were not transferred and were observed in culture until they reached the blastocyst stage or for 8 days if blastocyst stage was not reached earlier. The decision not to transfer spermatid-derived embryos was based upon our earlier experience with testicular ROSI that yielded no clinical pregnancies in 33 TESE cycles.

Statistics
The rate of progression of spermatid-derived embryos to the blastocyst stage was compared with that of supernumerary embryos derived from intracytoplasmic injection of testicular spermatozoa. Paired Student's t-test, {chi}2 and Fisher's exact test were used when applicable for data analysis. A P-value < 0.05 was considered as significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In all subjects very few mature spermatozoa were recovered despite extensive testicular sampling (total of six to eight large biopsy samples from both testes) and a meticulous search for mature cells. Of the 912 MII oocytes recovered from the female partners, 556 (61.0%) were injected with testicular spermatozoa and 356 (39.0%) with round spermatids. The fertilization rate following intracytoplasmic injection of testicular spermatozoa was significantly higher than the fertilization rate obtained after ROSI (P < 0.05) (Table IGo). Furthermore, significantly more embryos cleaved after ICSI compared to ROSI. Of the total 400 embryos derived from ICSI, 247 were transferred and 153 were observed in culture until the blastocyst stage. Freezing of these excess embryos was undertaken on day 5 or 6. A total of 141 embryos was derived after ROSI and these were observed under in-vitro culture until the blastocyst stage. Progression of ICSI and ROSI embryos to the blastocyst stage is shown in Table IGo. Significantly more ICSI-derived embryos progressed to the blastocyst stage. This was true both for G1 + G2 and G3 + G4 embryos. None of the ROSI blastocysts spontaneously hatched compared to 39.6% of blastocysts derived from G1 + G2 ICSI embryos and 17.2% of blastocysts derived from G3 + G4 ICSI embryos.


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Table I. Results of intracytoplasmic injection of testicular spermatozoa versus testicular round spermatids and progression to the blastocyst stage in the two groups
 
The quality of the blastocysts obtained is shown in Table IIGo. While 75% of the blastocysts obtained from ICSI of spermatozoa were either B1 or B2, none of the blastocysts in the spermatid group was B1 or B2. The timing of blastocyst formation in the two groups is also shown in Table IIGo. Of the spermatid-derived embryos, none reached the blastocyst stage on day 5 but 75% reached the blastocyst stage on day 7–8. Conversely all the spermatozoa-derived embryos reached the blastocyst stage on day 5 or 6.


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Table II. Quality and timing of blastocysts derived from intracytoplasmic injection of testicular spermatozoa and testicular round spermatids
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Intracytoplasmic spermatid injection has been proposed as a novel treatment for azoospermic men who do not yield mature spermatozoa upon TESE. Enthusiasm for this approach was stimulated following animal experiments that showed its feasibility. Spermatid-derived mammalian embryos showed the potential to form male pronuclei, participate in syngamy, to develop beyond the 2-cell stage and to produce live offspring (Ogura and Yanagimachi 1993Go, 1995Go; Ogura et al., 1994Go). In humans, spermatid injections for men with non-obstructive azospermia were attempted (Sofikitis et al., 1995Go). Further reports indicated the feasibility of this technique, but poor fertilization, implantation and pregnancy rates were reported especially when round spermatids were utilized for ICSI. This also has been our experience as only one biochemical pregnancy was obtained in 33 ROSI cycles. The Ethics Committee of the American Hospital therefore decided not to propose such a treatment option to the couples. In this study, after obtaining written consent, round spermatids were utilized for ICSI as there were not enough spermatozoa to inject all retrieved oocytes. Resulting embryos were not transferred but observed in culture in order to follow their progression to advanced stages of embryonic development. Despite the fact that embryo quality on day 3 was similar after spermatozoa and spermatid injections, progression to blastocyst stage occurred at a much lower and slower rate in spermatid-derived embryos and none of these embryos showed spontaneous hatching. Furthermore, all blastocysts resulting from injection of round spermatids were of poor quality.

Intracytoplasmic injection of round spermatids has been associated with a lower fertilization rate when compared with injection of spermatogenic cells at more advanced stages of maturation (Vanderzwalmen et al., 1998Go). This is especially true when round spermatids are derived from azoospermic men with complete spermatogenic arrest. Our study group comprised men having mature spermatozoa and spermatids after TESE. These subjects most probably represented a more favourable subset regarding fertilization success. Round spermatids isolated from testes with incomplete or defective spermiogenesis may not be identical to those obtained from testes with complete spermatogenic arrest. The absence of elongated spermatids in all tubules, which negatively affects the quality of spermatid maturation, may have a genetic cause (Vanderzwalmen et al., 1998Go). It was suggested (Tesarik et al., 1998aGo) that spermatids in patients lacking spermatozoa, i.e. patients with complete spermiogenesis failure, are often deficient in the factors responsible for oocyte activation. This deficiency may result in abnormal fertilization patterns and impairment of further preimplantation and postimplantation development. Furthermore, patients with complete spermiogenesis failure have an unusually high frequency of spermatids undergoing apoptosis and consequent DNA degradation (Tesarik et al., 1998bGo). In a small series of patients, Vanderzwalmen et al. injected oocytes with round spermatids from men with normal spermatogenesis, partial spermatogenesis failure or complete spermatogenesis failure. Two pronuclei fertilization rates were 42, 27 and 8% respectively (Vanderzwalmen et al., 1998Go). Fishel et al. compared fertilization rate in sibling oocytes injected with testicular spermatozoa or testicular round or elongated spermatids (Fishel et al., 1997Go). The fertilization rate was 24% for spermatids and 79% for testicular spermatozoa. Similarly, it was reported (Kahraman et al., 1998Go) that a low fertilization rate of 25.6% was obtained with round spermatids. In our series, 2PN fertilization rate with ROSI was 50%. This is significantly lower than the 73.9% fertilization rate obtained with the injection of testicular spermatozoa.

It is not clear whether spermatid-derived embryos are endowed with the potential to progress to further stages of embryonic development. The recent introduction of blastocyst culture has enabled us to observe the in-vitro development of embryos to the advanced stages of cleavage. The scarcity of pregnancies reported after ROSI is most probably due to the lack of or inadequate progression of these embryos to the blastocyst stage. Our results prove that this is indeed so. Not only did spermatid-derived embryos progress to the blastocyst stage at a much lower and slower rate, but none showed spontaneous hatching in vitro. In conclusion, embryos derived from ROSI are impaired in their ability to reach advanced stages of embryonic growth. This appears to be responsible for the dismal results reported with the utilization of this technique.


    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


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on October 25, 1999; accepted on March 6, 2000.