Correlation Between Sperm Morphology Using Strict Criteria in Original Semen and Swim-Up Inseminate and Human in vitro Fertilization

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1 Archives of Andrology Journal of Reproductive Systems ISSN: (Print) (Online) Journal homepage: Correlation Between Sperm Morphology Using Strict Criteria in Original Semen and Swim-Up Inseminate and Human in vitro Fertilization Y. S. Yang, S. U. Chen, H. N. Ho, H. F. Chen, K. H. Chao, H. R. Lin, S. C. Huang & T. Y. Lee To cite this article: Y. S. Yang, S. U. Chen, H. N. Ho, H. F. Chen, K. H. Chao, H. R. Lin, S. C. Huang & T. Y. Lee (1995) Correlation Between Sperm Morphology Using Strict Criteria in Original Semen and Swim-Up Inseminate and Human in vitro Fertilization, Archives of Andrology, 34:2, , DOI: / To link to this article: Published online: 09 Jul Submit your article to this journal Article views: 1 View related articles Citing articles: 11 View citing articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 02 January 2018, At: 19:33

2 CORRELATION BETWEEN SPERM MORPHOLOGY USING STRICT CRITERIA IN ORIGINAL SEMEN AND SWIM-UP INSEMINATE AND HUMAN IN VITRO FERTILIZATION Keywords Y. S. YANG S. U. CHEN H. N. HO H. F. CHEN K. H. CHAO H. R. LIN S. C. HUANG T. Y. LEE Department of Obstetrics and Gynecology, College of Medicine and the Hospital, National Taiwan University, Taipei, Taiwan, ROC To study the value of sperm morphology using strict criteria in raw semen and in swim-up inseminate of human in vitro fertilization (IVF), 135 cycles of IVF with normal sperm concentration and motility were recruited. At least two mature oocytes were recovered in each cycle. The correlation between the percentages of normal forms and fertilization rates of mature oocytes was analyzed. The results demonstrate that the percentage of normal forms in both the raw semen and swim-up sample of patients with poor fertilization was significantly lower than in those with acceptable fertilization. The percentages of normal forms both in raw semen and in swim-up sample were significantly correlated with fertilization rates in vitro, however, the former seemed to have a better correlation (r =.51 and.19, respectively). Regarding the percentages of normal forms in raw semen, the fertilization rate in patients with normal forms <4% was 6 f 11%, for 414% it was 58 f 36%, and for >14% it was 88 f 20%. The fertilization rates were significantly different among these three groups of patients. The evaluation of sperm morphology using strict criteria in raw semen before IVF is predictive of fertilization outcome and may also help doctors to choose an optimal method of treatment for patients. sperm morphology, strict criteria, in vitro fertilization This project was supported in part by a grant from the Department of Health (DOH-83-TD-035) of the Republic of China. The authors thank Jane-Ru Lin for her laboratory assistance. Address correspondence to Yu-Shih Yang, Department of Obstetrics and Gynecology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan, ROC. ARCHIVES OF ANDROLOGY 34: (1995) Copyright Taylor & Francis /95 $ OO 105

3 106 Y. S. Yang et al. The role of sperm morphology in male infertility has been a controversial issue. During natural intercourse, the study of sperm morphology in terms of fertilization is confounded by the female cervical, tubal, and ovulatory conditions, etc. In addition, there is no consensus for the criteria of normal or abnormal sperm. The judgment of sperm morphology is thought to be subjective and comparisons are difficult to make among the different laboratories and sometimes even within the same laboratory. Bestofte et al. regarded sperm morphology to be the best semen parameter in the prediction of the fertilizing capacity of sperm in vivo [2]. Francavilla et al. found a significant correlation with pregnancy rate in intrauterine insemination (IUI) for sperm morphology [S]. However, none of these results were found by Check et al. [3]. In the model of the zona-free hamster egg for human sperm penetration, the debate also exists. Rogers et al. [24] showed a strong association between normal sperm morphology and the ability of sperm to penetrate zona-free hamster eggs in vitro. However, Hall et al. did not find a significant correlation between sperm morphology and the zona-free hamster egg assay P11. The advent of human in vitro fertilization (IVF) allows a direct observation of fertilization of human sperm and oocytes and provides a direct approach to evaluate the fertilizing capacity of sperm avoiding interference from other infertility factors. However, controversies between sperm morphology and the fertilizing ability of sperm remain, even when using WHO criteria [l, 13, 16, 18, 25, 321. Recently, some investigators have introduced stricter criteria in order to obtain more accurate and repeatable results in morphology and have found them to be predictive of the fertilization rate in vitro [14, 211. Most of these studies evaluate the morphology of sperm from raw semen. In fact, the actual sperm interacting with oocytes are in the samples after preparation. The procedures used for sperm preparation in IVF may change the percentages of normal and abnormal forms. The morphological evaluation of sperm from inseminate obtained by the swim-up method or other procedures might provide more direct investigation of its relation with fertilization. The purpose of this study was to ascertain the role of sperm morphology using strict criteria for the IVF in both the raw semen and swim-up sample in a prospective project. MATERIALS AND METHODS Patients. From June 1992 to December 1993, 135 couples participating in an IVF program at the National Taiwan University Hospital were recruited. All male patients in this study had to have a sperm concentration of 220 x 106/mL and a progressive motility of 250% to minimize the influence of these two variables on the fertilization rate [29, 301. Furthermore, to reduce the possible effect of oocyte quality and number on the outcome of fertilization, the female patients in whom less than two mature oocytes were recovered were excluded from this project. The indications for IVF included tubal factor for 61 cycles, unexplained infertility for 54 cycles, endometriosis for 15 cycles, and premature ovarian failure using donated oocytes for five cycles. Ovarian Stimulation and Oocyte Retrieval. Ovarian stimulation was performed by a combination of a gonadotropin-releasing hormone analogue (Buserelin, Supremon, Hoechst AG, Germany) from midluteal phase with FSH (Metrodin, Serono, Italy) and hmg (Pergonal, Serono, Italy). Details of the stimulation regimens have been described previously [313. Ovarian response was monitored by the ultrasound folliculometry and serum estradiol (E,) measurement starting from day 8. When at least two leading follicles reached a mean diameter of 18 mm or more with proportional serum E, levels, 10,000 IU of hcg (Pregnyl, Organon, Holland) was administered intramuscularly. Thirty-four to 36 h after hcg adminis-

4 Sperm Morphology in IVF 107 tration, oocyte retrieval was performed by transvaginal ultrasound aspiration of the follicles. The maturity of oocyte was assessed by the morphological appearance of cumulus-corona-oocyte complex [19]. The recovered oocytes were preincubated for 6 h before insemination. Sperm Preparation. Semen samples were obtained by masturbation after abstinence of 3-5 days and liquefaction was completed at room temperature within 1 h. The aliquots were taken to examine the sperm count and motility, and the smear for evaluation of morphology. Semen (1 ml) was added to the bottom of a sterile round bottom tube (Falcon 2003, 12 x 75 mm, Becton Dickinson, New Jersey, USA) containing human tuba1 fluid (HTF) medium (1.2 ml) [29]. The tube was then placed at a 45" angle to allow the motile sperm to swim up into the medium. After 40 min of incubation, the supernatant was collected, 4 ml HTF medium was added, and the supernatant was then centrifuged at 400g for 5 min. The supernatant was removed and the sperm pellet was resuspended in HTF medium (1 ml). Aliquots were also taken to examine the sperm count and motility, and a smear was taken for evaluation of morphology. In Vitro Fertilization. Approximately 100,000 motile sperm were added to each oocyte in HTF medium (1 ml). The occurrence of fertilization was recognized by the presence of 2 or more pronuclei and a second polar body I6 to 18 h after insemination, or by the growth of an embryo later. Fertilization rate of mature oocytes was defined as the number of fertilized mature eggs divided by the number of mature eggs. Once the fertilized oocyte in a pronucleate stage was visualized, it was then transferred to a fresh medium for further growth. The HTF medium used for sperm preparation and oocyte/embryo culture was supplemented with 10% heat-inactivated serum from the patient. The culture condition was placed in a humidified CO, incubator (5%) at 37 C. Evaluation of Sperm Morphologv. Aliquots from both the raw semen and swim-up samples were taken to perform thin and well-spread smears on the cleaned slides. The smears were then dried in air and fixed in ethanol (95%). The staining procedure was Papanicolaou's method. Each sperm, including acrosome, postacrosomal region, neck, midpiece, and tail, was clearly and individually observed. The sperm were examined under an optical microscope at a magnification of 1000~ to differentiate the morphology according to Kruger's strict criteria as normal forms (a), slightly amorphous forms of head or neck (bl, b,), severely amorphous forms of head (c~+~), and other abnormalities (small, large, round, tapered, or double heads, and double tails, etc.) [15]. The sperm was considered normal when the head had a smooth oval configuration, the length was 5 4 mm, and the width was pm, when a well-defined acrosome was occupying 4&70% of the sperm head, as well as when there was an absence of neck, midpiece, and tail defects. No cytoplasmic droplet more than half the size of the sperm head was present. In total, 200 sperm were examined for each smear. All smears were evaluated by a well-trained technician who did not know the results of IVF. Statistic Analysis. The variables were calculated as percentages of normal forms (a), slightly amorphous forms of head or neck (bl, bj, severely amorphous forms of head (c) and other abnormalities. The percentages of normal forms and abnormal forms in the samples after the swim-up procedure were compared with those in the raw semen by paired t test. The mean fertilization rate of mature oocytes was calculated; the fertilization rates below two standard deviations (SD) were defined as poor fertilization and the others as acceptable fertilization. The correlation between the fertilization rate of mature oocytes and the percentage of normal forms was analyzed by Pearson's linear regression. According to the percentage of normal forms in the raw semen, the patients were divided into three groups: <4%, 4-14%, and >14% [15]. The fertilization rates of mature oocytes in each group were calculated and compared among the three groups by Student's t test. A p value <.05 was considered significant.

5 ~ ~ ~ 108 Y.S.Yangetal. TABLE 1 Percentages of Normal (a) and Abnormal Forms (b,, b,, c, Others) in Raw Semen and in Samples after Swim-up Procedure Raw Semen Swim-up Sample (n = 135) (n = 135) Normal forms (a) 23 f 10% 44 f 17%" Slightly amorphous (head; b,) 14 f 8% 15 f 10% Slightly amorphous (neck; b2) 14 f 9% 11 f 9%" Severely amorphous (head; c) 21 f 15% 19 f 16% Severely amorphous (others) 28f 12% 12 f 9%" Note. Values are means f SD. "p <.05. RESULTS The mean percentage of normal forms in... e raw semen was 23 f 0%. This s,gnificani Y increased to 44 f 17% in the samples after swim-up preparation (Table 1). At the same time, the percentages of slightly amorphous neck and severely amorphous forms following swim-up treatment decreased significantly. The mean fertilization rate of mature oocytes was 80 f 28%. The patients (n = 10) with fertilization rates below 24% were regarded as poor fertilization and the remaining patients (n = 125) as achieving acceptable fertilization. The number of mature oocytes was not significantly different between the two groups of patients. The percentage of normal forms in patients with poor fertilization (10 f 12%) was significantly lower than in those with acceptable fertilization (30 f 15%) in raw semen. These percentages were also significantly different between the two groups of patients in the swim-up samples (30 f 21% vs. 45 f 16%) (Table 2). Using Pearson's correlation analysis, the percentages of normal forms in raw semen were significantly correlated with fertilization rates of mature oocytes (r =.51) (Figure 1). The percentages of normal forms in swim-up samples were also significantly correlated with fertilization rates of mature oocytes (Y = -19)(Figure 2). It seemed that the percentages of normal forms in raw semen had better correlation with fertilization rates than those in swim-up samples. TABLE 2 Comparison of Percentages of Normal Forms in the Raw Semen and Swim-up Samples and Number of Mature Oocytes in Groups of Acceptable Fertilization and Poor Fertilization Acceptable Fertilization Poor Fertilization (n = 125) (n = 10) Normal forms ("h) Raw semen 30* 15 lo* 12" Swim-up sample 45* f 21" Mature oocyte number 7*4 6*4 Note. Values are means f SD. "p.05.

6 Sperm Morphology in IVF.. n435 ~0.51 PcO s 40 'J ia 20 1OOr 90 ie e! 3 60 E < lo Pmnta~~ of normal fma in raw aemen (%) FIGURE 1 Correlation of percentages of normal forms in raw semen with fertilization rates of mature oocytes n=135 FO.19 PcO.05 A & - I I -1 I I I J Percentage of normal forms in swim-up samples (%) FIGURE 2 Correlation of percentages of normal forms in swim-up samples with fertilization rates of mature oocytes.

7 110 Y. S. Yang et al. TABLE 3 Fertilization Rates of Mature Oocytes in Groups of Various Percentages of Normal Forms of Raw Semen Normal Forms <4% 4-14% >14% (n = 3) (n = 25) (n = 107) Fertilization rate ( A) 6* f f 20 Note. Values are means f SD. p <.05. Based on the percentages of normal forms in raw semen, the patients were divided into three groups as normal forms of <4% (n = 3), 4-14% (n = 25), and >14% (n = 107). The fertilization rate was 6 f 11% when normal forms were <4%. It was 58 f 36% for 414% and 88 f 20% for >14% of normal forms (Table 3). The fertilization rates were significantly different among these three groups of patients. DISCUSSION To minimize the influence of the oocyte factor on fertilization, we calculated the fertilization rate of mature oocytes. Because the number of motile sperm inseminated was constant and the original sperm count and motility were within normal limits, we could assess the role of sperm morphology in fertilization without influence of these factors. The swim-up procedure used for sperm preparation in IVF selected sperm of better morphology were consistent with previous reports [20, 23, 261. The morphology of sperm in the swim-up sample that actually participated in the interaction with the oocytes was related with the fertilization rate in vitro. This result indicated that the morphology of sperm plays an important role in the fertilization. The morphology of sperm in raw semen, which may be a reflection of the quality of semen, was also related to the fertilization rate in vitro. The latter was actually a better predictor of fertilizing ability. Using WHO criteria, Liu et al. found good correlation between sperm morphology in both prepared sperm samples and initial semen and IVF, and the latter also had a better predictive value for fertilization rates [ 161. Thus, the evaluation of sperm morphology in raw semen is simple but powerful in the prediction of fertilization in the clinical IVF laboratory. Various criteria have been used to evaluate sperm morphology and difficulties may result when comparing the findings among various studies. Using WHO criteria, several authors have reported a good correlation between sperm morphology and the IVF outcome [6, 161. However, Alper et al. were unable to find this relationship [l]. Using strict criteria, Kruger et al. showed a good correlation between fertilization rates in vitro and the percentages of normal forms [14, 151. Enginsu et al. reported that the correlation with fertilization was better for morphology evaluation using strict criteria rather than using WHO criteria [7]. The present study confirms the value of strict criteria in the evaluation of sperm morphology. Determining the superiority of conventional WHO criteria or strict criteria will require more prospective and comparative studies.

8 Sperm Morphology in IVF 111 The possible mechanisms of abnormal morphology in poor fertilization have been discussed in the literature. Rogers et al. suggested that abnormal sperm morphology is a reflection of poor testicular physiology [24]. The human zona pellucida is highly selective for binding of sperm with normal morphology [ 171. Fukuda et al. demonstrated an association between normal sperm morphology and acrosomal function [9]. It would appear that the morphology of sperm may be mainly related with binding ability to the zona pellucida and acrosomal function. It seems reasonable to increase the sperm concentration of inseminate to increase the number of normal forms for patients with high percentages of abnormal morphology. Although, in animal studies or in normospermic man, significant decreases in fertilization rates in vitro with increasing numbers of inseminated sperm were found [ 12, 18, 271, some studies supported the assumption that insemination with a higher number of sperm could improve the poor fertilization for male factor patients [5, 281. With the advent of micromanipulative procedures such as partial zona dissection, subzonal insertion, and intracytoplasmic sperm injection for the treatment of patients with low fertilization rate or fertilization failure, the problem of abnormal morphology may be treated by microinsemination [4, 10, 221. Gordt et al. found subzonal insertion better than conventional insemination in abnormal morphology using strict criteria [lo]. How to determine the concentration of sperm for insemination or to use microinsemination according to the sperm parameters deserves more clinical studies. The sperm morphology using strict criteria both in swim-up inseminate, which actually acted with oocytes, and in raw semen, which may reflect the quality of semen, was related with the fertilization rate in vitro. The latter turned out to be a better predictor of fertilizing ability. The significance of sperm morphology may be associated with the binding of zona pellucida and the acrosomal function. The evaluation of sperm morphology using strict criteria in raw semen before IVF may help to predict the patient s prognosis. The patients in the groups of <14% or even <4% of normal forms possibly suffered from poor fertilization outcome, but should not accordingly be abandoned from IVF. Utilizing the reference of morphology, efforts to modify conventional insemination and to develop microinsemination treatments are necessary in the IVF laboratory. REFERENCES 1. Alper MM, Lee GS, Seibel MM, Smith D, Oskowitz SP, Ransil BJ, Taymor ML (1985): The relationship of semen parameters to fertilization in patients participating in a program of in vitro fertilization. J In Vitro Fert Embryo Transfer 2: Bestofte E, Serup J, Rebbe H (1982): Relation between morphologically abnormal spermatozoa and pregnancies obtained during a twenty year follow-up period. Int J Androl 5: Check JH, Bollendorf A, Press M, Blue T (1992): Standard sperm morphology as a predictor of male fertility potential. Arch Androl 28: Cohen J, Alikani M, Malter HE, Adler A, Talansky BE, Rosenwaks Z (1991): Partial zona dissection or subzonal sperm insertion: microsurgical fertilization alternatives based on evaluation of sperm and embryo morphology. Fertil Steril 56:69& Diamond MP, Rogers BJ, Vaughn WK, Wentz AC (1985): Effect of the number of inseminating sperm and the follicular stimulation protocol on in vitro fertilization of human oocytes in male factor and non-male factor couples. Fertil Steril 44: Duncan WW, Glew MJ, Wang XJ, Flaherty SP, Matthews CD (1993): Prediction of in vitro fertilization rates from semen variables. Fertil Steril 59: Enginsu ME, Dumoulin JC, Pieters MH, Bras M, Evers JL, Geraedts JP (1991): Evaluation of human sperm

9 112 Y. S. Yang et al. morphology using strict criteria after Diff-Quik staining: correlation of morphology with fertilization in vitro. Hum Reprod 6: Francavilla F, Romano R, Santucci R, Poccia G (1990): Effect of sperm morphology and motile sperm count on outcome of intrauterine insemination in oligozoospermia and/or asthenozoospermia. Fertil Steril Fukuda M, Morales P, Overstreet JWA (1989): Acrosomal function of human spermatozoa with normal and abnormal head morphology. Gamete Res Gordts S, Garcia G, Vercruyssen M, Roziers P, Camp0 R, Swinnen K (1993): Subzonal insemination: a prospective randomized study in patients with abnormal sperm morphology. Fertil Steril 60: Hall J (1981): Relationship between semen quality and human sperm penetration of zona-free hamster ova. Fertil Steril 35: Iwamatsu T, Chang MC (1971): Factors involved the fertilization of mouse eggs in vitro. J Reprod Fertil 26: Jeyendran RS, Schrader SM, van der Ven HH, Burg J, Perez-Pelaez M, Al-Hasani S, Zaneveld LJD (1986): Association of the in-vitro fertilizing capacity of human spermatozoa with sperm morphology as assessed by three classification systems. Hum Reprod 1 : Kruger TF, Menkveld R, Stander FSH, Lombard CJ, Van der Merwe JP, van Zyl JA, Smith K (1986): Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril 46: Kruger TF, Swanson RJ, Acosta AA, Matta JF, Simmons KF, Oehninger S (1988): Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 49:11& Liu DY, Du Plessis YP, Nayudu PL, Johnston WIH, Baker HWG, Nayudu PL (1988): The use of in vitro fertilization to evaluate putative tests of human sperm function. Fertil Steril 49: Liu DY, Baker HWG (1992): Morphology of spermatozoa bound to the zona pellucida of human oocytes that failed to fertilize in vitro. J Reprod Fertil 94: Mahadevan MM, Trounson A0 (1984): The influence of seminal characteristics on the success rate human invitro fertilization. Fertil Steril 42: Marrs RP, Saito H, Yee B, Sato F, Brown J (1984): Effect of variation of in vivo culture techniques upon oocyte fertilization and embryo development in human in vitro fertilization procedures. Fertil Steril 41: McDowell JS, Veeck LL, Jones HW Jr (1985): Analysis of human spermatozoa before and after processing for in vitro fertilization. J In Vitro Fert Embryo Transfer 2: Menkveld R, Stander FSH, Kotze TJvW, Kruger TF, van Zyl JA (1990): The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum Reprod 5: Palermo G, Joris H, Derde MP, Camus M, Devroey P, Van-Steirteghem A (1993): Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertil Steril 59: Pousette A, Akerlof E, Rosenborg L, Fredricsson B (1986): Increase in progressive motility and improved morphology of human spermatozoa following their migration through Percoll gradients. Int J Androl 9: Rogers BJ, Bentwood BJ, Van Campen H, Helmbrecht G, Soderdahl D, Hale RW (1983): Sperm morphology assessment as an indicator of human fertilizing capacity. J Androl Rosenborg L, Gustafson 0, Lunell NO, Nylund L, Pousette A, Slotte H, Akerlof E, Fredricsson B (1990): Morphology of seminal and swim-up spermatozoa and the outcome of in vitro fertilization and embryo transfer. Andrologia 22: Scott RT Jr, Oehninger SC, Menkveld R, Veeck LL, Acosta AA (1989): Critical assessment of sperm morphology before and after double wash swim-up preparation for in vitro fertilization. Arch Androl 23: n Talbot P, Franclin LE, Fussel EN (1974): The effect of the concentration of golden hamster spermatozoa on the: acrosome reaction and egg penetration in vitro. J Reprod Fertil 36: Wolf DP, Byrd W, Dandekar P, Quigley MM (1984): Sperm concentration and the fertilization of human eggs in vitro. Biol Reprod 31: World Health Organization. WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. Cambridge, UK: Cambridge University Press, 1992.

10 Sperm Morphology in IVF Yang YS, Chen SU, Hwang JL, Ho HN, Lin HR, Lee TY (1993): Analysis of human in vitro fertilization failure. J Formosan Med Assoc 92: Yang YS, Chen SU, Ho HN, Chen HF, Lien YR, Lin HR, Huang SC, Lee TY (1994): Acrosin activity of human sperm did not correlate with IVF. Arch Androl 32: Yovich JL, Stanger JD (1984): The limitations of in vitro fertilization from males with severe oligospermia and abnormal sperm morphology. J In Vitro Fert Embxyo Transfer 1: