1 MAR&Gen, Molecular Assisted Reproduction and Genetics, Gracia 36, 18002 Granada and 4 University of Granada, Campus Fuentenueva, 18004 Granada, Spain, 2 Laboratoire dEylau, 55 rue Saint-Didier, 75116 Paris, France and 3 European Hospital, Via Portuense 700, Rome, Italy
5 To whom correspondence should be addressed at: MAR&Gen, Gracia 36, 18002 Granada, Spain. e-mail: cmendoza@ugr.es
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Abstract |
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Key words: embryo/implantation/paternal effect/preimplantation development/sperm DNA fragmentation
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Introduction |
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The early paternal effect has been suggested to be mediated by centrosome dysfunction or deficiency of oocyte-activating factors (Tesarik et al., 2002). The implication of sperm chromatin packaging or DNA damage remains a controversial issue. In fact, the early paternal effect was observed before the major activation of embryonic genome expression which begins at the 4-cell stage in humans (Tesarik et al., 1986,
1988; Braude et al., 1988
). However, limited RNA synthesis can be detected in human pronuclei (Tesarik and Kopecny, 1989
), and failure of this early zygotic transcription is associated with abnormal pronuclear development (Tesarik and Kopecny, 1990
). On the other hand, the late paternal effect may involve sperm aneuploidy, DNA damage or abnormal chromatin packaging, which can influence the orderly activation of paternal gene expression.
Several studies have evaluated DNA structure and integrity in human ejaculated sperm samples and reported a negative effect of high percentages of spermatozoa with damaged DNA on pregnancy rates in ART (Filatov et al., 1999; Host et al., 2000
; Larson et al., 2000
; Morris et al., 2002
; Tomsu et al., 2002
; Benchaib et al., 2003
). These studies have suggested that DNA damage may serve as a marker for the diagnosis of an adverse paternal effect on human preimplantation development. However, no study dealing with the possible relationship between sperm DNA damage and the early paternal effect on zygote quality has yet been performed.
In this study, we tested potential paternal effects by comparing two groups. The experimental group consisted of infertile couples using donor oocytes who had failed three previous ICSI attempts. The control group included randomly selected infertile couples also using donor oocytes in their first ICSI attempt. Both groups used sibling oocytes from the same donors. The results of this analysis were related to the percentage of spermatozoa with fragmented DNA in the corresponding patient groups.
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Materials and methods |
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Based on the evaluation of early embryo morphology in the previous unsuccessful attempts, the 18 couples with a history of three previous ART failures were divided into two groups referred to as early paternal effect and late paternal effect, respectively.
Early paternal effect
This group was formed by couples in whom 5 oocytes (
50%) were fertilized in each of the three previous ART attempts, but <25% of the resulting zygotes were classified as having good morphology according to the previously described criteria (Tesarik and Greco, 1999
; Tesarik et al., 2002
).
Late paternal effect
This group was formed by couples in whom 5 oocytes were fertilized in each of the three previous ART attempts that resulted in implantation failure in spite of no apparent impairment of zygote and cleaving embryo morphology (
50% of zygotes were classified as having good morphology).
Design
In our oocyte donation programme, oocytes from each donor are shared by two infertile couples. Each of the two couples received half of the metaphase II oocytes recovered from the corresponding donor. If an odd number of oocytes was recovered, the couple receiving the extra oocyte was determined randomly.
The analyses described in this study concern the outcomes of a fourth ART attempt with donor oocytes performed for the couples showing either an early or a late paternal effect (experimental group). The data obtained were compared with those concerning randomly chosen infertile couples undergoing their first ICSI attempt (control group) who shared sibling oocytes with the couples involved in the experimental group. In parallel, sperm samples used for ART in each of these attempts were analysed for basic sperm parameters and for DNA fragmentation (see below).
Oocyte donor preparation
Oocyte donors were healthy volunteers aged between 20 and 28 years, without any apparent reproductive or other pathology and with a good ovarian reserve (basal serum FSH and inhibin B concentrations of <7 IU/l and >100 pg/ml, respectively). The general health status of each candidate for oocyte donation was assessed by the examinations defined by the Spanish Law of Assisted Reproduction, including karyotype and tests for human immunodeficiency virus (HIV), hepatitis B and C, cytomegalovirus, herpes virus, rubella, clamydia, toxoplasmosis and syphilis.
Ovarian stimulation of oocyte donors was performed with the use of a combination of recombinant and urinary human gonadotrophins after pituitary downregulation started in the mid-luteal phase as described (Tesarik and Mendoza, 2002). Ovulation was induced with 10 000 IU of HCG (Profasi; Serono, Rome, Italy) when there were at least five follicles of
18 mm in diameter in both ovaries. Ultrasound-guided transvaginal follicular aspiration was performed 36 h later.
Oocyte recipient preparation
Oocyte recipients were ovulating women in whom the recourse to oocyte donation was indicated because of a poor response to ovarian stimulation or poor oocyte morphology. The ovarian activity of the oocyte recipients was suppressed by pituitary desensitization with triptorelin (Decapeptyl; Ipsen Pharma, Madrid, Spain) started in the midluteal phase. When the serum estradiol concentration was <45 pg/ml and there was no cystic structure of >10 mm detectable by ultrasonography in the patients ovaries, the desensitization was considered complete, and the patient was ready for the beginning of endometrial growth stimulation. This condition was maintained until the respective oocyte donor was ready for ovarian stimulation. Accordingly, the period between the start of triptorelin treatment and embryo transfer varied between 32 and 45 days. Triptorelin was first applied in a single i.m. injection of a long-acting preparation (Decapeptyl LP, 3.75 mg) followed, when necessary, by daily s.c. injections of a short-acting preparation of triptorelin (Decapeptyl, 0.1 mg).
Stimulation of endometrial growth was started 1 day before the beginning of ovarian stimulation of the respective donor. It was performed by oral estradiol valerate (Progynova; Schering, Madrid, Spain) at daily doses of 2 mg on days 14, 4 mg on days 58 and 6 mg from day 9 until 1 day after the injection of HCG in the respective oocyte donor when the daily dose of estradiol valerate was reduced to 2 mg. On the same day, intravaginal application of natural micronized progesterone (Utrogestan; Laboratoires Besins-Iscovesco, Paris, France) was started, beginning with a daily dose of 200 mg which was augmented on the two following days to 400 and 600 mg, respectively. The daily dose of 600 mg micronized progesterone was maintained until the pregnancy test, and in the case of pregnancy, this medication was prolonged through the 3 months following embryo transfer. The treatment with 2 mg of estradiol valerate was maintained until the negative pregnancy test or, in the case of pregnancy, until the serum estradiol concentration had reached 1000 pg/ml.
Assisted reproduction techniques
Oocytes were prepared and subjected to ICSI using previously described techniques and equipment (Tesarik and Sousa, 1995). Fertilization was assessed 1416 h after ICSI. Oocytes were considered fertilized when they contained two pronuclei. Abnormally fertilized (three or more pronuclei) and parthenogenetically activated oocytes (one pronucleus) were discarded at this stage and are not taken into account. Zygotes were classified as having good morphology or poor morphology according to previously described criteria based on the number and distribution of nucleolar precursor bodies (Tesarik and Greco, 1999
; Tesarik et al., 2000
).
Embryo in vitro culture was performed in IVF medium (Vitrolife; Göteborg, Sweden) equilibrated with 5% CO2 in air up to day 3 after ICSI when three embryos were transferred to the patients uterus. Embryos that showed the best scores at the cleavage stages (see the section on ygote and cleaving embryo evaluation) were selected for transfer. Where possible, these embryos were selected from those that developed from good morphology pronuclear zygotes. If supernumerary embryos of adequate quality were available, they were cryopreserved for eventual later transfer. However, only treatment attempts with fresh embryos are involved in this study. Only clinical pregnancies, characterized by the presence of an embryonic heartbeat, are taken into account in this study.
Sperm evaluation
Basic evaluation. Basic evaluation of sperm samples used in this study was performed according to World Health Organization instructions (World Health Organization, 1999).
Evaluation of sperm DNA fragmentation. Sperm DNA fragmentation was evaluated in the swim-up sperm preparation used for ICSI by the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay using the in situ Cell Death Detection Kit with fluorescein isothiocyanate (FITC)-labelled dUTP (Roche, Monza, Italy). Samples of each sperm preparation were smeared on at least four microscope slides, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. The rest of the procedure was carried out according to the manufacturers instruction. Briefly, the specimens were incubated in TUNEL reaction mixture in the dark at 37°C for 1 h followed by evaluation in a fluorescence microscope. Four hundred spermatozoa were randomly analysed in two slides for each sample (200 spermatozoa per slide). The remaining two slides were used for the negative (omitting the enzyme terminal transferase) and positive (using DNase I, 1 mg/ml for 20 min at room temperature) controls.
Zygote and cleaving embryo evaluation
Zygotes were evaluated with the use of a previously described scoring system based on the examination of pronuclear morphology (Tesarik and Greco, 1999) in its simplified version (Tesarik et al., 2000
). Accordingly, zygotes with normal pronuclear patterns are classified as having good morphology, and the five originally described abnormal pronuclear patterns (Tesarik and Greco, 1999
) are grouped together as poor morphology zygotes.
The quality of cleaving embryos was scored using criteria based on the cleavage speed and morphology (blastomere regularity and the volume occupied by anucleate cell fragments). The latter was quantified with the use of previously described embryo morphology-grading criteria (Bolton et al., 1989).
Statistics
Paired Students t-test and Wilcoxon matched pairs test were used as applicable. Continuous outcomes and average percentage differences between matched groups are represented as mean ± SD. All analyses were performed using the Statistica 5.0 software (Statsoft Version 5.1, Hamburg, Germany).
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Results |
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One singleton pregnancy was established in the experimental group. This contrasted with seven pregnancies, of which four were twin, in the control group. The implantation rate was significantly lower in the former group as compared with the latter (Table I). Endometrial thickness on the day of embryo transfer was similar in both groups (Table I).
Late paternal effect
Similarly to the early paternal effect, there were 50% normally fertilized oocytes in this group of patients as well as in the group of randomly chosen patients who shared sibling oocytes from the same donors. However, in contrast to the early paternal effect, no difference in the percentage of good morphology zygotes was observed between these two groups of patients (Table II).
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There was no pregnancy in the experimental group. This contrasted with six clinical pregnancies, of which three were twin, obtained in the control group. The difference in the implantation rate between the two groups was significant (Table II). Endometrial thickness on the day of embryo transfer was similar in both groups (Table II).
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Discussion |
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On the other hand, the results of this study show that high percentages of spermatozoa with fragmented DNA are often found in cases of repeated ART failures without any apparent impairment of zygote and cleaving embryo morphology, a condition referred to as late paternal effect in this study. The absence of a relationship between sperm DNA fragmentation and cleaving embryo morphology has also been reported by another recent study (Benchaib et al., 2003). Because embryos were transferred on day 3 after ICSI in this study, it is not clear whether this late paternal effect could influence blastocyst development if in vitro culture were prolonged to days 56 after ICSI or whether this effect becomes manifest later during implantation and post-implantation development.
No differences in endometrial thickness on the day of embryo transfer were found between the experimental and the control group. Moreover, all oocyte recipients had normal serum concentration of insulin, TSH and prolactin, none of them showed elevated serum concentrations of anti-cardiolipin, anti-phospholipid and anti-nuclear antibodies, and none suffered from endometriosis. Even though the existence of a hidden female factor, related to uterine receptivity, cannot be completely excluded, the fact that a sperm-related anomaly (sperm-derived impairment of zygote morphology or sperm DNA fragmentation) was detected in all cases included in the experimental group strongly suggests that implantation failure in the experimental group was caused by a paternal rather than a maternal effect.
The principal clinical message of this study can be derived from the observation that the results of ART using ICSI for fertilization can be compromised by paternal effects acting at different times of embryo development and detectable with the use of different diagnostic means. Analysis of sperm DNA fragmentation by the TUNEL assay is a valuable diagnostic method to reveal the paternal origin of unexplained repeated ICSI failures. However, this study also shows that normal values of the TUNEL assay do not exclude the existence of an adverse paternal effect. If the percentage of TUNEL-positive spermatozoa is not increased compared with values found in fertile individuals, the finding of high frequencies of zygote and cleaving embryo morphological abnormalities is sufficient to suspect a paternal origin of ART failure, especially if this condition occurs repeatedly or with oocytes of supposedly good quality, such as in an oocyte donation programme.
Moreover, this study also shows that a (late) adverse paternal effect on embryo development can be present even if no morphological abnormalities are apparent at the zygote stage. It is important to note that it is in just such cases that increased percentages of spermatozoa with fragmented DNA can often be detected by the TUNEL assay.
In conclusion, sperm DNA fragmentation should be evaluated in cases of unexplained ICSI failure without any apparent impairment of embryo morphology. If embryo morphology is impaired, a paternal factor is to be suspected even if the analysis of sperm DNA fragmentation shows normal values. The validity of these findings for conventional IVF remains to be evaluated. With regard to the development of future treatment strategies for sperm deficiencies responsible for sperm-borne embryo demise, the possibility of the existence of an early and a late paternal factor as two different pathological entities should be taken into consideration.
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References |
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Submitted on: August 22, 2003 ccepted on November 19, 2003