Mayada Abdul-Aziz, M.D., James Meriano, B.Sc., R.T., Robert F. Casper, M.D.

Objective:  To determine whether absence of fertilization in IVF associated with an acrosomal enzyme defect (hyaluronidase deficiency) results from a simple mechanical block to sperm penetration or from a more serious sperm abnormality.

Design:  Nonrandomized, prospective study.

Setting:  Toronto Center for Advanced Reproductive Technology, a tertiary referral center for infertility associated with The University of Toronto.

Patients:  One hundred twenty-two couples about to undergo intracytoplasmic sperm injection (ICSI) were selected. Thirty-six of the studied couples had failed to fertilize in prior IVF cycles.

Interventions:  Hyaluronidase activity was measured in the semen samples provided for ICSI using a zymogenic assay. Intracytoplasmic sperm injection was performed in all couples using standard techniques.

Results:  Forty-eight of 122 semen samples had poor or absent semen hyaluronidase activity. All 48 samples resulted in successful fertilization with ICSI in the present study. The average fertilization rate per oocyte was 59.43% in couples in whom the male partner had low semen hyaluronidase activity and 55.85% in whom the male had normal hyaluronidase activity. The ET rate per cycle was 100% and 95% and pregnancy rates per cycle were 26% and 25% in cycles with poor and normal semen hyaluronidase activity, respectively. Unlike routine IVF, no statistical correlation was found between semen hyaluronidase activity and the fertilization rate in ICSI.

Conclusion:  Our results indicate that semen hyaluronidase deficiency is associated with a simple mechanical block to fertilization. In addition, the measurement of semen hyaluronidase activity can provide a reliable means for selecting couples who would benefit from ICSI.
Fertil Steril 1996;65:977-80

Key Words:  Hyaluronidase, sperm, ICSI, male factor infertility

During the process of fertilization, acrosomal enzymes facilitate the penetration of sperm through the oocyte's vestments (1, 2). Hyaluronidase is an acrosomal enzyme that is considered to be important in the process of fertilization through its participation in the dissolution of the extracellular matrix the cumulus oophorus, containing hyaluronic acid (3, 4). The possibility that low acrosomal enzyme activity may be the cause of reduced fertilizing capability of sperm has been suggested (5). We have shown previously that semen hyaluronidase activity correlates with sperm concentration, motility, and the percentage of normal forms (6). We also have demonstrated that semen samples with low or absent hyaluronidase activity failed to fertilize in routine IVF (6). We hypothesized that the failure of such samples to fertilize in IVF was related to the inability of sperm to penetrate through the oocyte's vestments.

With the development of intracytoplasmic sperm injection (ICSI) (7, 8), direct injection of sperm into the oocyte's cytoplasm bypasses the zona pellucida and the oocyte membrane, thus overcoming the need for acrosomal enzymes (9). The objective of the present study was to determine whether intracytoplasmic injection of sperm from semen samples with low or absent hyaluronidase activity results in fertilization and cleavage rates similar to sperm from samples with apparently normal hyaluronidase activity. If so, these results would confirm the relationship of acrosomal enzyme deficiency with a simple mechanical block to fertilization.

MATERIALS AND METHODS

Patients

One hundred twenty-two couples about to undergo ICSI were selected for this study. The indications for ICSI were severe oligoasthenospermia (<500,000 motile sperm recovered on sperm wash and swim-up) or previous failure to fertilize in vitro. Forty-eight men had low semen hyaluronidease activity defined as a ring diameter of < 3 mm in the zymogenic assay we described previously (6). Of these 48 men, 21 had demonstrated failure to fertilize in prior IVF cycles (representing 58% of previous IVF fertilization failures) and 27 were diagnosed as severe male factor infertility based on their semen analysis and swim-up assessments. The remaining 74 couples studied underwent ICSI for similar indications, such as previous failure to fertilize in IVF or low motility of sperm but with apparently normal semen hyaluronidase activity.

Semen Hyaluronidase Activity

Hyaluronidase activity was measured using the method we described previously (6). In this method, petri dishes were used to place a mixture of agar and hyaluronic acid (bacterial sodium hyaluronate; Lifecore Biomedical, Chaska, MN) and a well was cut in the center of each dish. Semen was treated with Nonidet P-40 (Sigma Chemical Co., St. Louis, MO) for 6 hours and 30 mL of the mixture was placed into the well. After incubating the dishes 24 hours at 37 degrees C, the reaction was stopped and hyaluronidase activity was detected by the presence of clear rings of hydrolyzed substrate surrounding the well. The ring diameters were measured in millimeters and, after subtracting 7 mm, which represented the diameter of the well, the ring size was determined. A ring size of < 3 mm was considered to represent deficient hyaluronidase activity.

Controlled Ovarian Stimulation, Oocyte Retrieval, and ICSI

Controlled ovarian hyperstimulation consisted of a long protocol, using daily SC GnRH agonist (GnRH-a, 1 mg leuprolide acetate, Lupron; Abbott Pharmaceutical Company Ltd. Pointe Claire, Quebec, Canada) starting in the midluteal phase until day 3 of the subsequent menses, as we have described previously (10). If serum E2 concentration was <45 pg/mL (150 pmol/L) on cycle day 3, the dose of GnRH-a was reduced to half and hMG (Pergonal; Serono, Mississauga, Ontario, or Humegon; Organon, Toronto, Ontario) or highly purified human FSH (Fertinorm; Serono) was administered daily. The standard protocol began with three ampules of hMG or FSH in women <35 years and four ampules in women >35 years.

Vaginal ultrasound (US) for follicular measurement was performed starting on the 5th day of gonadotropin administration. Blood samples for measurement of serum E2 concentration were drawn serially. Human chronic gonadotropin (10,000 IU, Profasi; Serono) was administered when the mean diameter of more than two follicles measured > 18 mm, and the E2 level approximated 280 pg/mL (1,000 pmol/L) per mature follicle. Transvaginal US-guided oocyte retrieval was performed 36 hours after hCG administration. Oocyte and sperm preparation and sperm injection were carried out using the method of Van Steirteghem et al. (7), and the method we have described previously (10).

Statistics

The fertilization rate was calculated as the percentage of injected oocytes that had two pronuclei 24 hours after the injection and the number of embryos suitable for transfer in each couple was calculated. The group t-test and x2 test were used to compare patient parameters and oocyte fertilization rates. Spearman rank correlation was used to determine the relationship between semen hyaluronidase activity and the fertilization rate in ICSI.

RESULTS

The total number of oocytes in the studied cycles was 851, with an average 6.97 oocytes per patient. The total number of oocytes in the couples with male partners having poor semen hyaluronidase activity was 352, with an average of 7.33 oocytes per couple (ranging from 1 to 22). The total oocyte number in the group with good semen hyaluronidase activity was 499, with an average of 6.74 oocytes per couple (ranging from 1 to 21, not significant). Table 1 shows a comparison between the oocyte number, female age, and serum E2 levels in the two groups.

*No significant differences were found between the groups in any of the parameters.
⁺Conversion factor to SI unit: hCG, 1.00.

Our results show that 118 of 122 couples included in the study had successful fertilization in ICSI, resulting in an overall mean fertilization rate of 98.36% per couple. The average fertilization rate per oocyte was 57.26%. Table 2 shows the average fertilization rate in patients with poor semen hyaluronidase activity as compared with those with good enzyme activity. No correlation was found between semen hyaluronidase activity and the fertilization rate in ICSI in either group. Similarly, the pregnancy rate was similar in both groups (Table 2).

DISCUSSION

The crucial role of sperm quality in human reproduction is well established (1) and is highlighted by the introduction of assisted reproductive technologies whereby the capability of spermatozoa to fertilize the oocyte can be assessed directly. In vitro fertilization programs report reduced fertilization and pregnancy rates when insemination is performed with subnormal spermatozoa (11, 12). With the development of ICSI for male factor infertility, it is important to be able to identify patients who have a low probability of successful fertilization in routine IVF.

In a previous study (13), we defined severe male factor infertility as the recovery of <500,000 motile sperm after semen preparation by sperm wash and swim-up, based on a mean in vitro oocyte fertilization rate in these patients of <25%. On the basis of this cut-off value for motile sperm recovery, we now suggest ICSI as first-line therapy for infertile couples with severe male factor infertility in our center. We also have shown in a recent report (6) that men with poor semen hyaluronidase activity are unlikely to fertilize in routine IVF, regardless of sperm count or motility. The measurement of semen hyaluronidase activity provides presumptive information about acrosomal function and the fertilizing capability of sperm.

The present study was designed to compare fertilization rates with ICSI between sperm from men with semen hyaluronidase deficiency and-those from men with apparently normal enzyme activity. Based on previous work with globozoospermia (14), it appears that the fertilizing potential of sperm with an absent acrosome is diminished, even with ICSI. Liu et al. (14) performed 11 cycles of ICSI with round-headed sperm injected into 85 eggs. Only 14 embryos were formed in 4 of 11 cycles, for a fertilization rate per oocyte injected of 16.4% and an ET rate per cycle of 36.4%. These results suggest that the acrosomal defect in globozoospermia may be only part of the problem. It is possible that there may be other abnormalities of the round-headed sperm to account for the low fertilization and cleavage rate when the mechanical block to fertilization is overcome by ICSI. More ICSI cycles with globozoospermic men are required to confirm these preliminary observations.

In contrast, the results of the present study show no difference in the fertilization rate in ICSI between patients with good semen hyaluronidase activity, suggesting normal acrosomal function, compared with those with deficient semen hyaluronidase activity and presumably poor acrosomal function. The fertilization rate per oocyte injected was 56% and 59% for men with good or poor semen hyaluronidase activity, respectively. The ET rate per cycle was 97% and 100% in these two groups, respectively, and the pregnancy rate per cycle was 25% and 26%, respectively. Our data, therefore, suggest that sperm from men with deficient or absent semen hyaluronidase activity are otherwise normal and are appropriate for use with ICSI.

In conclusion, our results show that acrosomal enzyme deficiency, indicated by poor semen hyaluronidase activity, can be overcome by ICSI. Fertilization and pregnancy rates in couples where the male partner has poor semen hyaluronidase activity appear to be similar with ICSI compared with couples with normal semen hyaluronidase activity. The present study also supports our previous conclusion (6) that the measurement of semen hyaluronidase activity provides useful information for selection of couples who would benefit from ICSI rather than routine IVF.

*Values in parentheses are ring sizes.

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