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Poor sperm quality affects clinical outcomes of intracytoplasmic sperm injection in fresh and subsequent frozen–thawed cycles: potential paternal effects on pregnancy outcomes
Poor sperm quality affects clinical outcomes of intracytoplasmic sperm injection in fresh and subsequent frozen–thawed cycles: potential paternal effects on pregnancy outcomes Sun-Hee Lee, M.Sc.,a Haengseok Song, Ph.D.,a,b Yong-Seog Park, Ph.D.,a,b Mi Kyoung Koong, M.D., Ph.D.,c In Ok Song, M.D., Ph.D.,c and Jin Hyun Jun, Ph.D.a,b a Laboratory of Reproductive Biology and Infertility and Departments of bMedicine and of cObstetrics and Gynecology, Cheil General Hospital and Women’s Healthcare Center, Kwandong University College of Medicine, Seoul, South Korea
Objective: To evaluate objectively whether poor sperm quality affects sequential events from fertilization to delivery in fresh intracytoplasmic sperm injection (ICSI) and subsequent frozen–thawed embryo transfer (ET) cycles. Design: Retrospective study. Setting: University-based centers for reproductive medicine. Patient(s): For unbiased comparison, 206 cycles were chosen from 1,999 cycles of patients who underwent ICSIET and/or subsequent frozen–thawed ET. Cycles met the following criteria: day 3 ET; female age, <40 y; number of retrieved oocytes, R5; no split insemination; and no female factors but tubal factor. Intervention(s): None. Main Outcome Measure(s): The rates of fertilization, embryo implantation, clinical pregnancy, and delivery and sequential embryonic score (SES) were compared between normal-spermatogenesis patients (NSPs) and defectivespermatogenesis patients (DSPs). Result(s): Although sum SES, mean SES, and top SES of transferred embryos on day 3 were similar between NSPs and DSPs, the rates of implantation, clinical pregnancy, and delivery of NSPs were significantly higher than those of DSPs. Furthermore, subsequent ET cycles with frozen–thawed embryos in NSPs and DSPs who failed to achieve pregnancy in their fresh cycles showed that rates of implantation and clinical pregnancy also were significantly lower in DSPs. Conclusion(s): Quality of sperm may influence embryo implantation and subsequent pregnancy outcomes without impairment of embryo quality. (Fertil Steril 2009;91:798–804. 2009 by American Society for Reproductive Medicine.) Key Words: Defective spermatogenesis, paternal effect, clinical outcomes, ICSI, fresh ET, frozen-thawed ET
Conventional sperm parameters of semen analysis, such as count, motility, and morphology, used to be considered critical factors for fertilization and subsequent clinical outcomes in assisted reproductive technologies (ART). Poor outcomes were achieved in ART cycles with severe male factors because fertilization rates were inadequately low (1). Introduction of intracytoplasmic sperm injection (ICSI) has dramatically increased fertilization and pregnancy outcomes in these cycles and become the treatment of choice in male infertility, regardless of the severity of the condition (2). Although ICSI has overcome male infertility as defined by conventional parameters, allowing fertilization, some characteristics are still considered important for the clinical outcomes of ICSI.
Recent studies with oocyte donation cycles have suggested that consequences of the actions of sperm-derived factors on preimplantation embryo development, referred to as paternal effects, are responsible for repeated failure of ART cycles (3–5). Early paternal effect may cause delay of cleavage speed and increase the fragmentation of developing embryos (6). Late paternal effect may involve sperm aneuploidy, DNA damage, or abnormal chromatin packaging, which can influence the orderly activation of paternal gene expression (7). These works suggested that aberrant zygotic gene activation as a result of defective sperm may be a main cause of repeated failure of ART cycles, especially for those patients without impairment of zygote and cleaving embryo morphology (3, 8).
Received September 17, 2007; revised and accepted December 19, 2007. Authors S.-H.L. and H.S. contributed equally to the work and both should be considered to be the first author. Reprint requests: Jin Hyun Jun, Ph.D., Laboratory of Reproductive Biology and Infertility, Cheil General Hospital and Women’s Healthcare Center, 1-19 Mukjeong-dong, Jung-gu, Seoul 100-380, South Korea (FAX: 82-2-2265-5621; E-mail: [email protected]).
There have been several reports that male-factor infertility such as oligoasthenoteratozoospermia (OATS) resulted in decreased fertilization rate, poor embryo quality, and poor pregnancy outcomes (9, 10). In contrast, poor quality of sperm, such as in nonobstructive azoospermia (NOA) and severe OATS, does not affect clinical outcomes (fertilization
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rate, embryo quality, implantation, and/or pregnancy rate) in ICSI cycles (11, 12). Miller and Smith (1) reported that severe male factor is not related to fertilizing ability but is associated with a higher rate of developmental arrest at the five- to eight-cell stage on day 3 and with decreased blastocyst formation on day 5 in these cycles. Thus, it is still unclear whether poor sperm quality could lead to detrimental effects on fertilization, subsequent embryogenesis, implantation, and clinical pregnancy. In this study, for unbiased comparison, we selected ICSI cycles in which the embryo was transferred on day 3 and there were not genetic and/or female factors except tubal factor. A total of 206 selected ICSI cycles was divided into those of normal spermatogenesis patients (NSPs; normal ejaculated and obstructive azoospermic patients, n ¼ 149) and defective spermatogenesis patients (DSPs; severe OATS and nonobstructive azoospermic patients, n ¼ 57). Although the fertilization rate of DSPs was significantly lower than that of NSPs, the overall quality of early cleaving embryos in NSPs and DSPs was quite comparable. However, pregnancy outcomes (implantation, clinical pregnancy, and delivery) were significantly low in DSPs compared with in NSPs. Moreover, subsequent embryo transfer (ET) cycles with frozen–thawed embryos in NSPs and DSPs who failed to achieve pregnancy in their fresh cycles showed that the pregnancy outcomes were considerably lower in DSPs. Collectively, the quality of sperm could influence implantation and subsequent pregnancy outcome, as well as fertilization, in ICSI cycles. The poor clinical pregnancy outcomes of DSPs in fresh ET cycles and in those after frozen-thawed ET cycles may be derived from paternal effects of defective spermatogenesis in these patients. MATERIALS AND METHODS Study Design and Patients Between January 2003 and December 2004, 1,999 ET cycles after ICSI were performed in our center. To more objectively evaluate potential adverse effects of poor sperm quality on fertilization to implantation, pregnancy, and delivery in this retrospective study, ICSI cycles that met the following criteria were selected from a total of 1,999 ICSI-ET cycles carrying various infertility factors (Fig. 1): day 3 ET, with female age of <40 years and number of retrieved oocytes of R5 and without split insemination and any genetic factors. In addition, ICSI cycles with any female factors but tubal factor also were excluded from this study, because female factors may significantly influence oocyte quality, preimplantation embryo development, and/or implantation. Ejaculated and testicular sperms were evaluated on the basis of World Health Organization criteria and histological diagnosis, respectively (13–15). The selected 206 cycles were classified into NSPs (n ¼ 149) and DSPs (n ¼ 57). The NSP group consisted of normal ejaculated patients and those with obstructive azoospermia (OA), and the DSP group included patients with severe OATS (<5 million sperm per mL and/or <10% sperm motility and/or <4% normal sperm morphology) and nonobFertility and Sterility
FIGURE 1 A schematic diagram for patient selection to evaluate adverse effects of defective sperm quality on embryo implantation and clinical pregnancy outcomes in ICSI and subsequent frozen–thawed ET cycles. S-OATS ¼ severe OATS.
Lee. Paternal effects on clinical outcomes. Fertil Steril 2009.
structive azoospermia. Histological analysis showed that patients with nonobstructive azoospermia had defective spermatogenesis (13 hypospermatogenesis, 1 maturation arrest, and 3 germ cell aplasia) and that all patients with OA had normal spermatogenesis. Because of the retrospective nature of the study, institutional review board approval was not required. Sperm Preparation, Oocyte Retrieval, ICSI, and In Vitro Culture Sperm preparation was performed by routine protocols, as described elsewhere (16). Ovarian stimulation was performed by using GnRH agonist/antagonist, hMG, and human recombinant FSH. Human chorionic gonadotropin was administered when optimal follicle development was achieved, as evaluated by serial transvaginal ultrasound and estrogen determinations. Oocyte retrieval was performed via a transvaginal approach with sonographic guidance, 34 hours after hCG injection. Retrieved oocytes were incubated in G-Fert medium (VitroLife, Kungsbacka, Sweden) that was supplemented with 10% recombinant human serum albumin (VitroLife) at 37 C, 6% CO2 in air. Three to five hours after oocyte retrieval, cumulus cell mass and corona radiata of the oocytes were removed by incubation for 1 minute in medium with 799
0.1% hyaluronidase (Sigma, St. Louis, MO). Denuded oocytes were rinsed several times in fresh medium and evaluated under the microscope at 200 magnification. Intracytoplasmic sperm injection was performed on metaphase II oocytes, and fertilization was assessed 16–18 hours after ICSI. Fertilized zygotes were cultured with G1.3 and G2.3 sequential media (VitroLife) until ET. Sequential Embryonic Scoring Sequential embryonic score (SES) was established on the basis of the sum of the pronuclear (PN) score and embryonic score. The PN scoring method was modified from reports published elsewhere (17, 18). The PN score was monitored twice, at 18–20 hours and at 24–26 hours after ICSI. At 18–20 hours after ICSI, PN morphology was determined by position of the pronuclei and by morphology and orientation of the nucleoli. It was assessed by the following criteria: A is the ideal pattern (3 points), in which PN of approximately equal size had three or four equal-sized nucleoli beginning to align with systemic and polarized patterns on the borderline of the pronuclei. B is a moderate pattern (2 points), in which the PN had about 10 small-sized nucleoli, scattered or aligned. C is a nonideal pattern (1 point), in which PN of unequal size had different sizes and number of nucleoli. At 24–26 hours, the PN scoring was as follows: early cleavage embryo was 3 points, one cell was 2 points, syngamy was 1 point, and the other patterns were 0 points. Embryonic score represents the quality of cleaving embryos. It was scored by using criteria based on the cleavage speed, the number of blastomeres, and patterns of fragmentation (19). A grade I embryo was 5 points, grade I-1 was 4 points, grade II was 3 points, grade II-1 was 2 points, and grade III was 1 point. On day 3, eight cells or more was 3 points, four to seven cells was 2 points, and three cells or fewer was 1 point. Embryonic score was calculated on the basis of points for grade points for cell stage. Embryo Transfer and Assessment of Pregnancy The embryos were transferred into the uterine cavity on day 3 after oocyte retrieval. Pregnancy was determined if serial serum b-hCG level was >5 mIU/mL at 12 days after the oocyte retrieval. Clinical pregnancy was defined as the presence of a G-sac by ultrasonography at approximately 5 weeks of pregnancy. Supernumerary embryos were frozen at the PN or cleavage stage by using the slow-freezing method, using propylene glycol and sucrose (20). Freezing and thawing solutions were prepared in Dulbecco’s phosphate-buffered saline (Gibco-BRL, Grand Island, NY), supplemented with 20% synthetic serum substitute (Irvine Scientific, Irvine, CA). The embryos were exposed in a stepwise fashion to increasing concentrations of 1.5 M propylene glycol þ 0.1 M sucrose in the freezing solution. The embryos then were loaded into a 0.25 mL sterile straw (Bicef, L’Aigle, France), and then the straw was loaded into a programmable, controlled-rate freezing machine (Cryo-magic; Miraebiotech, 800
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Seoul, Korea) and kept at 20 C. The straw subsequently was cooled from 20 C to 7 C at a rate of 2 C/min, held at this temperature for 5 minutes, and then seeded manually. It then was cooled to 30 C at a rate of 0.3 C/min, cooled to 150 C at 30 C/min, and plunged into liquid nitrogen. Frozen embryos were thawed by the rapid thawing method (20). For thawing of the frozen human embryos, the straws were warmed by holding them in air for 40 seconds before being plunged them into a water bath at 37 C for 1 minute. The cryoprotectant then was removed by a reverse stepwise dilution. In frozen–thawed ET cycles, a daily dose of 6 mg of oral estradiol valerate was initiated on menstrual day 2. Estrogen and progesterone were administered, and the endometrial thickness was monitored. Embryo transfer was performed 3–5 days after the endometrial thickness reached >8 mm. After ET, luteal support with progest (Samil, Seoul, Korea) was continued for 14 days, until a urine pregnancy test was performed. Statistical Analysis Chi-square test or t-test was used for statistical analysis, and P values of < .05 were considered significant. RESULTS Selection and profiles of ICSI cycles grouped as NSPs and DSPs are summarized in Figure 1 and Table 1. The mean age of female partners of DSPs was younger than that of partners of NSPs, reflecting the higher number of retrieved oocytes with higher E2 value on the hCG day in DSPs. The matured MII oocyte rate was comparable between the groups. However, the normal fertilization rate with 2PN in DSPs was significantly lower than that in NSPs (67.6% vs. 80.3%, P<.05). Although the fertilization rate of DSPs was lower than that of NSPs, the overall quality of transferred embryos on day 3 was quite similar between both groups (Table 2). The mean number of transferred embryos was similar between NSPs and DSPs (3.3 0.7 vs. 3.4 0.9). To assess the quality of early developing embryos, SES was measured and analyzed between the groups. The sum SES of transferred embryos in each ET cycle was very similar for NSPs and DSPs. In particular, the mean SES of transferred embryos (11.6 3.3 vs. 11.6 3.4) and the SES of top-quality embryos (14.7 3.9 vs. 14.5 4.2) in each cycle were quite comparable between NSPs and DSPs. Collectively, this implied that embryos derived from sperms of DSPs developed to the eight-cell stage on day 3 as competently as did those from NSPs. Considering the similar characteristics between NSPs and DSPs of transferred embryos on day 3, an important observation is that pregnancy outcomes in DSPs were significantly decreased (Table 3). The implantation rate in DSPs was significantly lower than that in NSPs (15.5% vs. 24.3%, P<.05). The clinical pregnancy rate in DSPs also was significantly lower than that in NSPs (33.3% vs. 53.0%, P<.05). Consequently, whereas 70 (47.0%) of 149 cycles reached delivery in NSPs, 17 (29.8%) of 57 cycles resulted in delivery in Vol. 91, No. 3, March 2009
TABLE 1 The cycle characteristics of patients who underwent ICSI with spermatozoa from NSPs and DSPs. Parameter Mean of female age (y) E2 value on hCG day (pg/mL) No. of retrieved oocytes Oocyte maturation rate (%) Fertilization rate (%)
NSPs (n [ 149)
DSPs (n [ 57)
P value
32.3 3.3 2,077 1,222 14.1 7.8 1,611/2,093 (77.0) 1,295/1,611 (80.3)
30.6 2.7 2,930 2,012 19.4 9.6 863/1,108 (77.9) 583/863 (67.6)
< .01 < .01 < .01 NS < .05
Note: Values are mean SD or are n (%). NS ¼ not significant. Lee. Paternal effects on clinical outcomes. Fertil Steril 2009.
DSPs (P<.05). Although there was a significant difference in overall pregnancy outcomes between the two groups, they had similar miscarriage rates. To evaluate whether potential detrimental effects of controlled ovarian hyperstimulation on implantation and pregnancy contribute, at least partly, to lower implantation and clinical pregnancy rates in DSPs, we thus analyzed subsequent ET cycles with frozen–thawed embryos in NSPs and DSPs who failed to achieve pregnancy in their fresh cycles. Twenty-seven of 84 NSPs and 19 of 40 DSPs have continued their ET cycles with embryos that were cryopreserved at the PN or cleavage stage during their fresh cycles. Consistent with data from fresh cycles (Table 3), the data showed that rates of implantation (31.9% vs. 14.5%, P<.05) and clinical pregnancy (66.7% vs. 31.6%, P<.05) also were significantly reduced in DSPs (Table 4). The delivery rate also was lower in DSPs (26.3%) compared with in NSPs (55.6%), even though the difference did not reach statistical significance. Collectively, it clearly was shown that poor sperm quality may contribute to detrimental effects on embryo implantation and subsequent clinical pregnancy outcomes in ICSI-ET cycles. DISCUSSION With the current state of the art in ART, fertilization and early preimplantation development of multiple embryos can be accomplished successfully in most cases. However, the implan-
tation potential of the embryos after transfer to the uterine cavity still remains relatively low (21). Implantation and early pregnancy can be influenced by viability of the transferred embryos, by quality of oocyte and sperm, as well as by several factors responsible for uterine receptivity. Paternal effects, the inadequate contribution of paternal factors to preimplantation embryo development, have been shown to be responsible for repeated failure of ART cycles (4, 22, 23). In contrast, it also has been reported that sperm parameters are not associated with an impaired implantation or pregnancy potential (11, 12). This debate about the potential effect of sperm quality on these events may be associated with the multifactorial causes of infertility in retrospective studies. Thus, to objectively analyze all the clinical outcomes examined, we selected cycles in which severe male factor was the sole cause of infertility, except for female tubal factor, from total ICSI cycles. Our analysis of fresh ICSI-ET and subsequent frozen–thawed ET cycles clearly showed that DSPs have poor implantation rates and have poor pregnancy and delivery outcomes, without any distinct defects in early embryo development. This study, as well as that of Goker et al. (12), reported low fertilization rate in cycles with poor sperm quality. These results may come from an early paternal effect that possibly is mediated by centrosome dysfunction or by deficiency of oocyte activating factor (4). There have been many reports that early paternal effects are associated with early embryo development as well. Early paternal effect leads to delay of
TABLE 2 Comparison of embryo quality and number of transferred embryos between NSPs and DSPs. Parameter No. of transferred embryos Sum SES of transferred embryos Mean SES of transferred embryos Top SES
NSPs (n [ 149)
DSPs (n [ 57)
3.3 0.7 38.3 11.2 11.6 3.3 14.7 3.9
3.4 0.9 38.0 12.0 11.6 3.4 14.5 4.2
Note: Values are mean SD. Comparisons of the two groups were not significant on all parameters. Lee. Paternal effects on clinical outcomes. Fertil Steril 2009.
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TABLE 3 Comparison of clinical pregnancy outcomes between NSPs and DSPs in fresh ICSI cycles.
Implantation rate (%) Clinical pregnancy rate (%) Delivery rate (%) Miscarriage rate (%)
NSPs (n [ 149)
DSPs (n [ 57)
P value
121/498 (24.3) 79 (53.0) 70 (47.0) 9 (11.4)
30/193 (15.5) 19 (33.3) 17 (29.8) 2 (10.5)
< .05 < .05 < .05 NS
Note: NSP ¼ normal spermatogenesis patients; DSP ¼ defective spermatogenesis patients; NS ¼ not significant. Lee. Paternal effects on clinical outcomes. Fertil Steril 2009.
cleavage speed and increases the fragmentation rate of cleaving embryos (6). Up to 25% of the nondividing eggs are in fact fertilized but succumb to cell division defects (24). However, our data showed that development potentials of embryos from DSPs and NSPs are similar, at least up to the eight-cell stage on day 3, whereas fertilization rate in DSPs is significantly lower than that in NSPs. This suggests that reduced fertilization potential of poor quality sperm in DSPs is not directly associated with early paternal effects. The application of SES for selection of good embryos improved implantation and subsequent clinical pregnancy outcomes in ART (25). Because multiple embryos usually were transferred in ART cycles, average SES and highest SES of transferred embryos are critical factors in comparing the potential of embryos for implantation and further pregnancy. We observed that these scores were very similar between NSPs and DSPs, suggesting that the morphological quality of embryos from DSPs is as good as that of embryos from NSPs. Goker et al. (12) also reported a comparable good embryo rate in ICSI cycles with defective ejaculated sperms. Although ours as well as previous works showed that morphologically good embryos can be derived from defective sperms, we clearly showed that DSPs had poor implantation, pregnancy, and delivery outcomes without an impairment in quality of embryos transferred on day 3, in both their fresh
and subsequent frozen–thawed cycles. Our data suggests that morphological evaluation for embryo quality may not fully represent embryo potentials for blastocyst formation and implantation. In fact, there has been a recent observation that a large proportion of morphologically normal embryos on day 3 is chromosomally abnormal (26). This is consistent with a report that poor quality sperms were correlated significantly with increased rate of developmental arrest at the fiveto eight-cell stage and with poor blastocyst development (1). When ICSI is used, these abnormal spermatozoa are capable of achieving fertilization, but sperm DNA from DSPs, such as in the case of severe OATS, may possess anomalies such as loose packaging of the chromatin and DNA strand breaks (27), possibly resulting in developmental arrest during the preimplantation phase (28, 29). Sperm DNA fragmentation may affect postimplantation development in the ICSI procedure, and it could compromise embryo viability, resulting in pregnancy loss (7, 30). There is growing evidence that significant DNA damage in poor quality sperm may be caused by high levels of reactive oxygen species in male reproductive tracts and seminal plasma (31). Decreasing seminal plasma antioxidant levels have been reported in seminal plasma of males with impaired sperm function (32). Collectively, detrimental paternal effects of a defective paternal genome could contribute to aberrant zygotic gene activation, leading to development arrest at the eight-cell stage, poor blastocyst
TABLE 4 Comparison of embryo quality and clinical pregnancy outcomes between NSPs and DSPs in subsequent frozen–thawed embryo transfer cycles. Parameter Mean female age (y) Good embryo rate (%) No. of transferred embryos Implantation rate (%) Clinical pregnancy rate (%) Delivery rate (%) Miscarriage rate (%)
NSPs (n [ 27)
DSPs (n [ 19)
P value
32.2 3.2 72/127 (56.7) 3.4 0.8 29/91 (31.9) 18 (66.7) 15 (55.6) 3 (16.7)
30.1 1.6 52/90 (57.8) 3.6 0.8 10/69 (14.5) 6 (31.6) 5 (26.3) 1 (16.7)
< .05 NS NS < .05 < .05 NS NS
Note: Values are mean SD or n (%). NS ¼ not significant. Lee. Paternal effects on clinical outcomes. Fertil Steril 2009.
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development, and subsequent poor implantation and pregnancy outcome in vitro and in vivo. There is a spectrum of factors that may influence ART outcomes, such as age of the female partner, number of retrieved oocytes, performance of ICSI, etiology of infertility, and choice of embryos transferred. Among them, age of the female partner is one of the most significant factors influencing clinical outcomes of ART cycles. Considering that younger female partners, in general, have better clinical outcomes in ART cycles (33), it is even more noteworthy that implantation and clinical pregnancy rates for DSPs with younger female partners are significantly lower than those for NSPs in fresh ICSI and subsequent frozen–thawed ET cycles. Controlled ovarian hyperstimulation may significantly affect clinical outcomes as well as embryo implantation in ART cycles, too. Supraphysiological levels of estrogen resulting from controlled ovarian hyperstimulation may alter endometrial homeostasis and uterine receptivity, leading to poor clinical outcomes (34–36). Adverse effects of controlled ovarian hyperstimulation on clinical outcomes in ART are very prominent in aged female partners but are counterbalanced in younger ones (33). Once more, this suggests that DSPs with younger female partners have an advantage over NSPs in terms of clinical outcomes. Thus, those previous reports collectively reinforced our notion that defective spermatogenesis could contribute to poor embryo implantation and clinical pregnancy outcomes in both fresh ICSI-ET and subsequent frozen–thawed ET cycles. In summary, our results from fresh ICSI cycles and subsequent frozen–thawed cycles clearly demonstrate that detrimental paternal effects caused by defective spermatogenesis significantly influence implantation and subsequent clinical outcomes, possibly via aberrant embryonic gene activation that occurs on day 3.
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Objective: To evaluate objectively whether poor sperm quality affects sequential events from fertilization to delivery in fresh intracytoplasmic sperm injection (ICSI) and subsequent frozen–thawed embryo transfer (ET) cycles. Design: Retrospective study. Setting: University-based centers for reproductive medicine. Patient(s): For unbiased comparison, 206 cycles were chosen from 1,999 cycles of patients who underwent ICSIET and/or subsequent frozen–thawed ET. Cycles met the following criteria: day 3 ET; female age, <40 y; number of retrieved oocytes, R5; no split insemination; and no female factors but tubal factor. Intervention(s): None. Main Outcome Measure(s): The rates of fertilization, embryo implantation, clinical pregnancy, and delivery and sequential embryonic score (SES) were compared between normal-spermatogenesis patients (NSPs) and defectivespermatogenesis patients (DSPs). Result(s): Although sum SES, mean SES, and top SES of transferred embryos on day 3 were similar between NSPs and DSPs, the rates of implantation, clinical pregnancy, and delivery of NSPs were significantly higher than those of DSPs. Furthermore, subsequent ET cycles with frozen–thawed embryos in NSPs and DSPs who failed to achieve pregnancy in their fresh cycles showed that rates of implantation and clinical pregnancy also were significantly lower in DSPs. Conclusion(s): Quality of sperm may influence embryo implantation and subsequent pregnancy outcomes without impairment of embryo quality. (Fertil Steril 2009;91:798–804. 2009 by American Society for Reproductive Medicine.) Key Words: Defective spermatogenesis, paternal effect, clinical outcomes, ICSI, fresh ET, frozen-thawed ET
Conventional sperm parameters of semen analysis, such as count, motility, and morphology, used to be considered critical factors for fertilization and subsequent clinical outcomes in assisted reproductive technologies (ART). Poor outcomes were achieved in ART cycles with severe male factors because fertilization rates were inadequately low (1). Introduction of intracytoplasmic sperm injection (ICSI) has dramatically increased fertilization and pregnancy outcomes in these cycles and become the treatment of choice in male infertility, regardless of the severity of the condition (2). Although ICSI has overcome male infertility as defined by conventional parameters, allowing fertilization, some characteristics are still considered important for the clinical outcomes of ICSI.
Recent studies with oocyte donation cycles have suggested that consequences of the actions of sperm-derived factors on preimplantation embryo development, referred to as paternal effects, are responsible for repeated failure of ART cycles (3–5). Early paternal effect may cause delay of cleavage speed and increase the fragmentation of developing embryos (6). Late paternal effect may involve sperm aneuploidy, DNA damage, or abnormal chromatin packaging, which can influence the orderly activation of paternal gene expression (7). These works suggested that aberrant zygotic gene activation as a result of defective sperm may be a main cause of repeated failure of ART cycles, especially for those patients without impairment of zygote and cleaving embryo morphology (3, 8).
Received September 17, 2007; revised and accepted December 19, 2007. Authors S.-H.L. and H.S. contributed equally to the work and both should be considered to be the first author. Reprint requests: Jin Hyun Jun, Ph.D., Laboratory of Reproductive Biology and Infertility, Cheil General Hospital and Women’s Healthcare Center, 1-19 Mukjeong-dong, Jung-gu, Seoul 100-380, South Korea (FAX: 82-2-2265-5621; E-mail: [email protected]).
There have been several reports that male-factor infertility such as oligoasthenoteratozoospermia (OATS) resulted in decreased fertilization rate, poor embryo quality, and poor pregnancy outcomes (9, 10). In contrast, poor quality of sperm, such as in nonobstructive azoospermia (NOA) and severe OATS, does not affect clinical outcomes (fertilization
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Fertility and Sterility Vol. 91, No. 3, March 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
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rate, embryo quality, implantation, and/or pregnancy rate) in ICSI cycles (11, 12). Miller and Smith (1) reported that severe male factor is not related to fertilizing ability but is associated with a higher rate of developmental arrest at the five- to eight-cell stage on day 3 and with decreased blastocyst formation on day 5 in these cycles. Thus, it is still unclear whether poor sperm quality could lead to detrimental effects on fertilization, subsequent embryogenesis, implantation, and clinical pregnancy. In this study, for unbiased comparison, we selected ICSI cycles in which the embryo was transferred on day 3 and there were not genetic and/or female factors except tubal factor. A total of 206 selected ICSI cycles was divided into those of normal spermatogenesis patients (NSPs; normal ejaculated and obstructive azoospermic patients, n ¼ 149) and defective spermatogenesis patients (DSPs; severe OATS and nonobstructive azoospermic patients, n ¼ 57). Although the fertilization rate of DSPs was significantly lower than that of NSPs, the overall quality of early cleaving embryos in NSPs and DSPs was quite comparable. However, pregnancy outcomes (implantation, clinical pregnancy, and delivery) were significantly low in DSPs compared with in NSPs. Moreover, subsequent embryo transfer (ET) cycles with frozen–thawed embryos in NSPs and DSPs who failed to achieve pregnancy in their fresh cycles showed that the pregnancy outcomes were considerably lower in DSPs. Collectively, the quality of sperm could influence implantation and subsequent pregnancy outcome, as well as fertilization, in ICSI cycles. The poor clinical pregnancy outcomes of DSPs in fresh ET cycles and in those after frozen-thawed ET cycles may be derived from paternal effects of defective spermatogenesis in these patients. MATERIALS AND METHODS Study Design and Patients Between January 2003 and December 2004, 1,999 ET cycles after ICSI were performed in our center. To more objectively evaluate potential adverse effects of poor sperm quality on fertilization to implantation, pregnancy, and delivery in this retrospective study, ICSI cycles that met the following criteria were selected from a total of 1,999 ICSI-ET cycles carrying various infertility factors (Fig. 1): day 3 ET, with female age of <40 years and number of retrieved oocytes of R5 and without split insemination and any genetic factors. In addition, ICSI cycles with any female factors but tubal factor also were excluded from this study, because female factors may significantly influence oocyte quality, preimplantation embryo development, and/or implantation. Ejaculated and testicular sperms were evaluated on the basis of World Health Organization criteria and histological diagnosis, respectively (13–15). The selected 206 cycles were classified into NSPs (n ¼ 149) and DSPs (n ¼ 57). The NSP group consisted of normal ejaculated patients and those with obstructive azoospermia (OA), and the DSP group included patients with severe OATS (<5 million sperm per mL and/or <10% sperm motility and/or <4% normal sperm morphology) and nonobFertility and Sterility
FIGURE 1 A schematic diagram for patient selection to evaluate adverse effects of defective sperm quality on embryo implantation and clinical pregnancy outcomes in ICSI and subsequent frozen–thawed ET cycles. S-OATS ¼ severe OATS.
Lee. Paternal effects on clinical outcomes. Fertil Steril 2009.
structive azoospermia. Histological analysis showed that patients with nonobstructive azoospermia had defective spermatogenesis (13 hypospermatogenesis, 1 maturation arrest, and 3 germ cell aplasia) and that all patients with OA had normal spermatogenesis. Because of the retrospective nature of the study, institutional review board approval was not required. Sperm Preparation, Oocyte Retrieval, ICSI, and In Vitro Culture Sperm preparation was performed by routine protocols, as described elsewhere (16). Ovarian stimulation was performed by using GnRH agonist/antagonist, hMG, and human recombinant FSH. Human chorionic gonadotropin was administered when optimal follicle development was achieved, as evaluated by serial transvaginal ultrasound and estrogen determinations. Oocyte retrieval was performed via a transvaginal approach with sonographic guidance, 34 hours after hCG injection. Retrieved oocytes were incubated in G-Fert medium (VitroLife, Kungsbacka, Sweden) that was supplemented with 10% recombinant human serum albumin (VitroLife) at 37 C, 6% CO2 in air. Three to five hours after oocyte retrieval, cumulus cell mass and corona radiata of the oocytes were removed by incubation for 1 minute in medium with 799
0.1% hyaluronidase (Sigma, St. Louis, MO). Denuded oocytes were rinsed several times in fresh medium and evaluated under the microscope at 200 magnification. Intracytoplasmic sperm injection was performed on metaphase II oocytes, and fertilization was assessed 16–18 hours after ICSI. Fertilized zygotes were cultured with G1.3 and G2.3 sequential media (VitroLife) until ET. Sequential Embryonic Scoring Sequential embryonic score (SES) was established on the basis of the sum of the pronuclear (PN) score and embryonic score. The PN scoring method was modified from reports published elsewhere (17, 18). The PN score was monitored twice, at 18–20 hours and at 24–26 hours after ICSI. At 18–20 hours after ICSI, PN morphology was determined by position of the pronuclei and by morphology and orientation of the nucleoli. It was assessed by the following criteria: A is the ideal pattern (3 points), in which PN of approximately equal size had three or four equal-sized nucleoli beginning to align with systemic and polarized patterns on the borderline of the pronuclei. B is a moderate pattern (2 points), in which the PN had about 10 small-sized nucleoli, scattered or aligned. C is a nonideal pattern (1 point), in which PN of unequal size had different sizes and number of nucleoli. At 24–26 hours, the PN scoring was as follows: early cleavage embryo was 3 points, one cell was 2 points, syngamy was 1 point, and the other patterns were 0 points. Embryonic score represents the quality of cleaving embryos. It was scored by using criteria based on the cleavage speed, the number of blastomeres, and patterns of fragmentation (19). A grade I embryo was 5 points, grade I-1 was 4 points, grade II was 3 points, grade II-1 was 2 points, and grade III was 1 point. On day 3, eight cells or more was 3 points, four to seven cells was 2 points, and three cells or fewer was 1 point. Embryonic score was calculated on the basis of points for grade points for cell stage. Embryo Transfer and Assessment of Pregnancy The embryos were transferred into the uterine cavity on day 3 after oocyte retrieval. Pregnancy was determined if serial serum b-hCG level was >5 mIU/mL at 12 days after the oocyte retrieval. Clinical pregnancy was defined as the presence of a G-sac by ultrasonography at approximately 5 weeks of pregnancy. Supernumerary embryos were frozen at the PN or cleavage stage by using the slow-freezing method, using propylene glycol and sucrose (20). Freezing and thawing solutions were prepared in Dulbecco’s phosphate-buffered saline (Gibco-BRL, Grand Island, NY), supplemented with 20% synthetic serum substitute (Irvine Scientific, Irvine, CA). The embryos were exposed in a stepwise fashion to increasing concentrations of 1.5 M propylene glycol þ 0.1 M sucrose in the freezing solution. The embryos then were loaded into a 0.25 mL sterile straw (Bicef, L’Aigle, France), and then the straw was loaded into a programmable, controlled-rate freezing machine (Cryo-magic; Miraebiotech, 800
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Seoul, Korea) and kept at 20 C. The straw subsequently was cooled from 20 C to 7 C at a rate of 2 C/min, held at this temperature for 5 minutes, and then seeded manually. It then was cooled to 30 C at a rate of 0.3 C/min, cooled to 150 C at 30 C/min, and plunged into liquid nitrogen. Frozen embryos were thawed by the rapid thawing method (20). For thawing of the frozen human embryos, the straws were warmed by holding them in air for 40 seconds before being plunged them into a water bath at 37 C for 1 minute. The cryoprotectant then was removed by a reverse stepwise dilution. In frozen–thawed ET cycles, a daily dose of 6 mg of oral estradiol valerate was initiated on menstrual day 2. Estrogen and progesterone were administered, and the endometrial thickness was monitored. Embryo transfer was performed 3–5 days after the endometrial thickness reached >8 mm. After ET, luteal support with progest (Samil, Seoul, Korea) was continued for 14 days, until a urine pregnancy test was performed. Statistical Analysis Chi-square test or t-test was used for statistical analysis, and P values of < .05 were considered significant. RESULTS Selection and profiles of ICSI cycles grouped as NSPs and DSPs are summarized in Figure 1 and Table 1. The mean age of female partners of DSPs was younger than that of partners of NSPs, reflecting the higher number of retrieved oocytes with higher E2 value on the hCG day in DSPs. The matured MII oocyte rate was comparable between the groups. However, the normal fertilization rate with 2PN in DSPs was significantly lower than that in NSPs (67.6% vs. 80.3%, P<.05). Although the fertilization rate of DSPs was lower than that of NSPs, the overall quality of transferred embryos on day 3 was quite similar between both groups (Table 2). The mean number of transferred embryos was similar between NSPs and DSPs (3.3 0.7 vs. 3.4 0.9). To assess the quality of early developing embryos, SES was measured and analyzed between the groups. The sum SES of transferred embryos in each ET cycle was very similar for NSPs and DSPs. In particular, the mean SES of transferred embryos (11.6 3.3 vs. 11.6 3.4) and the SES of top-quality embryos (14.7 3.9 vs. 14.5 4.2) in each cycle were quite comparable between NSPs and DSPs. Collectively, this implied that embryos derived from sperms of DSPs developed to the eight-cell stage on day 3 as competently as did those from NSPs. Considering the similar characteristics between NSPs and DSPs of transferred embryos on day 3, an important observation is that pregnancy outcomes in DSPs were significantly decreased (Table 3). The implantation rate in DSPs was significantly lower than that in NSPs (15.5% vs. 24.3%, P<.05). The clinical pregnancy rate in DSPs also was significantly lower than that in NSPs (33.3% vs. 53.0%, P<.05). Consequently, whereas 70 (47.0%) of 149 cycles reached delivery in NSPs, 17 (29.8%) of 57 cycles resulted in delivery in Vol. 91, No. 3, March 2009
TABLE 1 The cycle characteristics of patients who underwent ICSI with spermatozoa from NSPs and DSPs. Parameter Mean of female age (y) E2 value on hCG day (pg/mL) No. of retrieved oocytes Oocyte maturation rate (%) Fertilization rate (%)
NSPs (n [ 149)
DSPs (n [ 57)
P value
32.3 3.3 2,077 1,222 14.1 7.8 1,611/2,093 (77.0) 1,295/1,611 (80.3)
30.6 2.7 2,930 2,012 19.4 9.6 863/1,108 (77.9) 583/863 (67.6)
< .01 < .01 < .01 NS < .05
Note: Values are mean SD or are n (%). NS ¼ not significant. Lee. Paternal effects on clinical outcomes. Fertil Steril 2009.
DSPs (P<.05). Although there was a significant difference in overall pregnancy outcomes between the two groups, they had similar miscarriage rates. To evaluate whether potential detrimental effects of controlled ovarian hyperstimulation on implantation and pregnancy contribute, at least partly, to lower implantation and clinical pregnancy rates in DSPs, we thus analyzed subsequent ET cycles with frozen–thawed embryos in NSPs and DSPs who failed to achieve pregnancy in their fresh cycles. Twenty-seven of 84 NSPs and 19 of 40 DSPs have continued their ET cycles with embryos that were cryopreserved at the PN or cleavage stage during their fresh cycles. Consistent with data from fresh cycles (Table 3), the data showed that rates of implantation (31.9% vs. 14.5%, P<.05) and clinical pregnancy (66.7% vs. 31.6%, P<.05) also were significantly reduced in DSPs (Table 4). The delivery rate also was lower in DSPs (26.3%) compared with in NSPs (55.6%), even though the difference did not reach statistical significance. Collectively, it clearly was shown that poor sperm quality may contribute to detrimental effects on embryo implantation and subsequent clinical pregnancy outcomes in ICSI-ET cycles. DISCUSSION With the current state of the art in ART, fertilization and early preimplantation development of multiple embryos can be accomplished successfully in most cases. However, the implan-
tation potential of the embryos after transfer to the uterine cavity still remains relatively low (21). Implantation and early pregnancy can be influenced by viability of the transferred embryos, by quality of oocyte and sperm, as well as by several factors responsible for uterine receptivity. Paternal effects, the inadequate contribution of paternal factors to preimplantation embryo development, have been shown to be responsible for repeated failure of ART cycles (4, 22, 23). In contrast, it also has been reported that sperm parameters are not associated with an impaired implantation or pregnancy potential (11, 12). This debate about the potential effect of sperm quality on these events may be associated with the multifactorial causes of infertility in retrospective studies. Thus, to objectively analyze all the clinical outcomes examined, we selected cycles in which severe male factor was the sole cause of infertility, except for female tubal factor, from total ICSI cycles. Our analysis of fresh ICSI-ET and subsequent frozen–thawed ET cycles clearly showed that DSPs have poor implantation rates and have poor pregnancy and delivery outcomes, without any distinct defects in early embryo development. This study, as well as that of Goker et al. (12), reported low fertilization rate in cycles with poor sperm quality. These results may come from an early paternal effect that possibly is mediated by centrosome dysfunction or by deficiency of oocyte activating factor (4). There have been many reports that early paternal effects are associated with early embryo development as well. Early paternal effect leads to delay of
TABLE 2 Comparison of embryo quality and number of transferred embryos between NSPs and DSPs. Parameter No. of transferred embryos Sum SES of transferred embryos Mean SES of transferred embryos Top SES
NSPs (n [ 149)
DSPs (n [ 57)
3.3 0.7 38.3 11.2 11.6 3.3 14.7 3.9
3.4 0.9 38.0 12.0 11.6 3.4 14.5 4.2
Note: Values are mean SD. Comparisons of the two groups were not significant on all parameters. Lee. Paternal effects on clinical outcomes. Fertil Steril 2009.
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TABLE 3 Comparison of clinical pregnancy outcomes between NSPs and DSPs in fresh ICSI cycles.
Implantation rate (%) Clinical pregnancy rate (%) Delivery rate (%) Miscarriage rate (%)
NSPs (n [ 149)
DSPs (n [ 57)
P value
121/498 (24.3) 79 (53.0) 70 (47.0) 9 (11.4)
30/193 (15.5) 19 (33.3) 17 (29.8) 2 (10.5)
< .05 < .05 < .05 NS
Note: NSP ¼ normal spermatogenesis patients; DSP ¼ defective spermatogenesis patients; NS ¼ not significant. Lee. Paternal effects on clinical outcomes. Fertil Steril 2009.
cleavage speed and increases the fragmentation rate of cleaving embryos (6). Up to 25% of the nondividing eggs are in fact fertilized but succumb to cell division defects (24). However, our data showed that development potentials of embryos from DSPs and NSPs are similar, at least up to the eight-cell stage on day 3, whereas fertilization rate in DSPs is significantly lower than that in NSPs. This suggests that reduced fertilization potential of poor quality sperm in DSPs is not directly associated with early paternal effects. The application of SES for selection of good embryos improved implantation and subsequent clinical pregnancy outcomes in ART (25). Because multiple embryos usually were transferred in ART cycles, average SES and highest SES of transferred embryos are critical factors in comparing the potential of embryos for implantation and further pregnancy. We observed that these scores were very similar between NSPs and DSPs, suggesting that the morphological quality of embryos from DSPs is as good as that of embryos from NSPs. Goker et al. (12) also reported a comparable good embryo rate in ICSI cycles with defective ejaculated sperms. Although ours as well as previous works showed that morphologically good embryos can be derived from defective sperms, we clearly showed that DSPs had poor implantation, pregnancy, and delivery outcomes without an impairment in quality of embryos transferred on day 3, in both their fresh
and subsequent frozen–thawed cycles. Our data suggests that morphological evaluation for embryo quality may not fully represent embryo potentials for blastocyst formation and implantation. In fact, there has been a recent observation that a large proportion of morphologically normal embryos on day 3 is chromosomally abnormal (26). This is consistent with a report that poor quality sperms were correlated significantly with increased rate of developmental arrest at the fiveto eight-cell stage and with poor blastocyst development (1). When ICSI is used, these abnormal spermatozoa are capable of achieving fertilization, but sperm DNA from DSPs, such as in the case of severe OATS, may possess anomalies such as loose packaging of the chromatin and DNA strand breaks (27), possibly resulting in developmental arrest during the preimplantation phase (28, 29). Sperm DNA fragmentation may affect postimplantation development in the ICSI procedure, and it could compromise embryo viability, resulting in pregnancy loss (7, 30). There is growing evidence that significant DNA damage in poor quality sperm may be caused by high levels of reactive oxygen species in male reproductive tracts and seminal plasma (31). Decreasing seminal plasma antioxidant levels have been reported in seminal plasma of males with impaired sperm function (32). Collectively, detrimental paternal effects of a defective paternal genome could contribute to aberrant zygotic gene activation, leading to development arrest at the eight-cell stage, poor blastocyst
TABLE 4 Comparison of embryo quality and clinical pregnancy outcomes between NSPs and DSPs in subsequent frozen–thawed embryo transfer cycles. Parameter Mean female age (y) Good embryo rate (%) No. of transferred embryos Implantation rate (%) Clinical pregnancy rate (%) Delivery rate (%) Miscarriage rate (%)
NSPs (n [ 27)
DSPs (n [ 19)
P value
32.2 3.2 72/127 (56.7) 3.4 0.8 29/91 (31.9) 18 (66.7) 15 (55.6) 3 (16.7)
30.1 1.6 52/90 (57.8) 3.6 0.8 10/69 (14.5) 6 (31.6) 5 (26.3) 1 (16.7)
< .05 NS NS < .05 < .05 NS NS
Note: Values are mean SD or n (%). NS ¼ not significant. Lee. Paternal effects on clinical outcomes. Fertil Steril 2009.
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development, and subsequent poor implantation and pregnancy outcome in vitro and in vivo. There is a spectrum of factors that may influence ART outcomes, such as age of the female partner, number of retrieved oocytes, performance of ICSI, etiology of infertility, and choice of embryos transferred. Among them, age of the female partner is one of the most significant factors influencing clinical outcomes of ART cycles. Considering that younger female partners, in general, have better clinical outcomes in ART cycles (33), it is even more noteworthy that implantation and clinical pregnancy rates for DSPs with younger female partners are significantly lower than those for NSPs in fresh ICSI and subsequent frozen–thawed ET cycles. Controlled ovarian hyperstimulation may significantly affect clinical outcomes as well as embryo implantation in ART cycles, too. Supraphysiological levels of estrogen resulting from controlled ovarian hyperstimulation may alter endometrial homeostasis and uterine receptivity, leading to poor clinical outcomes (34–36). Adverse effects of controlled ovarian hyperstimulation on clinical outcomes in ART are very prominent in aged female partners but are counterbalanced in younger ones (33). Once more, this suggests that DSPs with younger female partners have an advantage over NSPs in terms of clinical outcomes. Thus, those previous reports collectively reinforced our notion that defective spermatogenesis could contribute to poor embryo implantation and clinical pregnancy outcomes in both fresh ICSI-ET and subsequent frozen–thawed ET cycles. In summary, our results from fresh ICSI cycles and subsequent frozen–thawed cycles clearly demonstrate that detrimental paternal effects caused by defective spermatogenesis significantly influence implantation and subsequent clinical outcomes, possibly via aberrant embryonic gene activation that occurs on day 3.
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