Abstract
Aim: It has been suggested that the position of the sperm after intracytoplasmic sperm injection (ICSI) has an effect on the development and quality of the embryo. In this study, we retrospectively examined whether pronucleus stage evaluation used through clinical studies in recent years has relevance with regard to sperm location.
Methods: From 2003 to 2005, 1285 oocytes from 459 patients (average age: 36 years) were retrospectively analyzed. The 459 patients underwent ICSI because of fertilization disorders and oligozoospermia. Follicle stimulation was via either Clomid or the long protocol. Human chorionic gonadotropin was administered to induce ovulation and oocyte retrieval was conducted 35 h later. After confirming the presence of a polar body, we immobilized the ovum at the 6 o’clock position, introduced the injection pipette at the 3 o’clock position and carried out ICSI.
Results: When a sperm was located at a position that was opposite to the polar body, both classifications of Scott and Tesarik regarding embryo quality were distinctly low. Furthermore, a good embryo classification ensued when the sperm was located adjacent to the polar body.
Conclusion: The zone in which the sperm was located did not always correlate with embryo quality; however, our study suggested that sperm location affects the synchronization of the nucleolus. When carrying out ICSI, it is important to take into consideration the insertion point of the sperm. (Reprod Med Biol 2007; 6: 171–174)
Keywords: intracytoplasmic sperm injection, pronuclear stage, sperm position
INTRODUCTION
CLINICAL APPLICATIONS OF in vitro fertilization have existed for approximately 25 years, and the procedure is currently an important modality for infertility treatment. Oocyte evaluation is used for the prediction of embryo quality. In recent years, evaluation of the pronucleus using the methods of Tesarik and Scott has been reported. 1 , 2 Furthermore, evaluation of the pronucleus and the nucleolus are important prognostic factors.
With regard to intracytoplasmic sperm injection (ICSI) in mammals, Uehara and Yanagimachi injected human sperm into an unfertilized hamster ovum in 1976, and were the first to report a subsequent male pronucleus formation. 3 Moreover, more than 15 years ago, pregnancy using ICSI was reported by Palermo et al. in humans. 4 For the past decade, ICSI has been a clinically successful method for infertility treatment.
Studies have reported that the fertilization rate for ICSI does not differ significantly from that of in vitro fertilization (IVF). 5 , 6 Evaluation of embryo quality has also included the physical relationship of the polar body and the spindle fibers. In the present study, we recorded sperm position in relation to the first polar body at the time of ICSI and we examined the generating pattern of the subsequent pronucleus.
MATERIALS AND METHODS
IN THE YEARS from 2003 to 2005, oligozoospermia was a common indicator for ICSI. To decrease the effect of age, we limited the study to women aged 37 years or less. The cytoplasm of 762 ova obtained from 210 cycles (average patient age: 33.7 years) was divided into three categories and the sperm position after ICSI was observed. The classification methods of Tesarik and Scott were used 19 h after ICSI and the presence of a pronucleus was confirmed. Ovulation was induced with either a GnRH‐analog (long protocol) or Clomid: ova were harvested 35 h after the administration of 10 000 U human chorionic gonadotropin (HCG). After precultivation, the cumulus was completely removed via 40 IU of hyaluronidase medium (Sage In‐Vitro Fertilization, Trumbull, CT, USA); ICSI was carried out on MII stage oocytes only.
The sperm were cultured for 30 min at room temperature and washed with 10% serum substitute supplement + P‐1 medium (Irvine Scientific, Santa Ana, CA, USA). After centrifugation for 10 min at 1200 g, the supernatant was removed. Sperm were collected using the swim‐up method. The sperm were transferred to a Petri dish in preparation for ICSI. The ICSI was microscopically carried out (Olympus Corporation, Tokyo, Japan) using an injection and holding needle (Humagen Fertility Diagnostic, Charlottesville, VA, USA). Using immobilized good‐quality sperm, the position of ovum's polar body was fixed at 6:00 o’clock and the puncture was made at 3:00 o’clock. One observer made all classifications of sperm location in relation to the polar body. The appearance of a pronucleus was confirmed with an inverted microscope 19 h after ICSI and ovum quality was assessed using the classification systems of both Tesarik and Scott. 1 , 2 A significant difference was determined using a χ2 test; P < 0.05 was considered statistically significant.
RESULTS
AFTER ICSI, THE relationship between the location of the sperm and the polar body was divided into three zones from top to bottom (zones A, B, C in Fig. 1). The percentage of Tesarik 0 ova was lowest in the A Group (12/28 [43%]) and differed significantly from that of the B Group (464/706 [66%]). The number of ova classified as Tesarik 5 was low: C Group 2/19 (11%) and A Group 10/28 (36%); the difference between the A Group and the C Group was statistically significant. In the Scott classification, a high percentage of Z1 was found in the C Group (13/20 [65%]) compared with the A Group (12/28 [43%]; Fig. 2).
Figure 1.
Ovum showing the location of zones A, B and C.
Figure 2.
Percentages of Tesarik's and Scott's criteria following intracytoplasmic sperm injection.
DISCUSSION
WITH ASSISTED REPRODUCTIVE technology (ART), laboratory culture of gametes and early embryos is a necessity; furthermore, morphological evaluation of the embryo during its growth process is mandatory. Good‐quality embryos must be selected in the clinical setting to facilitate the implantation rate. 1 , 2 Morphological evaluation is necessary for ART and ICSI.
In general, the puncture location for ICSI is chosen to be in the hemisphere opposite the first polar body; this technique avoids damage to the spindle. 7 However, because it is difficult to know the location of the spindle from the position of the first polar body, it is possible that the spindle body can be damaged using ICSI. 8 , 9 Furthermore, injecting the sperm into the cytoplasm is clinically difficult.
We previously reported a comparison of sperm position after ICSI with fertilization rate and pregnancy rate; we have also reported satisfactory results in an animal model. 10 From this study, we theorized that sperm location with ICSI might influence the synchroneity of the nucleolus and the subsequent Tesarik classification. 1 We also evaluated the pronucleus using the criteria of Scott et al. 2
Recently, it was reported that polarization of nucleolus precursor bodies in both pronuclei is as reliable a marker of implantation as embryo mophology on day 3. 11
In the present study, we found that with ICSI the sperm location is usually near the cell midline. Ovum quality was assessed by dividing the ovum into three zones (A, B and C) according to the distance between the sperm and the polar body. Moreover, because the synchronization of the nucleolus is important with regard to the pronucleus, the statistical analysis was conducted by paying particular attention to patterns 0 and 5 of Tesarik's classification. 1
The percentage of Tesarik 0 was the lowest in the A Group, and was significantly different from that of the B Group. The percentage of Tesarik 5 ova was lower than that of Tesarik 0 ova, and the majority were in the C Group; a statistically significant difference was found between the A group and the C group. With the Scott classification, which showed the relationship of the blastocyst formation, a high percentage of Z1 was found in the C group compared with the A group. In view of these findings, we theorized that the greater the distance between the sperm and the polar body the greater the degradation of the synchroneity of the nucleolus. Other investigators have reported a higher pregnancy rate if the sperm is inserted near the spindle. 12 , 13
Recent literature contains reports that show that using a PolScope facilitates observation of the spindle. The probability that the spindle is near the first polar body ranges from 20 to 30%, and in these cases it may be damaged during ICSI. 9 One investigator reported a higher fertilization rate when a spindle was noted at the time of ICSI. 14 Although only a few institutions possess a PolScope, they report that the instrument provides additional information with regard to sperm positioning. With the use of a PolScope, it has been suggested for the first time that the position of the sperm following ICSI affects the synchroneity of the sex pronucleus. It is possible that the distance of the spindle from the sperm is related to the synchroneity of the sex pronucleus; however, examination with a PolScope does not reflect the actual position of the spindle. In the future, by carrying out ICSI and viewing the spindle under non‐fluorescence, the physical relationship of the spindle and the sperm, and the relationship between the pronucleus and synchroneity, should become clearer. This procedure may lead to further development of ICSI technology.
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