A comparison of ICSI outcomes with fresh and cryopreserved epididymal spermatozoa from the same couples

Selahittin Cayan1, Douglas Lee2, Joseph Conaghan2, Carolyn A. Givens2, Isabelle P. Ryan2, Eldon D. Schriock2 and Paul J. Turek1,2,3

1 Departments of Urology and 2 Obstetrics and Gynecology and Reproductive Sciences, University of California San Francisco School of Medicine, San Francisco, CA, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The published experience with frozen–thawed epididymal spermatozoa and intracytoplasmic sperm injection (ICSI) suggests that fertilization and pregnancy success rates are comparable to those achieved with freshly retrieved spermatozoa. However, no study has exactly compared clinical outcomes between the two IVF/ICSI cycles in the same couples. To formally address this issue, we assessed ICSI outcomes in couples each of whom had had two IVF/ICSI cycles: one using fresh and the second using frozen–thawed epididymal spermatozoa obtained from a single aspiration procedure. From a pool of 101 consecutive patients undergoing IVF/ICSI with epididymal spermatozoa, 19 couples initially used fresh epididymal spermatozoa and subsequently underwent a second IVF/ICSI procedure with frozen–thawed spermatozoa from the same aspiration. Normal (2PN) oocyte fertilization rates, embryo quality and pregnancy rates were compared between the two IVF/ICSI cycles for each couple. In the fresh epididymal sperm group, 58.4% of the injected oocytes fertilized normally compared with 62.0% of the injected oocytes in the frozen–thawed epididymal sperm group, revealing no statistically significant difference. Graded embryo quality also did not differ significantly between the paired IVF/ICSI cycles. The clinical pregnancy rates were 31.6% (6/19) and 36.8% (7/19) in the first and second cycles respectively. All but one pregnancy were singletons. In summary, this study provides strong evidence to support the notion that motile, cryopreserved and thawed epididymal spermatozoa are equal to freshly retrieved spermatozoa for ICSI in couples with obstructive azoospermia.

Key words: cryopreservation/epididymal spermatozoa/ICSI/IVF


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of spermatozoa from an obstructed epididymis in conjunction with assisted reproductive techniques has been performed for 15 years (Temple-Smith et al., 1985Go). Although early experience revealed that pregnancies were possible with epididymal spermatozoa and IVF, fertilization efficiency and pregnancy rates were quite low (Silber et al., 1990Go; Bladou et al., 1991Go). The addition of intracytoplasmic sperm injection (ICSI) to IVF greatly augmented fertilization and pregnancy outcomes in cases of epididymal sperm retrieval, such that we currently enjoy success rates with epididymal spermatozoa that are equivalent to those achieved with ejaculated spermatozoa and IVF (Silber et al., 1994Go; Tsirigotis et al., 1996Go; Cha et al., 1997Go).

Given this success, several centres initiated the use of frozen-thawed epididymal spermatozoa in addition to freshly retrieved spermatozoa to achieve ICSI pregnancies. The apparent clinical benefits inherent in epididymal sperm cryopreservation were immediately obvious to both patients and providers: the use of frozen–thawed epididymal spermatozoa could reduce the number of male partner procedures required for pregnancy (Oates et al., 1996Go; Nudell et al., 1998Go) as well as minimize the inconvenience associated with sperm retrieval performed concurrently with ovulation induction (Oates et al., 1996Go). Furthermore, data that address the cryobiological behaviour of epididymal spermatozoa have demonstrated that it is quite feasible to consider frozen-thawed epididymal spermatozoa for ICSI procedures (Bachtell et al., 1999Go).

The most important issue on this matter, however, is whether frozen–thawed epididymal spermatozoa provide clinical outcomes equivalent to that of fresh spermatozoa. To date, the published experience with frozen–thawed epididymal spermatozoa and ICSI suggests that fertilization and pregnancy success rates are comparable to those achieved with freshly retrieved spermatozoa (Nagy et al., 1995Go; Oates et al., 1996Go; Cha et al., 1997Go; Holden et al., 1997Go; Friedler et al., 1998Go; Hutchon et al., 1998Go; Van Steirteghem et al., 1998Go; Tournaye et al., 1999Go). However, the majority of studies use historical or unmatched control patients to compare clinical outcomes. Three studies reported clinical outcomes in patients who underwent at least two IVF/ICSI cycles, one with fresh and a subsequent cycle with frozen–thawed epididymal spermatozoa (Friedler et al., 1998Go; Hutchon et al., 1998Go; Tournaye et al., 1999Go). These studies represent a more powerful design because female factor variables were better controlled. Despite better design, the aims of these studies varied from a comparison of results with different epididymal sperm retrieval techniques (Friedler et al., 1998Go) to an examination of clinical outcomes in couples who had subsequent but not necessarily consecutive IVF/ICSI cycles (Hutchon et al., 1998Go). To further clarify this issue, we sought to assess ICSI outcomes in couples each of whom underwent consecutive IVF/ICSI cycles, the first with fresh and the second with frozen–thawed epididymal spermatozoa obtained from a single aspiration procedure.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient selection
From 1995 to 1999, 101 azoospermic men underwent microsurgical epididymal sperm aspiration (MESA) in conjunction with IVF/ICSI cycle at a single institution. Among these couples, 19 underwent two separate IVF/ICSI cycles, the first performed with freshly retrieved epididymal spermatozoa and the second with frozen-thawed spermatozoa derived from the initial aspiration procedure. The outcomes from these 38 IVF/ICSI cycles were examined respectively and constitute the subject matter of this study. Outcomes examined included epididymal sperm quality, fertilization rates, embryo quality and pregnancy rates. Observed reasons for repeating IVF/ICSI cycles included failure to achieve a pregnancy on the first attempt or a desire for more children.

Microsurgical sperm aspiration procedure (mini-MESA)
Epididymal sperm aspiration was performed in all patients by a single surgeon (P.J.T.) as previously described (Nudell et al., 1998Go). Briefly, under local anaesthesia, a small scrotal incision was made and the epididymis exposed lateral to the testis. The epididymal tunic was incised over a dilated epididymal tubule using microsurgical technique. Individual epididymal tubules were entered with microscissors and fluid with spermatozoa aspirated into small syringes and subsequently into sterile 5 ml test tubes (Becton Dickinson, Franklin Lakes, NJ, USA) containing Earle's Medium (Gibco BRL, Grand Island, NY, USA) supplemented with 4 mmol/l sodium bicarbonate, 21 mmol/l HEPES (Calbiochem-Novabiochem Corp., La Jolla, CA, USA), 0.47 mmol/l pyruvate, and 10% v/v synthetic serum substitute (SSS; Irving Scientific, Santa Ana, CA, USA) and maintained at 37°C. At each epididymal site, 10 µl of extracted fluid was examined under x400 bright-field microscopy for the presence of motile spermatozoa. Epididymal sites chosen for aspiration progressed from the distal (cauda) to proximal (caput) epididymis until motile spermatozoa were observed in the aspirate. The epididymal tubules and tunic were closed with interrupted 10–0 and 9–0 nylon sutures respectively. Aspirated epididymal fluid was immediately transferred to the IVF laboratory for micromanipulation and processing.

Oocyte stimulation and retrieval, ICSI and embryo transfer
As described previously, female partners underwent pituitary desensitization with a gonadotrophin hormone-releasing agonist and ovarian stimulation with human menopausal gonadotrophins (Pergonal or Fertinex; Serono, Norwell, MA, USA) (Nudell et al., 1998Go). Retrieved oocytes were handled in the laboratory according to the technique of Hillier et al. (1984) and cultured in Earle's medium with 5% CO2 at 37°C. After 2 h incubation in medium, oocytes were denuded of cumulus cells using 80 U/ml hyaluronidase (Sigma Chemical Co., St Louis, MO, USA).

Motile epididymal spermatozoa were minimally processed and handled prior to micromanipulation. For ICSI, morphologically normal and progressively motile spermatozoa were recovered using a `swim-out' technique (Conaghan et al., 1997Go; Nudell et al., 1998Go). Briefly, 5 µl of undiluted sperm suspension was pipetted to the centre of a microdroplet (7 µl) of polyvinylpyrrolidine (PVP; Irvine Scientific). Spermatozoa reaching the microdroplet edge were collected with a finely drawn ICSI pipette (Pacific Andrology Inc., Montrose, CA, USA), transferred to a clean droplet of PVP for tail breaking, and injected into oocytes.

Following ICSI, oocytes were transferred into culture medium under mineral oil (Sigma) and incubated for 16 h. Normal fertilization was confirmed when two pronuclei were clearly visible in the oocyte cytoplasm. Embryos were transferred to the uterus 24 or 48 h after fertilization. Successful implantation was determined by appropriately rising serum ß-HCG concentrations. A clinical pregnancy was confirmed with the presence of a fetal sac and a heart beat on vaginal ultrasound 4–6 weeks later.

Sperm analysis and cryopreservation and thaw protocol
Aspirated spermatozoa not used for ICSI during the first IVF cycle were prepared for analysis and cryopreservation. Sperm concentration and motility analyses were performed on both fresh and frozen–thawed spermatozoa from each patient according to World Health Organization criteria (WHO, 1992Go) under light microscopy at 37°C after dilution and preparation on a microcell slide (Conception Technologies, San Diego, CA, USA). Motility progression was scored as follows: 0 = immotile spermatozoa; 1 = non-progressive, twitching spermatozoa; 2 = slow progressive or sluggish motility; 3 = rapid forward progressive motility; 4 = capacitated (hyperactivated) progression (Mortimer, 1994Go). In addition, the total motile sperm count (TMC: volumexconcentrationxmotile fraction) was determined for the entire retrieved specimen from each patient.

Aspirated spermatozoa to be cryopreserved were washed twice (centrifuged at 170 g, 10 min) in phosphate-buffered saline (Gibco) with 2% v/v SSS. The final pellet was resuspended in 0.5 ml of Earle's medium. Specimens were cryopreserved in a 1:1 dilution of sperm suspension to test yolk buffer (Irvine Scientific) in 1.0 ml vials by the slow freeze method (Mahadevan and Trounson, 1983Go) in a Planar Biomed controlled rate freezer (T.S. Scientific, Perkasie, PA, USA). Upon thawing for the subsequent IVF cycle, vials were thawed by incubation at 37°C for 10 min. The thawed spermatozoa were then gradually reintroduced into isotonic medium by adding an equal volume of Earle's medium to the vial over a 5 min period. This diluted specimen was centrifuged (170 g, 10 min). This dilution and wash procedure was repeated twice and the final pellet resuspended in 0.5 ml of Earle's buffered medium for both ICSI and reassessment of sperm parameters. Motile spermatozoa were used for all ICSI procedures in this study.

Assessment of embryo quality
Embryos derived from each IVF/ICSI cycle were assessed for quality according to an established grading system prior to embryo transfer. Embryos were observed under a zoom stereomicroscope (model SZH-ILLB: Olympus Optical Co. Ltd, Japan), at a magnification of x128, illuminated using a 6 V, 20 W halogen bulb. Embryo quality was assessed by a single embryologist (J.C.) at 72 h after retrieval of oocytes and rated on a scale of 1 = excellent to 5 = poor (Plachot et al., 1986Go; Scott et al., 1991Go).

Statistical analysis
Statistical analysis was performed using the paired t-test to compare fresh and frozen–thawed sperm parameters and the {chi}2-test to compare ICSI outcomes. P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clinical characteristics of female and male partners
The mean age of the 19 female partners at the time of the first IVF/ICSI cycle in which fresh epididymal spermatozoa was used was 34.3 ± 5.6 years (range 23–42). The mean age of these female partners at the time of the subsequent IVF/ICSI cycle in which frozen–thawed epididymal spermatozoa was used was 35.5 ± 5.4 years (range 24–43). Thus, the mean difference in age of the women from the first to second IVF cycles was 12.2 months (range 2–51). IVF cycles were repeated for failure to achieve a pregnancy on the first attempt in 13 (68%) couples and a desire for more children in six (32%) couples.

The mean age of the male partners at the time of epididymal sperm retrieval was 45.9 ± 12.9 years (range 29–71). The causes of obstruction were congenital bilateral absence of the vas deferens (CBAVD) in 10 patients (53%), vasectomy or failed vasectomy reversal in seven patients (37%) and idiopathic epididymal obstruction in two patients (10%). Epididymal sperm retrieval was successful in all cases in which it was attempted.

Fresh and frozen–thawed epididymal sperm parameters
Epididymal sperm parameters before and after cryopreservation are given in Table IGo. As expected, there was a significant difference in sperm motility when the fresh and frozen–thawed epididymal spermatozoa were compared (P = 0.004). However, no significant differences were observed in forward progression of motility between the two specimens.


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Table I. Fresh and frozen–thawed epididymal sperm parameters from 19 male partners. Values are mean ± SD
 
ICSI outcomes
Table IIGo outlines the outcome of ICSI with both fresh and frozen–thawed epididymal spermatozoa. A total of 238 metaphase II oocytes were injected with fresh epididymal spermatozoa in 19 IVF cycles, and 313 metaphase II oocytes were injected with frozen–thawed epididymal spermatozoa from the second IVF cycles in these same couples. No statistically significant differences were found in oocyte fertilization rates between the two groups. In the fresh epididymal sperm group, 58.4% of the injected oocytes fertilized normally compared to 62.0% of the injected oocytes in the frozen–thawed epididymal sperm group. The mean number of embryos transferred was 3.0 ± 1.6 (range 0–6) and 3.4 ± 1.5 (range 0–7) for first and second IVF cycles respectively.


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Table II. ICSI Results with fresh and frozen–thawed epididymal spermatozoa. Values are mean ± SD
 
Figure 1Go summarizes the association between embryo quality and type of epididymal spermatozoa used in paired ICSI cycles. There were no statistically significant differences in the grades of embryo quality between the two groups. The clinical pregnancy rates were 31.6% (6/19) and 36.8% (7/19) in the first and second cycles, respectively. In the fresh epididymal sperm group, 2/19 pregnancies miscarried; no miscarriages were observed in the frozen–thawed epididymal sperm group (Table IIGo). All but one pregnancy were singletons.



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Figure 1. The association of embryo quality and type of epididymal spermatozoa in couples undergoing paired ICSI cycles.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although pregnancy rates with ICSI and epididymal spermatozoa have improved remarkably in the last several years, many couples still require multiple IVF cycles to achieve a pregnancy (Cha et al., 1997Go). Thus sperm aspiration techniques should be not only reliable and have low morbidity, but also have potential to harvest sufficient spermatozoa to enable multiple attempts at IVF and ICSI without repeated surgical procedures on the male partner. Despite convincing evidence that freshly aspirated epididymal spermatozoa are associated with excellent fertilization and pregnancy success with ICSI, data supporting the feasibility of frozen and thawed epididymal spermatozoa are still accumulating. The goal of this simple study was to further clarify our understanding on this matter.

A summary of the ICSI experience with fresh and frozen–thawed epididymal spermatozoa is given in Table IIIGo. Most of the prior studies on this issue generally lack control groups or use controls derived from historical series or parallel but different groups of patients. In a study that reviewed a large early experience with epididymal spermatozoa and ICSI, Van Steirteghem et al. (1998) reported higher fertilization (64.8% and 54.7%), and pregnancy rates (44.3 and 33.8%) with fresh versus frozen–thawed epididymal spermatozoa. Conversely, Shibahara et al. (1999) reported very significant differences in fertilization rates (68.6 versus 45.3%) and pregnancy rates (60 versus 23.1%) when fresh and frozen–thawed epididymal spermatozoa were compared among different couples. Importantly, however, in cases in which only motile spermatozoa were used for ICSI, oocyte fertilization rates between fresh (68.6%) and frozen–thawed (68.4%) epididymal spermatozoa were virtually identical. In general, oocyte fertilization rates after ICSI with frozen–thawed epididymal spermatozoa ranged from 37 to 72%, which compares favourably to that found with fresh epididymal spermatozoa (range 20–73%). Thus, this body of work suggests that excellent ICSI fertilization rates are achievable with frozen and thawed epididymal spermatozoa.


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Table III. ICSI outcomes with fresh and frozen–thawed epididymal spermatozoa in the literature
 
Three studies have assessed the comparative efficacy of fresh and frozen–thawed epididymal spermatozoa in ICSI by examining couples undergoing paired IVF cycles. Hutchon et al. (1998) compared results in 17 ICSI cycles with fresh epididymal spermatozoa to 15 cycles using frozen–thawed spermatozoa from the same couples (Hutchon et al., 1998Go). Epididymal spermatozoa were retrieved either percutaneously or microscopically. They detected no difference in normal oocyte fertilization rates between these two cohorts of ICSI cycles. However, they observed fewer top grade embryos in the frozen–thawed sperm group. The time from the first to subsequent ICSI cycles is not given in their study, nor is it clear whether the two couples who did not proceed from the fresh sperm cycle to the frozen–thawed cycle would have significantly altered the findings. Similarly, Friedler et al. (1998) compared outcomes in a cohort of 24 patients with obstructive azoospermia who underwent percutaneous or microscopic epididymal sperm retrieval and ICSI with fresh spermatozoa to 21 subsequent cycles in which frozen–thawed spermatozoa were used (Friedler et al., 1998Go). Oocyte fertilization rates did not differ between these two groups. The authors concluded that ICSI outcomes with both types of spermatozoa after percutaneous retrieval are similar to that after microscopic sperm retrieval, and that epididymal sperm cryopreservation is feasible and efficient. Finally, Tournaye et al. (1999) found no difference in ICSI outcomes in 67 couples who underwent ICSI cycles with both fresh and frozen–thawed epididymal sperm retrieved during a single procedure (Tournaye et al., 1999Go). They reported a 60.1% normal fertilization rate and a 32.1% pregnancy rate in patients with fresh epididymal spermatozoa and a 53% oocyte fertilization rate and a 35.2% pregnancy rate with frozen–thawed epididymal spermatozoa. In summary, studies that attempt to control for oocyte variables also suggest that ICSI outcomes are no different with fresh and frozen–thawed epididymal spermatozoa.

In the current study, we selected couples each of whom underwent two consecutive IVF/ICSI cycles, one with fresh and frozen and the next with frozen–thawed epididymal spermatozoa. In this way, each couple served as its own control and the time between the IVF cycles was minimized to reduce the effect of age-related changes in maternal reproductive potential on the results. Similar to the majority of previous studies, we conclude that fresh and frozen–thawed epididymal spermatozoa produce similar ICSI oocyte fertilization rates. Furthermore, in our observations of early embryo development, we noted no significant differences in early embryo quality with the two types of epididymal spermatozoa, unlike the observations made by Hutchon et al. (1998).

Despite the repeated measures design of this retrospective study, there are inherent limitations to our findings. Certain confounding variables may have influenced our results but the effects of these variables are difficult to analyse. The average increase in maternal age between the first and second IVF cycles was 1 year and therefore differences in maternal reproductive potential may exist. In addition, a comparison of pregnancy rates between the first and second IVF cycles may not accurately reflect differences in sperm quality because of an obvious, inherent selection bias: most couples proceeded to a second IVF cycle because of pregnancy failure in the initial cycle. Moreover, the higher pregnancy rate obtained with frozen–thawed epididymal spermatozoa in the second cycle may simply directly reflect the application of knowledge about the female partner gained from the clinical experience of the first IVF cycle. This is exemplified by the fact that, on average, more oocytes were retrieved and more embryos were transferred in the second than the first IVF cycles. Despite these caveats, the similarities in oocyte fertilization rates and embryo quality between the two kinds of motile epididymal spermatozoa suggest that they are equal with respect to early ICSI outcomes.

In summary, this study provides strong evidence to support the notion that motile, cryopreserved and thawed epididymal spermatozoa are equal to freshly retrieved spermatozoa for ICSI in couples with obstructive azoospermia. It is hoped that these findings raise clinical awareness of the value and priority of sperm cryopreservation in all epididymal sperm aspiration procedures.


    Notes
 
3 To whom correspondence should be addressed at: Department of Urology, University of California San Francisco, 2330 Post Street, 6th Floor, San Francisco, CA 94115–1695, USA. E-mail: mrvas{at}itsa.ucsf.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on August 29, 2000; accepted on November 7, 2000.