Blastocyst transfer following intracytoplasmic injection of ejaculated, epididymal or testicular spermatozoa

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

Assisted Reproduction Unit, American Hospital of Istanbul, Turkey


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recent studies indicate a strong paternal influence on embryo development and progression of the embryo to the blastocyst stage. The aim of this study was to compare, during extended culture, the in-vitro development of embryos resulting from intracytoplasmic sperm injection (ICSI) of ejaculated spermatozoa (group 1, n = 347), epididymal (group 2, n = 22) or testicular (group 3, n = 18) spermatozoa from obstructive azoospermic and testicular spermatozoa from non-obstructive azoospermic (group 4, n = 31) subjects. Fertilization and blastocyst formation rates were significantly lower in group 4 (P < 0.05). The incidence of expanded and hatching blastocysts was significantly lower in group 4 (P < 0.05). Overall in 93.2% ejaculate ICSI cycles, blastocysts were transferred on day 5. This was significantly higher than the 62% day 5 transfers in the non-obstructive azoospermic group (P < 0.05). Implantation rate per embryo was significantly higher in the ejaculate ICSI group compared with the other groups (P < 0.05). Clinical pregnancy per transfer was similar between groups; however, significantly fewer multiple pregnancies were encountered in the non-obstructive azoospermic group (P < 0.01). In conclusion, the source of the spermatozoa, most likely to be indicative of the severity of spermatogenic disorder, affects the rate of blastocyst formation and blastocyst implantation. Spermatozoa from non-obstructive azoospermic subjects, when utilized for ICSI, result in embryos that progress to the blastocyst stage at a lower and slower rate and implant less efficiently.

Key words: azoospermia/blastocysts/ICSI/testicular spermatozoa


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Intracytoplasmic sperm injection (ICSI) has gained broad acceptance since its introduction (Palermo et al., 1992Go). It is now being used to treat all forms of male infertility ranging from mild oligoasthenoteratozoospermia to azoospermia of different aetiology. Furthermore, ICSI provides the only solution to idiopathic fertilization failure in assisted reproduction.

The source and quality of spermatozoa utilized for intracytoplasmic injection differ tremendously. It is therefore not surprising that different outcomes in terms of fertilization, cleavage, blastocyst formation, implantation, and pregnancy were reported for male infertility of varying severity. In the light of recent studies, there appears to be a paternal effect on the implantation potential of the human embryo. Lower rates of blastocyst formation on feeder cells when impaired semen was used for IVF have been reported (Janny and Ménézo, 1994Go). Similarly it has been shown that embryos derived from men with poor semen parameters were of lower quality (Ron-El et al., 1991Go).

Further extending the above observations, it has been shown that blastocysts were produced at a higher rate from spermatozoa that showed progressive motility (Shoukir et al., 1998Go). They also reported a higher blastocyst formation rate in IVF cycles compared with ICSI cycles, suggesting a paternal effect on embryo viability. However, in this study the authors used a single culture medium throughout the entire culture period that may account for the low rate of blastocyst formation in ICSI cycles. The progression of IVF embryos to the blastocyst stage has been compared with ICSI embryos (Dumoulin et al., 2000Go). These workers demonstrated a lower rate of blastocyst formation in ICSI embryos regardless of the culture medium used or different culture conditions showed decreased fertilization and pregnancy rates in men with non-obstructive azoospermia when compared with men undergoing epididymal sperm aspiration for obstructive azoospermia (Palermo et al., 1999Go). Spermatozoa from testicular biopsies fertilized 57% of the retrieved oocytes compared with 80.5% of the oocytes being fertilized with spermatozoa from non-obstructive cases (Palermo et al., 1999Go). These findings collectively suggest that embryo cleavage, quality and implantation is paternally influenced. The severity of the spermatogenic defect appears to be the crucial factor, with more severe spermatogenic defect probably having the greatest impact.

A paternal effect on embryo development is most likely to manifest itself after embryonic genome activation, i.e. after the appearance of first paternal transcriptional products. Besides acting at the genetic level, negative paternal effect on embryo development can also be observed mediated by other components of the spermatozoa. Incomplete formation of the sperm aster due to a centrosome defect can lead to fertilization or cleavage failure (Asch et al., 1995Go). Unfavourable paternal influence on embryonic development and viability, therefore, should be expected in men with more severe spermatogenic disorders.

Blastocyst formation appears to be an indicator of embryo quality and viability. Unfavourable paternal influence affecting embryo quality may manifest itself clearly during the phase of extended embryo growth up to the blastocyst phase. We speculated that severity of the spermatogenic defect would hinder the potential of the cleavage stage embryo to develop into a blastocyst. Therefore we aimed to compare the rate of blastocyst formation and pregnancy in couples undergoing ICSI with ejaculated spermatozoa, epididymal spermatozoa, and testicular spermatozoa.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 418 oocyte retrieval-ICSI cycles with the intention to transfer at the blastocyst stage were included in the study. Of these, 347 were performed for moderate to severe oligoasthenoteratozoospermia (OAT) and 71 for azoospermia. Azoospermic men were further divided into three groups according to the source of spermatozoa and spermatogenic defect causing azoospermia. Therefore, four different groups were evaluated: group 1: men with moderate to severe OAT; group 2: men with obstructive azoospermia in whom spermatozoa were retrieved from the epididymis by percutaneous epididymal sperm aspiration (PESA); group 3: men with obstructive azoospermia in whom spermaotozoa were retrieved by percutaneous testicular sperm aspiration (PTSA) or testicular sperm extraction (TESE); and group 4: men with non-obstructive azoospermia in whom spermatozoa were retrieved by PTSA or TESE.

Ovarian stimulation, oocyte retrieval and embryo transfer procedures
In the majority of treatment cycles, ovarian stimulation was undertaken using s.c. buserelin acetate (Suprefact proinjection; Hoechst AG, Frankfurt am Main, Germany) in a long protocol combined with pure FSH (Metrodin, 75; I.F. Serono, Rome, Italy). Buserelin acetate (0.3 mg/day) was commenced on day 20 or 21 of the preceding cycle and continued until the day of human chorionic gonadotrophin (HCG). In cycles where the female was predicted to respond poorly to ovarian stimulation, a flare gonadotrophin-releasing hormone (GnRH) analogue protocol was used. FSH was initiated on the third day of the menstrual cycle with 2–6 ampoules depending on the patients' previous or anticipated response. The treatment was then individualized in a step-down fashion. When the leading follicle reached 20 mm in mean diameter with a serum oestradiol level of 200–300 pg/ml per mature follicle, 10 000 U HCG (Profasi HP 5000; I.F. Serono) was administered. Oocyte retrieval was performed 36 h after the injection of HCG. ICSI was performed only on metaphase II oocytes. All available oocytes were injected with spermatozoa showing at least twitching motility. Embryo transfer was carried out 5–6 days after oocyte retrieval. A serum pregnancy test was performed 9–11 days after embryo transfer. Clinical pregnancy was defined as the presence of gestational sac(s) with a viable embryo shown on vaginal ultrasonography performed ~24 days after embryo transfer.

Testicular sperm aspiration and extraction procedures
Procedures were performed under general anaesthesia or local anaesthesia with i.v. sedation. All procedures were performed 24–48 h prior to oocyte retrieval. In men with obstructive azoospermia, PESA was attempted first. The epididymis was secured between the thumb and index finger of the left hand while a 21 gauge butterfly needle was gently inserted into the epididymal body and aspiration was affected through the microtubing attached to a 20 ml syringe. If aspiration failed, PTSA was performed. This was undertaken with a 21 gauge butterfly needle that was inserted into the testes and moved up and down to sample a wide area. An artery forceps was secured across the attached microtubing set before the needle was withdrawn. The aspirate located in the tubing was washed into a Falcon tube with a small volume of media. The presence of spermatozoa was sought under x200 magnification. Percutaneous aspiration was attempted from three different areas of the testis and, if spermatozoa were not observed, TESE was performed. In men with non-obstructive azoospermia, PTSA was attempted first and TESE was performed if the former failed. Tissue samples measuring in size from 0.5x0.5x0.5 cm to 1x1x1 cm were removed until spermatozoa were identified or four or five biopsy pieces were extracted from each testis. Testicular tissue samples obtained in this manner were placed into Falcon tubes (Becton Dickinson, Franklin Lakes, NJ, USA) containing 2 ml of preincubated IVF-100 (IVF Science Scandinavia, Gothenburg, Sweden) medium. The samples were then transferred to Petri dishes (Falcon) containing 2 ml of the same preincubated medium. The tissue was crushed with sterile needles and subsequently with glass slides in order to separate the seminiferous tubules. The crushed tissue was then vortexed for a few seconds to facilitate the dispersal of spermatozoa into the medium. The presence of spermatozoa was sought under an inverted microscope at x20 magnification. The medium of the mixture was then treated with Pure Sperm (Nidacon International AB, Gothenburg, Sweden) gradient system. A two step washing procedure was then undertaken. The sample was first washed with Pure Sperm gradients of 90 and 50% at 500 g for 20 min. Subsequently washing was performed with 10 ml IVF-100 at 800-1000 g for another 10 min. The pellet formed was placed in the cover of a Petri dish, prepared as a swim-out droplet and covered with Ovoil 100 (IVF Science). The final solution was kept in the incubator until the ICSI procedure. ICSI was performed according to standard techniques previously described (Palermo et al., 1999Go). Spermatozoa from non-obstructive azoospermic subjects were co-incubated with recombinant FSH supplemented medium. This has been shown to increase the motility of testicular spermatozoa and induce motility in non-motile cells (Urman et al., 1998Go; Balaban et al., 1999Go).

In-vitro culture of embryos to the blastocyst stage and embryo grading
In-vitro culture of embryos was undertaken as previously described (Balaban et al., 1998Go; Schoolcraft et al., 1999Go). All cycles, regardless of the number of oocytes collected, were programmed for blastocyst transfer. Sequential media system (G1 and G2 media; Scandinavian Science AB Products, Gothenburg, Sweden) designed for further embryonic development was used. Embryos were group-cultured in 4-well dishes until day 3 in G1 media. After the evaluation of the embryo cell number and morphology they were transferred to G2 medium for further culture up to the blastocyst stage. 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 previously published criteria (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.

Statistics
Numerical variables were compared using analysis of variance with Bonferroni post-hoc test. Categorical variables were compared using {chi}2 or Fisher exact tests. P < 0.05 was accepted as significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient characteristics and stimulation variables are shown in Table IGo. Mean female age, duration of infertility mean number of oocytes retrieved were similar throughout the groups (P > 0.05). Total fertilization rate and the two-pronuclear fertilization rate were significantly decreased in the group with non-obstructive azoospermia compared to the ejaculate ICSI and obstructive azoospermia group (P < 0.001). These variables were similar when ejaculate ICSI and obstructive azoospermia groups were compared. In men with obstructive azoospermia the site of sperm retrieval (epididymal versus testicular) did not change the fertilization rate. Cleavage rate was similar in all groups (P > 0.05).


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Table I. Stimulation and laboratory variables in the treatment groups
 
Of the cleaved embryos, significantly fewer reached the blastocyst stage in the group with non-obstructive azoospermia (P < 0.001) (Table IIGo). The rate of progression to the blastocyst stage was similar in the ejaculate ICSI and obstructive azoospermia (epididymal and testicular) groups (P > 0.05). When the timing of blastocyst formation was studied (day 5 versus day 6), more embryos in the ejaculate ICSI group reached the blastocyst stage on day 5 compared to the other groups (P < 0.0001 for all groups) (Table IIGo). In this regard the obstructive azoospermia (epididymal and testicular) group was also significantly different from the non-obstructive azoospermia group (P < 0.01). The percentage of hatching and expanded blastocysts was significantly decreased in the non-obstructive azoospermia group when compared with the other three groups (P < 0.001). Of the cleavage stage embryos reaching the blastocyst stage there was no difference among the groups regarding blastocyst grading (Table IIGo).


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Table II. Blastocyst characteristics of the groups (for group definitions, see Table IGo)
 
Of the 347 initiated cycles with ejaculate ICSI, 310 (89.3%) culminated in blastocysts available for transfer (Table IIIGo). There were no blastocysts available for transfer in 37 cycles. Of these, 26 had fewer than five oocytes retrieved and 11 had only grade 4 cleavage stage embryos on day 3. These were excluded from the analysis of results. Rates for blastocyst availability for transfer were similar in the other groups.


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Table III. Embryo transfer and clinical outcome of the groups (for group definitions, see Table IGo)
 
In the ejaculate ICSI group, 93% of embryo transfers were performed on day 5. The rate of day 5 embryo transfers was significantly higher in this group compared with the other three groups. Clinical pregnancy rate and abortion rates were similar throughout the groups. Significantly more embryos implanted in the ejaculate ICSI group compared with the other groups (P < 0.05). However, there was no statistically significant difference in the implantation rate when obstructive and non-obstructive azoospermia groups were compared.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of this study clearly demonstrate a paternal effect on embryo development. Increasing severity of spermatogenetic defect was associated with decreased fertilization rates, and lower and slower rate of blastocyst formation. The above was especially true when testicular spermatozoa from non-obstructive azoospermic men were used for ICSI. Furthermore, in the latter group the incidence of hatching and expanded blastocysts was significantly decreased. Although clinical pregnancy rate per transfer was similar between the groups, implantation rate per transferred embryo was significantly increased in the group where the source of spermatozoa was the ejaculate.

The above are in concordance with the results of previous studies. In one study (Parinaud et al. 1993Go) on the influence of sperm parameters on embryo quality, it was concluded that the spermatozoon was involved in embryo quality starting from the early stages of development; there was also an association between abnormal sperm morphology and poor embryo quality. Another study (Janny and Ménézo, 1994Go) compared 334 IVF cycles performed with normal semen, cryopreserved donor semen, and abnormal fresh semen. All embryos obtained were co-cultured with Vero cells up to the blastocyst stage. The cleavage rate and blastocyst formation was highest in the group with normal fresh semen. Furthermore, 87.5% of the patients in the normal fresh semen group had at least one blastocyst available for transfer. Respective rates for patients with cryopreserved donor semen and fresh abnormal semen were 67.4 and 63.2%. The authors performed a regression analysis and found a linear correlation between cleavage rates and blastocyst formation. Finally a higher pregnancy rate was achieved in the group with normal fresh semen, although this did not reach statistical significance. The results of this study suggest a relationship between semen parameters and subsequent markers of embryo competence and viability.

In contrast to these studies, the paternal effect on implantation and pregnancy outcome in IVF/ICSI cycles was investigated (Oehninger et al., 1998). Although a poorer implantation and pregnancy outcome was noted in OAT versus normozoospermic subjects undergoing IVF, this was attributed to the high insemination concentration that was used. This conclusion was based on the findings of an earlier study undertaken by this group that showed higher implantation rates in teratozoospermic subjects treated with ICSI compared to IVF.

In a study of the development of blastocysts from supernumerary embryos after ICSI (Shoukir et al., 1998Go), the rate of blastocyst formation after ICSI (26.8%) was significantly lower than that after IVF (47.3%). This was partly affected by the higher quality and increased number of day 3 embryos in the IVF group. In this study, sperm concentration and morphology did not appear to influence blastocyst formation. However, in the ICSI group a higher rate of blastocyst formation was observed when spermatozoa from samples with higher progressive motility were used. More recently, the in-vitro development of embryos originating from either conventional in-vitro fertilization or ICSI was compared (Dumoulin et al., 2000Go). Culture of surplus embryos derived from ICSI progressed to the blastocyst stage at a significantly lower rate when compared to embryos originating from IVF.

In a study of the factors affecting the success of blastocyst development following IVF (Jones et al., 1998Go), besides other parameters such as the number of retrieved oocytes, number of cleavage stage embryos, and embryo quality, male factor aetiology in the absence of identifiable female infertility significantly influenced the rate of blastocyst formation up to day 7 after insemination.

Fertilization, pregnancy, and abortion rates after ICSI of ejaculated and surgically retrieved spermatozoa have been compared (Ghazzawi et al., 1998Go). Fertilization and pregnancy rates were similar when spermatozoa from the ejaculate, epididymis, or testes were used. However, the abortion rate was significantly higher in the latter, suggesting a diminished developmental competence of testicular sperm-derived embryos.

The above studies collectively demonstrate that embryo development and implantation are under paternal influence. In accordance with the above, our results indicate that severity of the spermatogenic disorder is associated with the success of ICSI. Blastocyst formation that is commonly accepted as an indicator of embryo quality and viability is affected by the source of the spermatozoa utilized for ICSI. When spermatozoa are retrieved from the testes as in non-obstructive azoospermia, a lower rate of blastocyst formation and implantation per blastocyst should be anticipated.


    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
 
Asch, R., Simerly, C. and Schatten, G. (1995) The stages at which fertilization arrests in humans: defective sperm centrosomes and sperm asters as causes of human infertility. Hum. Reprod., 10, 1897–1906.[Abstract]

Balaban, B., Urman, B., Sertac, A. et al. (1998) Progression of excess embryos to the blastocyst stage predicts pregnancy and implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 13, 2564–2567.[Abstract]

Balaban, B., Urman B., Sertac, A. et al. (1999) In-vitro culture of spermatozoa 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]

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

Dumoulin, J.C.M., Coonen, E., Bras, M. et al. (2000) Comparison of in-vitro development of embryos originating from either conventional in-vitro fertilization or intracytoplasmic sperm injection. Hum. Reprod., 15, 402–409.[Abstract/Free Full Text]

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Shoukir, Y., Chardonnens, D., Campana, A. and Sakkas, D. (1998) Blastocyst development from supernumerary embryos after intracytoplasmic sperm injection: a paternal influence. Hum. Reprod., 13, 1632–1637.[Abstract]

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Submitted on July 21, 2000; accepted on October 11, 2000.