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Intracytoplasmic Spermatid Injection Can Result in the Delivery of Normal Offspring

IBRAHIM M. FAHMY^*,

From the ^* Egyptian IVF-ET Center, Hadayek El-Maadi, Maadi, Cairo, Egypt; and the Andrology Department, Faculty of Medicine, Cairo University, Cairo, Egypt.

Correspondence to: Ragaa Mansour, MD, PhD, the Egyptian IVF-ET Center, 3 Street 161, Hadayek El-Maadi, Maadi, Cairo 11431, Egypt (e-mail: ivf{at}link.net).

Received for publication April 2, 2003; accepted for publication May 13, 2003.

	Abstract

Top Abstract Material and Methods Results Discussion References

 
Almost one-third of all patients with nonobstructive azoospermia undergoing testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) have cancelled cycles due to failure to find spermatozoa. For these patients, every attempt should be made to rescue the cycles by searching for spermatids. In this retrospective study, we report our experience in using elongating (stage Sb2) and elongated (stage Sc and Sd1) spermatids for ICSI. The study included 488 consecutive ICSI and TESE cycles performed for 452 patients with nonobstructive azoospermia. In 179 (36.7%) cycles, neither spermatozoa nor mature spermatids (stage Sd2) suitable for injection were found. After an extensive search only Sb2, Sc, and Sd1 spermatids were found in 22 of these 179 cycles (12.3%). These spermatids were used for injection of retrieved oocytes. The fertilization rate was 33.2%, and 19 patients (86.4%) reached the embryo transfer stage. In 6 cycles a chemical pregnancy occurred, and 3 clinical pregnancies were established, resulting in the delivery of 3 healthy boys with normal karyotypes. When normal living spermatozoa or mature spermatids (stage Sd2) cannot be found during TESE, late spermatids (stage Sb2, Sc, and Sd1) can be used successfully and result in the delivery of healthy offspring.

     Key words: Nonobstructive azoospermia, TESE, ICSI

Spermatids are a unique source of a haploid number of chromosomes (Edwards et al, 1994). Animal experiments showed the potential of round spermatids or round spermatid nuclei to achieve fertilization and pregnancy in hamsters, mice (Ogura and Yanagimachi, 1993; Ogura et al, 1993, 1994), rabbits (Sofikitis et al, 1994, 1996), and rhesus monkeys (Hewitson et al, 2002). This success with animal experiments led to the suggestion of using spermatids for ICSI in humans when spermatozoa are completely absent (Edwards et al, 1994). Fertilization and pregnancies after the use of ejaculated round spermatids (Fishel et al, 1995; Tesarik et al, 1995, 1996;) and testicular round or elongated spermatids (Vanderzwalmen et al, 1995; Chen et al, 1996; Fishel et al, 1996; Mansour et al, 1996; Amer et al, 1997; Antinori et al, 1997; Araki et al, 1997; Fishel et al, 1997; Vanderzwalmen et al, 1997; Barak et al, 1998; Bernabeu et al, 1998; Kahraman et al, 1998; Sofikitis et al, 1998; Sousa et al, 1999, 2002) have been reported in humans.

The classification of different types of spermatogenic cells including spermatids is based on the work of Clermont (1963) and de Kretser (1969). Spermatids are classified under light or electron microscopy into 4 stages: Golgi phase, cap phase, acrosomal phase, and mature phase. Because of difficulty in visualizing the acrosomal structure in hematoxylin-and-eosin-stained histopathological sections, spermatids were classified into 6 phases according to nuclear shape and position, namely: Sa, Sb1, Sb2, Sc, Sd1, and Sd2 (Clermont, 1963) (Figure 1). Under the inverted microscope, during ICSI, identification of these forms of spermatids is difficult. Because human spermatozoa exhibit high percentages of abnormal forms, one would expect to find an increased percentage of abnormal spermatids not fulfilling the classical criteria for classification. This difficulty is further increased in patients with nonobstructive azoospermia due to pathological spermatogenesis.

View larger version (127K): [in this window] [in a new window]   Figure 1. Diagrammatic presentation of spermatids showing various stages of spermatids. Reprinted with permission from De Kretser and Kerr, 1994.

From a practical and clinical viewpoint, Vanderzwalmen et al (1998) suggested a simple classification on the basis of the size and shape of the spermatid nuclei under the inverted microscope. According to their classification, spermatids are classified into 4 categories: round spermatids (stage Sa and Sb1), elongating spermatids (stage Sb2), elongated spermatids (stage Sc and Sd1), and mature spermatids, also called immature spermatozoa (Sd2). This classification corresponds well with our own experience in identifying different types of spermatids. Sousa et al (1999) introduced another classification on the basis of the position of the nucleus and the length of the emerging tail.

The aim of this study is to report our experience in using specific types of spermatids, namely the elongating spermatids (stage Sb2) and the elongated spermatids (Sc, Sd1), for ICSI in nonobstructive azoospermia when no normal living spermatozoa, or mature spermatids (stage Sd2), could be retrieved during TESE.

	Material and Methods

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This retrospective study includes 488 consecutive cycles of TESE/ICSI performed for 452 patients with nonobstructive azoospermia between January 1996 and December 2000 at our institution. In 179 (36.7%) of these cycles, no spermatozoa or mature spermatids (stage Sd2) suitable for injection were found. After an extensive search only spermatids, stages Sb2, Sc, and Sd1, were found in 22 of these 179 cycles (12.3%). All the patients were counseled and consented to the use of spermatids for injection in an attempt to rescue the cycle. They were informed about the novelty of the technique, and the possible risk of transmitting infertility problems to their male offspring.

The routine andrological investigations for patients undergoing surgical retrieval of spermatozoa included conventional semen analysis, an endocrine profile, urine analysis, and prostatic examination. Other investigations like transrectal and scrotal ultrasonography, diagnostic testicular biopsy, and karyotyping were done, as indicated.

As for the spouses, general examination, pelvic examination, and transvaginal ultrasonography were routinely performed. Routine laboratory tests included liver and kidney functions. Ovulation induction was performed using a gonadotropin releasing hormone—a long protocol. Ten thousand units (10 000 IU) of human chorionic gonadotropin (hCG) were given intramuscularly when the 3 leading follicles reached 18 mm in mean diameter. Oocyte pickup was performed 36 hours later through transvaginal ultrasonography as previously described (Mansour et al, 1994).

TESE Procedure

Open testicular biopsies were performed under local infiltration anesthesia using a mixture of 1:1 bupovacaine and lidocaine as previously described (Fahmy et al, 1997). A search for spermatozoa was done and if no spermatozoa suitable for injection were found another biopsy was taken either from the same site (one large biopsy from 5 to 10 mm diameter when the testicular tissue looked homogenous in a moderate- or normal-sized testicle) or from other sites (multiple smaller biopsies). If spermatozoa were still not found, another biopsy was performed on the other testis. An extra piece of testicular tissue was obtained, fixed in Bouin's solution, and used later to prepare 4-µm-thin paraffin sections stained with hematoxylin and eosin. The collected pieces of testicular tissues were minced in a 200-500 µL droplet of HEPES-buffered Earle's medium (Medicult, Copenhagen, Denmark) in a Petri dish (Falcon, cat. no. 3001, Becton Dickinson, Plymouth, United Kingdom) using two G28 needles. Microdroplets from the suspension of minced testicular tissues were transferred directly to the injection dish and placed carefully in the microdroplets previously prepared under mineral oil in an injection dish. The microdroplets containing the suspension of the minced testicular tissue were examined under the inverted phase microscope. If no spermatozoa were found after a rapid search, the rest of the testicular tissue suspension was distributed in the injection dish and examined for 2-3 hours by at least 2 embryologists for spermatozoa. During this prolonged search for spermatozoa, elongating or elongated spermatids were collected and transferred to a separate microdroplet.

Spermatid Identification and Injection

On the basis of the illustrations of spermatids by Clermont (1963) and de Kretser and Kerr (1994) (Figure 1), and the classification of Vanderzwalmen et al (1998), our embryologists practiced the identification of different types of spermatids on testicular biopsies taken from patients with obstructive azoospermia. Elongating spermatids corresponding to stage Sb2 have a dark, condensed nucleus, which resembles the head of a spermatozoon. The cytoplasm is shed from around the nucleus at one side, giving the nucleus an eccentric location and leaving the elongating spermatid with a very characteristic shape, resembling an ice-cream cone, as described by Mansour (1998) (Figure 2a, b). Elongated spermatids corresponding to stage Sc and Sd1 (Figure 2c, d) have a prominent oval nucleus protruding partially from the cytoplasm, which surrounds the early-formed tail. This retrospective study did not include the cycles in which early rounded spermatids or mature spermatids with long tails not covered by cytoplasm and corresponding to stage Sd2 (Figure 2e) were used for injection of all or some of the retrieved oocytes.

View larger version (106K): [in this window] [in a new window]   Figure 2. Different types of late spermatids as they appear under the inverted microscope: Sb2 (a, b), Sc (c), Sd1 (d), and Sd2 (e). The size of the late spermatid can be compared with the size of a red blood cell and with the diagrammatic presentation of spermatids in the previous figure. Spermatid Sb2 (a, b) show the characteristic "ice-cream cone appearance." Spermatid heads (Sd2) (e) are seen within Sertoli cell cytoplasm, showing long tails of variable lengths.

After identification, late spermatids were picked up by a microinjection pipette (Humagen, Boston, Mass) of ordinary size (inner diameter 7-8 µm), and injected through the cytoplasm. The tip of the microinjection pipette was used to touch the cytoplasm surrounding the emerging tail of the Sd1 spermatid to be removed. No extra techniques were applied to induce cytoplasmic activation. The oocytes were checked the next morning for pronuclear (PN) formation and the embryo transfer was done on day 2 or 3 after ovum pickup.

	Results

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Of the 488 cycles of TESE performed for patients with nonobstructive azoospermia, 274 cases contained adequate numbers of normal spermatozoa or mature spermatids (stage Sd2), 179 (36.7%) did not contain spermatozoa suitable for injection. In 22 of these cycles (12.3%) only Sb2, Sc, and Sd1 spermatids were found and used for ICSI. In 16 of these 22 cycles, a few abnormal or nonviable spermatozoa were found, but they were not suitable for injection. In the remaining 6 cycles, neither spermatozoa nor mature spermatids were found. Interestingly, in all 6 patients in whom no spermatozoa could be found at day of ICSI, a few spermatozoa had appeared occasionally in previous semen reports, and in 1 patient they were retrieved during a previous TESE/ICSI cycle (Table 1). In the remaining 35 cycles, very few spermatozoa were found for injection and the rest of the oocytes were injected with spermatids. Data of these 35 cycles were excluded.

In total, 184 metaphase II (MII) oocytes were injected and 2PN was observed in 61 oocytes, resulting in a fertilization rate of 33.2%. Nineteen cycles (86.4%) reached the embryo transfer stage. In 6 cycles, a positive ß-hCG test was obtained after 2 weeks. Three clinical pregnancies were established and resulted in the delivery of 3 healthy boys with normal karyotyping. A regular followup of the 3 boys was done at 6-month intervals. Now, the boys are 6 years, 5 years, and 2.5 years old, respectively.

The results of the ICSI in the 22 cycles with only Sb2, Sc, and Sd1 spermatids were compared with the data of cycles in which testicular spermatozoa were used for injection in patients with nonobstructive (274 cycles) and obstructive (285 cycles) azoospermia, which were performed in our center during the same period of time. When spermatids were used for injection, the fertilization and clinical pregnancy rates were significantly lower than the results obtained using testicular spermatozoa in both obstructive and nonobstructive azoospermia (Table 2). The testicular biopsies were evaluated qualitatively and quantitatively by using the late spermatid score (Silber and Rodriguez-Rigau, 1981).

The results of the histopathology of the testicular biopsies of the 22 cycles in which spermatids were used for injection are also shown in Table 1. Tubular hyalinization was diagnosed in two cycles and Sertoli cell syndrome was diagnosed in one. In 14 cycles, incomplete spermatogenic arrest was diagnosed on the basis of the presence of a few late spermatid nuclei, which are indistinguishable from sperm heads in paraffin sections (late spermatid score <18). In the remaining five cycles (3, 6, 15, 16, 16R), many late spermatid nuclei were seen in most tubules (late spermatid score 18), giving the impression of normal spermatogenesis. However, since no, or very few, spermatozoa could be retrieved despite the presence of many late spermatids, late spermatid arrest was diagnosed.

	Discussion

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In this study, which included a large number of ICSI cycles performed for nonobstructive azoospermia, no spermatozoa or mature spermatids (stage Sd2) were found in about one-third of the cases after an extensive search. In 12.3% of these cycles, intermediate stages, namely elongating and elongated spermatids, were found and were used for injection. Otherwise, the cycles would have been cancelled after performing ovum pickup and the testicular biopsy. In the majority of those cases, very few grossly abnormal immotile spermatozoa were found. In an attempt at offering better chances for these patients, we preferred to inject intermediate spermatids with normal-looking nuclei rather than injecting abnormal sperm. In 6 cases, no spermatozoa were found though they were occasionally present in previous semen analyses, or during a previous TESE. These data suggest that complete late spermatid arrest is rare. Similar findings were reported by Sousa et al (1999), who found complete arrest in stage Sb1-Sc2 spermatids in only 2 patients. These data support the assumption of Silber and Johnson (1998) that complete spermatogenic arrest at the spermatid level is extremely rare.

When no late spermatids are found it is logical to search for round spermatids. However, because of many concerns about the safety of injecting round spermatids (Sa and Sb1), and the problem of their identification (Silber et al, 2000), many centers, including ours, are conservative about using round spermatids during oocyte injection. These concerns include genomic imprinting, DNA stability, spermatid oocyte cell cycle asynchronization, the cytosolic sperm factor for oocyte activation, and the role of paternal centrioles (Tsai et al, 2000). Since elongating and elongated spermatids are more mature than rounded ones, such spermatids might be considered safer when used for ICSI, even though congenital abnormalities have been recently associated with late spermatid injection (Zech et al, 2000). Nevertheless, the birth of 3 normal boys in our study, plus the other reported healthy babies delivered after late spermatid injection, support the assumption that late spermatid injection is relatively safe. However, the safety of the procedure cannot be truly assessed until a larger amount of data accumulate.

A review of the literature shows that the fertilization rates and pregnancy rates after spermatid injection varied considerably among different studies. Generally, there is a remarkably low success rate reported after round spermatid injections (Balaban et al, 2000; Levran et al, 2000; Khalili et al, 2002; Urman et al, 2002). In addition, the fertilization rate was better using elongated spermatids as compared to round spermatids (Tesarik et al, 1995, 1996; Amer et al, 1997; Vanderzwalmen et al, 1997; Sousa et al, 2002). The reported fertilization rate among different studies in which only elongated spermatids were used for injection varied considerably from 24% (Fishel et al, 1997) to 71% (Kahraman et al, 1998). The reported pregnancy rate varied from 15.4% (Sofikitis et al, 1998) to 50% (Vanderzwalmen et al, 1997). In a large series including 17 cycles of elongated spermatid injection, the fertilization and pregnancy rates were 57.5% and 17.6% respectively (Antinori et al, 1997). This marked variation might be related to the type of spermatid injected. The reported low fertilization rate associated with spermatid injection may be related to a high frequency of apoptosis among postmeiotic germ cells. In vitro culture of testicular tissue may improve the results (Tesarik et al 2000).

The majority of the published studies provided no strict criteria for the type of late spermatid injection used. Reports giving higher fertilization rates may have injected more mature stages, namely the Sd2 spermatids. In the present study, lower fertilization and pregnancy rates were achieved. This may be explained by the fact that we injected a combination of relatively earlier stages (Sb2 and Sc).

Identification of different types of spermatids used during ICSI settings represents a major problem. There is still a controversy about the proper classification of unstained spermatids under the inverted microscope. For example, spermatids belonging to stage Sc under the classification suggested by Sousa et al, may be considered Sd1 or Sd2 under the classification suggested by Vanderzwalmen et al (1997). According to Sousa et al (1999), the length of the tail was considered the basis for the classification of intermediate stages of spermatids. However, de Kretser (1969) and Clermont (1963) have demonstrated that the shedding of the cytoplasm and the appearance of a bare visible tail occurs only during the late stages (Sd1 and Sd2) of spermatogenesis. For this reason, we preferred to adopt the classification suggested by Vanderzwalmen et al (1997).

By definition, cells attached to Sertoli cells are spermatids. The release of mature spermatids into the lumen of the seminiferous tubules is referred to as spermiation. At this point the cell is referred to as a spermatozoon. Mechanical dissection during TESE may release late spermatids (stage Sd2) from their attachment to Sertoli cells. Being very similar to spermatozoa, this may cause their confusion with spermatozoa. For this reason we did not include them in our spermatid study.

In the present study, the main histopathological pattern was incomplete spermatogenic arrest, during which few late spermatids or sperm heads were seen in some tubules. Similar data were reported by Sousa et al (2002). Retrieval of late spermatids is unlikely only in severe histopathological patterns such as Sertoli cell only and tubular hyalinization.

In conclusion, when normal living spermatozoa or mature spermatids (stage Sd2) cannot be found during TESE, spermatids (stage Sb2, Sc, and Sd1) can be used successfully, and result in the delivery of healthy offspring even though further research is needed to determine the long term effect of spermatid injection.

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