ORIGINAL ARTICLE
Correlation analysis of the progesterone-induced sperm acrosome reaction rate and the fertilisation rate in vitro T. Jiang1, Y. Qin2, T. Ye3, Y. Wang1, J. Pan1, Y. Zhu4, L. Duan1, K. Li1 & X. Teng1 1 2 3 4
Center of Reproductive Medicine, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, China; Department of Gynaecology and Obstetrics, Ninth People’s Hospital of Wuxi City, Wuxi, Jiangsu Province, China; Department of Obstetrics and Gynaecology, University of Hong Kong, Hong Kong, China; Department of Gynaecology, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, China
Keywords Acrosome reaction—in vitro fertilisation— progesterone—tripronuclear—unexplained infertility Correspondence Kunming Li, Center of Reproductive Medicine, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, #536 of Changle Road, Jing An District, Shanghai 200040, China. Tel./Fax: +86 021 20261157; E-mail: [email protected] Ting Jiang and Yaping Qin are regarded as co-first author. Accepted: August 15, 2014 doi: 10.1111/and.12361
Summary In this study, we aimed to investigate whether progesterone-induced acrosome reaction (AR) rate could be an indicator for fertilisation rate in vitro. Twenty-six couples with unexplained infertility and undergoing in vitro fertilisation (IVF) treatment were involved. On the oocytes retrieval day after routine IVF, residual sperm samples were collected to receive progesterone induction (progesterone group) or not (control group). AR rate was calculated and fertilisation rate was recorded. The correlation between progesterone-induced AR and fertilisation rate and between sperm normal morphology and 3PN (tripronuclear) were analysed using the Spearman correlation analysis. The AR rate of progesterone group was statistically higher than that of the control group (15.6 5.88% versus 9.66 5.771%, P < 0.05), but not significantly correlated with fertilisation rate (r = 0.053, P > 0.01) or rate of high-quality embryo development (r = 0.055, P > 0.01). Normal sperm morphology also showed no significant correlation with the amount of 3PN zygotes (r = 0.029, P > 0.01), rate of 3PN zygotes production (r = 0.20, P > 0.01), rate of 3PN embryo development (r = 0.406, P > 0.01), fertilisation rate (r = 0.148, P > 0.01) or progesterone-induced AR rate (r = 0.214, P > 0.01). Progesterone can induce AR in vitro significantly; however, the progesterone-induced AR may not be used to indicate fertilisation rate.
Introduction Gnoth et al. (2003) defined infertility as no pregnancy after six cycles, no matter of age, without contraception. In China, unexplained infertility (UI) was commonly defined as no obvious causes for infertility through the testing means for the couples who have regular sexual activity without contraception but cannot conceive in 1 year. In vitro fertilisation (IVF) is a common approach to treat UI. However, there still exists the risk of low fertilisation or nonfertilisation (Karimzadeh et al., 2009). The failure of IVF may be attributed to the disordered zona pellucida (ZP)-induced sperm acrosome reaction (DZPIAR) that reduces the sperm-ZP penetration and leads to nonfertilisation (Liu & Baker, 1994). Although the human ZP-induced AR can be potentially used for fertilisation rate test, it is inconvenient to prepare the human ZP (Liu & Baker, 1994). In addition, follicular fluidinduced AR has also been reported to be related with © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
fertilisation rate in vitro (Calvo et al., 1989, 1994a,b). However, the AR-inducing activity of individual human follicular fluid samples is highly variable (Morales et al., 1992), which makes its application in diagnosis less attractive. It has been reported that calcium ion ionophore A23187 may be used to predict the fertilisation rate of IVF for the couples with male infertility (Yovich et al., 1994). As a natural composition, high level of progesterone can induce AR in humans and many other species (Osman et al., 1989; Saaranen et al., 1993). The combination of progesterone and sperm plasma membrane receptor induces calcium ion channels to open, triggering a variety of calcium ions-dependent physiological reactions (Blackmore et al., 1990; Harper et al., 2004; Publicover et al., 2007), such as capacitation, AR and the tendency to egg (Lishko et al., 2011). Moreover, little information is available for the progesterone-induced AR and the relationship between the progesterone-induced AR and fertilisation rate. Until now, only two related studies have 1
Relationship of AR and IVF rate
been reported, which indicated a positive correlation between the progesterone-induced AR and the fertilisation rate among randomly selected patient population (Krausz et al., 1995, 1996). Thus, an assumption that progesterone may be an indicator of fertilisation rate was proposed and evaluated in this study. In addition, tripronuclear (3PN) zygotes can be observed in the course of routine IVF treatment cycle, which results in abnormal embryologic development. It has been reported that high human chorionic gonadotrophin or polycystic ovary may be associated with 3PN zygotes (Kim et al., 2011). Several reports have shown that the causes of abnormality fertilisation were retention of the second polar body, then developing the third pronucleus after intracytoplasmic sperm injection (ICSI) (Flaherty et al., 1995a,b, 1998). The abnormal fertilisation rate (> or = 3PN) increased with oocyte ageing after ICSI (Nagy et al., 1995). However, whether normal sperm morphology is related to abnormal fertilisation rate (3PN zygotes) in vitro remains unclear. In this study, we analysed whether progesteroneinduced AR rate by semen analysis could be a predictive indicator for fertilisation rate in vitro. Besides, we also investigated the relation between sperm normal morphology and 3PN fertilisation rate. Materials and methods Patients Twenty-six couples of UI undergoing IVF treatment in Shanghai First Maternity and Infant Hospital Reproductive Center were involved in this study. Diagnostic criteria of UI was in accordance with the fifth edition of the World Health Organization (WHO) diagnostic criteria. For females, hormones tests on the third day of the menstrual cycle, ovulation detection, hysteroscope and laparoscopy were performed. No abnormalities were observed. Computer-aided sperm analysis (CASA) was performed for males, showing that sperm concentration > 15 9 106 ml 1, grade a+b motility > 32%, sperm normal morphology >4% and absence of antisperm antibody. The females accepted conventional luteal phase controlled ovarian hyperstimulation (COH). All couples signed informed consents, and experiments were approved by ethic committee of Shanghai First Maternity and Infant Hospital Reproductive Center. IVF treatment The couples accepted standard IVF treatment as previous report (Zhu et al., 2011). Women were treated with a stimulation protocol using gonadotropin-releasing 2
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hormone agonist triptorelin and recombinant follicularstimulating hormone. Oocytes retrieval and fertilisation were performed according to conventional IVF. Embryo transfer was performed on day 6 after oocyte retrieval. Semen preparation and analysis On the oocytes retrieval day after the routine IVF, the residual sperm samples were collected and spermatozoa was selected by a direct ‘swim-up’ technique according to the WHO laboratory manual (Mortimer, 1994a,b). Then, the concentration of sample was counted by the improved Neubauer haemocytometer and adjusted to 5 9 106 ml using sperm preparation medium (Origio company, Malov, Denmark). Finally, the sample was placed at 37 °C for 3 h. Sperm morphology was assessed by modified papanicolaou staining according to WHO criteria (Organization, 2010). At least 200 spermatozoa were assessed using strict criteria (Menkveld & Kruger, 1995). Progesterone-induced AR The sperm samples described above were divided into two groups, with a final volume of 125 ll in each group: one as the progesterone group with the addition of 0.5 ll progesterone solution (2.5 mg progesterone was dissolved in 1 ml anhydrous ethanol) to induce AR and the other one as the control group with the addition of 0.5 ll anhydrous ethanol. Both of the two groups were inculcated at 37 °C for 1 h. After that, sperm samples were washed with Earle’s balanced salt solution (EBSS) buffer for three times and the concentration was adjusted to 4 9 106 ml 1. Then, 50 ll reacted semen solution was lied in the semen ring on the glass slide, which was then dried up at 70 °C for 30 min, added with vinyl alcohol on the semen ring and incubated at 4 °C overnight away from light. Immunohistochemistry and assessment of AR Fluorescein isothiocyanate-labelled pisum sativum agglutinin (FITC-PSA; Sigma, St Louis, Mo., USA) is widely used for assessing the AR (Ho et al., 1994; Porter et al., 2003). First, 2 mg FITC-PSA was added in 1 ml phosphate-buffered saline (PBS) buffer to prepare the stain solution. Second, 2 ll stain solution was added to each sperm ring, and 50 ll PBS solution was added to spread over the smears. After 1 h of dyeing, smears were washed with distilled water for three times, then sealed and fixed with mounting medium. The sperm smears were viewed at 9400 magnification under oil immersion using a fluorescence microscope (Nikon, Tokyo, Japan) at 450–490 nm excitation. A © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
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minimum of 200 spermatozoa were examined and categorised as follows: 1 Acrosome intact (AI): 50–75% of the spermatozoa head is brightly and uniformly fluorescing (Fig. 1). 2 Acrosome reacted (AR): Only a tiny band of fluorescence was localised at the equatorial segment or no fluoresce in the acrosome region (Fig. 1). Detection of fertilisation rate The fertilisation rate was calculated as the number of oocytes fertilised divided by the number of retrieved oocytes. The normal fertilisation rate (2PN) and the abnormal fertilisation rate (3PN) were both detected. Statistical analysis Results were expressed as means standard deviation (SD) or standard error of mean (SEM). Data were analysed by SPSS 16.0 (Chicago, IL, USA). The results of both the progesterone and control groups in the progesteroneinduced AR follow the non-normal distribution, and the comparison between the two groups was performed with the nonparametric test and P < 0.05 indicates statistically significance. Correlation analysis was performed with the Spearman correlation analysis test. P < 0.01 in a double side test was considered statistically significant. Results The basic characteristics of 26 couples with UI and undergoing IVF treatment are shown in Table 1.
Relationship of AR and IVF rate
Table 1 Basic characteristics of 26 couples Parameters
Value (mean SD)
Time of infertility (year) Female age (year) Retrieved oocytes Male age (year) Semen density Semen live rate (%) a% b% Normal sperm morphology rate (%) Fertilised oocytes Number of high-quality embryos Number of multipronuclear zygotes
5.25 31.23 10.50 33.35 38.26 67.56 17.80 15.74 8.65 7.65 5.92 0.77
2.88 3.43 4.70 4.91 21.138 11.26 6.196 3.468 7.52 4.60 3.95 0.95
Statistical analysis showed that male (r = 0.152, P = 0.459) and female age (r = 0.183, P = 0.37) had no effect on fertilisation rate. Although the AR rate of progesterone group was significantly higher than that of control group (15.6 5.88% versus 9.66 5.77%, P < 0.05, data not shown), AR rate of progesterone group had no correlation with the fertilisation rate, rate of good-quality embryo development, number of 3PN zygotes, rate of 3PN zygotes production or 3PN development embryo rate in the progesterone group (Table 2). The normal sperm morphology rate was divided into six intervals based on its value. The parameters of number of 3PN zygotes, rate of 3PN zygotes production (3PN zygotes/retrieved eggs) and rate of 3PN embryo development (3PN zygotes/retrieved eggs) at each interval were calculated and shown in Table 3. Using Spearman correlation analysis, we found that no significant correlation between the normal sperm morphology rate and the number of 3PN zygotes, rate of 3PN zygotes production or rate of 3PN embryo development (Table 4). Discussion
Fig. 1 View of spermatozoa stained with fluorescein isothiocyanatelabelled pisum sativum agglutinin (FITC-PSA) at 9400 magnification under oil immersion using a fluorescence microscope at 450–490 nm excitation. Spermatozoa were classified as either acrosome intact or acrosome reacted. AI, Acrosome intact; AR, acrosome reacted.
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
Progesterone has been widely used for induction of AR in vitro (Osman et al., 1989; Saaranen et al., 1993), and progesterone-induced AR rate has been reported to be correlated with fertilisation rate in vitro (Calvo et al., 1994a). In this study, AR rate in the progesterone group was higher than control group, but AR rate of progesterone group was not significantly correlated with fertilisation rate or rate of high-quality embryo development. Besides, we also observed that sperm normal morphology was not significantly related with amount of 3PN zygotes, rate of 3PN zygotes production, rate of 3PN embryo development, fertilisation rate or the progesterone-induced AR rate. In this study, progesterone can specifically induce sperm AR, but no simple correlation between AR and the 3
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Table 2 Spearman correlation analysis between age, AR rate and fertilisation rate-related parameters in the progesterone group Fertilisation rate of IVF (%) Male age r P Female age r P AR rate r P
Good-quality embryo development rate (%)
Number of 3PN zygotes (n)
Rate 3PN zygotes production (%)
Rate of 3PN embryo development (%)
0.152 0.459
0.301 0.135
0.132 0.519
0.069 0.738
0.075 0.715
0.183 0.370
0.037 0.858
0.13 0.948
0.031 0.879
0.006 0.978
0.053 0.798
0.055 0.789
0.140 0.494
0.174 0.395
0.156 0.446
1 ≤ r ≤ 1; P < 0.05 indicates significant difference.
Table 3 Number of 3PN zygotes, rate of 3PN zygotes production or rate of 3PN embryo development at each interval of normal sperm morphology rate Normal morphology sperm rate (%)
Number of 3PN zygotes
4–5 5–6 6–7 7–8 8–9 >9
1.00 0.17 1.17 1.33 1.00 0.50
1.00 0.41 1.17 1.15 1.41 0.84
Rate of 3PN zygotes production 0.12 0.01 0.08 0.08 0.11 0.05
0.13 0.02 0.09 0.07 0.16 0.08
Rate of 3PN embryo development 0.13 0.01 0.10 0.11 0.25 0.07
0.14 0.03 0.10 0.09 0.35 0.11
Table 4 Spearman correlation analysis test between normal sperm morphology rate and fertilisation rate-related parameters
Fertilisation rate
P4-induced AR rate
Number of 3PN zygotes (n)
Normal sperm morphology rate interval r 0.184 0.214 0.029 P 0.369 0.293 0.957
Rate of 3PN zygotes production (%)
0.200 0.704
Rate of 3PN embryo development (%)
0.406 0.425
1 ≤ r ≤ 1; P < 0.01 indicates significant difference.
fertilisation rate in vitro was found, which was not inconsistent with previous studies (Krausz et al., 1995, 1996). In addition to progesterone, there is a variety of hormones involved in AR regulation. By the nuclear receptors and the cytoplasm receptors, estradiol has been found to be able to react in a variety of cells including sperm (Hess et al., 1997; Baldi et al., 2009). Angiotensin II in bovine and human can induce AR through the expression of AT1 receptor in sperm (Gur et al., 1998; K€ ohn et al., 1998). Atrial natriuretic peptide (ANP), a hormone produced by myocardial cell, has been 4
repeatedly found in mammalian reproductive system organisation, such as fallopian tube (Zhang et al., 2006) and follicular fluid (Anderson et al., 1994). There is evidence indicating that ANP can induce AR for many kinds of mammals, including humans (Anderson et al., 1994, 1995). Besides, prolactin (Hashimoto et al., 1988), relaxin (Miah et al., 2006), insulin (Lampiao & Du Plessis, 2008), catecholamine, epinephrine and norepinephrine in high concentration in the mammalian oviduct (Way et al., 2001) have been shown to be able to associate with mammalian AR. So, we think that fertilisation is a complex and delicate process, affected and constrained by multiple factors, such as the sperm function, internal environment and endocrine environment. Thus, it is difficult to use single factor to induce the sperm AR in in vitro experiments to simulate the in vivo process. Our research showed that, among the 26 couples with UI, 12 couples had zygotes with 3PN, accounting for 46.15% (12/26). In addition, there are 20 3PN fertilised oocytes, which accounts for 7.33% of the total number of eggs (data not shown). The 3PN incidence of 7.33% is close to that reported elsewhere (Porter et al., 2003). There is no clear correlation between sperm morphology and 3PN in this study. Similarly, our results showed that normal sperm morphology was not correlated with amount of 3PN zygotes and 3PN rate of embryo development. It has been reported that use of frozen semen was significantly correlated with the risk of recurrence comparing with patients having no tripronucleate zygotes in IVF (Plachot & Crozet, 1992). Polyspermic fertilisation has been reported to be related with sperm concentration (Wolf et al., 1984). In this study, we firstly report the correlation between sperm morphology and 3PN fertilisation rate. In conclusion, an assumption that progesteroneinduced AR rate could alternatively be used to diagnose fertilisation rate with greatly increased feasibility was proposed and evaluated in this study by analysing a sample © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
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consist of 26 couples of UI undertaking the treatment of IVF. However, no statistically significant correlation was found either between the progesterone-induced AR rate and the fertilisation rate in vitro, or between normal sperm morphology and the amount of 3PN zygotes, refuting the proposed assumption. Thus, progesterone rate may not indicate the fertilisation rate in vitro, and the sperm morphology may not reflect the 3PN fertilisation rate. However, correlation between progesteroneinduced AR and fertilisation rate and between normal sperm morphology and 3PN fertilisation rate need to be investigated by analysing a larger patients population. Acknowledgement This study was supported by Shanghai Municipal Bureau of Health Projects (Bureaus). References Anderson RA, Feathergill KA, Drisdel RC, Rawlins RG, Mack SR, Zaneveld LJ (1994) Atrial natriuretic peptide (ANP) as a stimulus of the human acrosome reaction and a component of ovarian follicular fluid: correlation of follicular ANP content with in vitro fertilization outcome. J Androl 15:61–70. Anderson RA, Feathergill KA, Rawlins RG, Mack SR, Zaneveld LJ (1995) Atrial natriuretic peptide: a chemoattractant of human spermatozoa by a guanylate cyclase-dependent pathway. Mol Reprod Dev 40:371–378. Baldi E, Luconi M, Muratori M, Marchiani S, Tamburrino L, Forti G (2009) Nongenomic activation of spermatozoa by steroid hormones: facts and fictions. Mol Cell Endocrinol 308:39–46. Blackmore P, Beebe S, Danforth D, Alexander N (1990) Progesterone and 17 alpha-hydroxyprogesterone. Novel stimulators of calcium influx in human sperm. J Biol Chem 265:1376–1380. Calvo L, Vantman D, Banks S, Tezon J, Koukoulis G, Dennison L, Sherins R (1989) Follicular fluid-induced acrosome reaction distinguishes a subgroup of men with unexplained infertility not identified by semen analysis. Fertil Steril 52:1048. Calvo L, Dennison-Lagos L, Banks SM, Dorfmann A, Thorsell LP, Bustillo M, Schulman JD, Sherins RJ (1994a) Andrology: acrosome reaction inducibility predicts fertilization success at in-vitro fertilization. Hum Reprod 9:1880–1886. Calvo L, Dennison-Lagos L, Banks SM, Sherins RJ (1994b) Andrology: characterization and frequency distribution of sperm acrosome reaction among normal and infertile men. Hum Reprod 9:1875–1879. Flaherty SP, Dianna P, Swann NJ, Matthews CD (1995a) Aetiology of failed and abnormal fertilization after
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
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intracytoplasmic sperm injection. Hum Reprod 10:2623–2629. Flaherty SP, Payne D, Swann NJ, Matthews CD (1995b) Assessment of fertilization failure and abnormal fertilization after intracytoplasmic sperm injection (ICSI). Reprod Fertil Dev 7:197–210. Flaherty SP, Payne D, Matthews CD (1998) Fertilization failures and abnormal fertilization after intracytoplasmic sperm injection. Hum Reprod 13:155–164. Gnoth C, Godehardt D, Godehardt E, Frank-Herrmann P, Freundl G (2003) Time to pregnancy: results of the German prospective study and impact on the management of infertility. Hum Reprod 18:1959–1966. Gur Y, Breitbart H, Lax Y, Rubinstein S, Zamir N (1998) Angiotensin II induces acrosomal exocytosis in bovine spermatozoa. Am J Physiol-Endocrinol Metabol 275:E87–E93. Harper CV, Barratt CL, Publicover SJ (2004) Stimulation of human spermatozoa with progesterone gradients to simulate approach to the oocyte. Induction of [Ca2+]i oscillations and cyclical transitions in flagellar beating. J Biol Chem 279:46315–46325. Hashimoto H, Izumi S, Mori C, Nakane P, Hoshino K (1988) Immunohistochemical observation of prolactin receptor in rat epididymal spermatozoa by backscattered electron imaging. In: Prolactin Gene Family and Its Receptors Molecular Biology to Clinical Problems, Excerpta Medica International Congress Series. Hoshino K (ed.). Elsevier, Amsterdam, pp. 341–344. Hess RA, Bunick D, Lee K-H, Bahr J, Taylor JA, Korach KS, Lubahn DB (1997) A role for oestrogens in the male reproductive system. Nature 390:509–512. Ho P-C, Yeung W, Chan Y-F, So W, Chan S (1994) Factors affecting the incidence of polyploidy in a human in vitro fertilization program. Int J Fertil Menopausal Stud 39:14–19. Karimzadeh MA, Ayazi Rozbahani M, Tabibnejad N (2009) Endometrial local injury improves the pregnancy rate among recurrent implantation failure patients undergoing in vitro fertilisation/intra cytoplasmic sperm injection: a randomised clinical trial. Aust N Z J Obstet Gynaecol 49:677–680. Kim YJ, Ku SY, Jee BC, Suh CS, Kim SH, Choi YM, Kim JG, Moon SY (2011) Increased responsiveness may be associated with tripronuclear embryos in both conventional in vitro fertilization and intracytoplasmic sperm injection cycles using gonadotropin-releasing hormone agonist long protocols: a self-matched observational study. J Obstet Gynecol Res 37:37–44. K€ ohn FM, M€ uller C, Drescher D, Neukamm C, Mulla KE, Henkel R, H€agele W, Hinsch E, Habenicht U, Schill WB (1998) Effect of angiotensin converting enzyme (ACE) and angiotensins on human sperm functions. Andrologia 30:207–215. Krausz C, Bonaccorsi L, Luconi M, Fuzzi B, Criscuoli L, Pellegrini S, Forti G, Baldi E (1995) Intracellular calcium increase and acrosome reaction in response to progesterone
5
Relationship of AR and IVF rate
in human spermatozoa are correlated with in-vitro fertilization. Hum Reprod 10:120–124. Krausz C, Bonaccorsi L, Maggio P, Luconi M, Criscuoli L, Fuzzi B, Pellegrini S, Forti G, Baldi E (1996) Andrology: two functional assays of sperm responsiveness to progesterone and their predictive values in in-vitro fertilization. Hum Reprod 11:1661–1667. Lampiao F, Du Plessis SS (2008) Insulin and leptin enhance human sperm motility, acrosome reaction and nitric oxide production. Asian J Androl 10:799–807. Lishko PV, Botchkina IL, Kirichok Y (2011) Progesterone activates the principal Ca2+ channel of human sperm. Nature 471:387–391. Liu DY, Baker HG (1994) Andrology: disordered acrosome reaction of spermatozoa bound to the zona pellucida: a newly discovered sperm defect causing infertility with reduced sperm-zona pellucida penetration and reduced fertilization in vitro. Hum Reprod 9:1694–1700. Menkveld R, Kruger T (1995) Advantages of strict (Tygerberg) criteria for evaluation of sperm morphology. Int J Androl 18:36. Miah A, Tareq K, Hamano K, Kohsaka T, Tsujii H (2006) Effect of relaxin on acrosome reaction and utilization of glucose in boar spermatozoa. J Reprod Dev 52:773–779. Morales P, Llanos M, Gutierrez G, Kohen P, Vigil P, Vantman D (1992) The acrosome reaction-inducing activity of individual human follicular fluid samples is highly variable and is related to the steroid content. Hum Reprod 7:646– 651. Mortimer D (1994a) Laboratory standards in routine clinical andrology. Reprod Med Rev 3:97–111. Mortimer D (1994b) Practical Laboratory Andrology. Oxford University Press, New York. Nagy Z, Staessen C, Liu J, Joris H, Devroey P, Van Steirteghem A (1995) Prospective, auto-controlled study on reinsemination of failed-fertilized oocytes by intracytoplasmic sperm injection. Fertil Steril 64:1130–1135. Osman RA, Andria ML, Jones AD, Meizel S (1989) Steroid induced exocytosis: the human sperm acrosome reaction. Biochem Biophys Res Commun 160:828–833.
6
T. Jiang et al.
Plachot M, Crozet N (1992) Fertilization abnormalities in human in-vitro fertilization. Hum Reprod 7:89–94. Porter R, Han T, Tucker MJ, Graham J, Liebermann J, Sills ES (2003) Estimation of second polar body retention rate after conventional insemination and intracytoplasmic sperm injection: in vitro observations from more than 5000 human oocytes. J Assist Reprod Genet 20:371–376. Publicover S, Harper CV, Barratt C (2007) [Ca2+] i signalling in sperm-making the most of what you’ve got. Nat Cell Biol 9:235–242. Saaranen M, Calvo L, Dennison L, Banks S, Bustillo M, Dorfmann A, Goldstein M, Thorsell L, Schulman J, Sherins R (1993) Andrology: acrosome reaction inducing activity in follicular fluid correlates with progesterone concentration but not with oocyte maturity or fertilizability. Hum Reprod 8:1448–1454. Way A, Barbato G, Killian G (2001) Identification of norepinephrine in bovine oviductal fluid by high performance liquid chromatography. Life Sci 70:567–576. Wolf D, Byrd W, Dandekar P, Quigley M (1984) Sperm concentration and the fertilization of human eggs in vitro. Biol Reprod 31:837–848. World Health Organization (2010) WHO Laboratory Manual for the Examination and Processing of Human Semen. Cambridge University Press, Cambridge. Yovich J, Edirisinghe W, Yovich J (1994) Use of the acrosome reaction to ionophore challenge test in managing patients in an assisted reproduction program: a prospective, double-blind, randomized controlled study. Fertil Steril 61:902. Zhang M, Hong H, Zhou B, Jin S, Wang C, Fu M, Wang S, Xia G (2006) The expression of atrial natriuretic peptide in the oviduct and its functions in pig spermatozoa. J Endocrinol 189:493–507. Zhu D, Zhang J, Cao S, Zhang J, Heng BC, Huang M, Ling X, Duan T, Tong GQ (2011) Vitrified-warmed blastocyst transfer cycles yield higher pregnancy and implantation rates compared with fresh blastocyst transfer cycles-time for a new embryo transfer strategy? Fertil Steril 95: 1691–1695.
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Correlation analysis of the progesterone-induced sperm acrosome reaction rate and the fertilisation rate in vitro T. Jiang1, Y. Qin2, T. Ye3, Y. Wang1, J. Pan1, Y. Zhu4, L. Duan1, K. Li1 & X. Teng1 1 2 3 4
Center of Reproductive Medicine, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, China; Department of Gynaecology and Obstetrics, Ninth People’s Hospital of Wuxi City, Wuxi, Jiangsu Province, China; Department of Obstetrics and Gynaecology, University of Hong Kong, Hong Kong, China; Department of Gynaecology, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, China
Keywords Acrosome reaction—in vitro fertilisation— progesterone—tripronuclear—unexplained infertility Correspondence Kunming Li, Center of Reproductive Medicine, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, #536 of Changle Road, Jing An District, Shanghai 200040, China. Tel./Fax: +86 021 20261157; E-mail: [email protected] Ting Jiang and Yaping Qin are regarded as co-first author. Accepted: August 15, 2014 doi: 10.1111/and.12361
Summary In this study, we aimed to investigate whether progesterone-induced acrosome reaction (AR) rate could be an indicator for fertilisation rate in vitro. Twenty-six couples with unexplained infertility and undergoing in vitro fertilisation (IVF) treatment were involved. On the oocytes retrieval day after routine IVF, residual sperm samples were collected to receive progesterone induction (progesterone group) or not (control group). AR rate was calculated and fertilisation rate was recorded. The correlation between progesterone-induced AR and fertilisation rate and between sperm normal morphology and 3PN (tripronuclear) were analysed using the Spearman correlation analysis. The AR rate of progesterone group was statistically higher than that of the control group (15.6 5.88% versus 9.66 5.771%, P < 0.05), but not significantly correlated with fertilisation rate (r = 0.053, P > 0.01) or rate of high-quality embryo development (r = 0.055, P > 0.01). Normal sperm morphology also showed no significant correlation with the amount of 3PN zygotes (r = 0.029, P > 0.01), rate of 3PN zygotes production (r = 0.20, P > 0.01), rate of 3PN embryo development (r = 0.406, P > 0.01), fertilisation rate (r = 0.148, P > 0.01) or progesterone-induced AR rate (r = 0.214, P > 0.01). Progesterone can induce AR in vitro significantly; however, the progesterone-induced AR may not be used to indicate fertilisation rate.
Introduction Gnoth et al. (2003) defined infertility as no pregnancy after six cycles, no matter of age, without contraception. In China, unexplained infertility (UI) was commonly defined as no obvious causes for infertility through the testing means for the couples who have regular sexual activity without contraception but cannot conceive in 1 year. In vitro fertilisation (IVF) is a common approach to treat UI. However, there still exists the risk of low fertilisation or nonfertilisation (Karimzadeh et al., 2009). The failure of IVF may be attributed to the disordered zona pellucida (ZP)-induced sperm acrosome reaction (DZPIAR) that reduces the sperm-ZP penetration and leads to nonfertilisation (Liu & Baker, 1994). Although the human ZP-induced AR can be potentially used for fertilisation rate test, it is inconvenient to prepare the human ZP (Liu & Baker, 1994). In addition, follicular fluidinduced AR has also been reported to be related with © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
fertilisation rate in vitro (Calvo et al., 1989, 1994a,b). However, the AR-inducing activity of individual human follicular fluid samples is highly variable (Morales et al., 1992), which makes its application in diagnosis less attractive. It has been reported that calcium ion ionophore A23187 may be used to predict the fertilisation rate of IVF for the couples with male infertility (Yovich et al., 1994). As a natural composition, high level of progesterone can induce AR in humans and many other species (Osman et al., 1989; Saaranen et al., 1993). The combination of progesterone and sperm plasma membrane receptor induces calcium ion channels to open, triggering a variety of calcium ions-dependent physiological reactions (Blackmore et al., 1990; Harper et al., 2004; Publicover et al., 2007), such as capacitation, AR and the tendency to egg (Lishko et al., 2011). Moreover, little information is available for the progesterone-induced AR and the relationship between the progesterone-induced AR and fertilisation rate. Until now, only two related studies have 1
Relationship of AR and IVF rate
been reported, which indicated a positive correlation between the progesterone-induced AR and the fertilisation rate among randomly selected patient population (Krausz et al., 1995, 1996). Thus, an assumption that progesterone may be an indicator of fertilisation rate was proposed and evaluated in this study. In addition, tripronuclear (3PN) zygotes can be observed in the course of routine IVF treatment cycle, which results in abnormal embryologic development. It has been reported that high human chorionic gonadotrophin or polycystic ovary may be associated with 3PN zygotes (Kim et al., 2011). Several reports have shown that the causes of abnormality fertilisation were retention of the second polar body, then developing the third pronucleus after intracytoplasmic sperm injection (ICSI) (Flaherty et al., 1995a,b, 1998). The abnormal fertilisation rate (> or = 3PN) increased with oocyte ageing after ICSI (Nagy et al., 1995). However, whether normal sperm morphology is related to abnormal fertilisation rate (3PN zygotes) in vitro remains unclear. In this study, we analysed whether progesteroneinduced AR rate by semen analysis could be a predictive indicator for fertilisation rate in vitro. Besides, we also investigated the relation between sperm normal morphology and 3PN fertilisation rate. Materials and methods Patients Twenty-six couples of UI undergoing IVF treatment in Shanghai First Maternity and Infant Hospital Reproductive Center were involved in this study. Diagnostic criteria of UI was in accordance with the fifth edition of the World Health Organization (WHO) diagnostic criteria. For females, hormones tests on the third day of the menstrual cycle, ovulation detection, hysteroscope and laparoscopy were performed. No abnormalities were observed. Computer-aided sperm analysis (CASA) was performed for males, showing that sperm concentration > 15 9 106 ml 1, grade a+b motility > 32%, sperm normal morphology >4% and absence of antisperm antibody. The females accepted conventional luteal phase controlled ovarian hyperstimulation (COH). All couples signed informed consents, and experiments were approved by ethic committee of Shanghai First Maternity and Infant Hospital Reproductive Center. IVF treatment The couples accepted standard IVF treatment as previous report (Zhu et al., 2011). Women were treated with a stimulation protocol using gonadotropin-releasing 2
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hormone agonist triptorelin and recombinant follicularstimulating hormone. Oocytes retrieval and fertilisation were performed according to conventional IVF. Embryo transfer was performed on day 6 after oocyte retrieval. Semen preparation and analysis On the oocytes retrieval day after the routine IVF, the residual sperm samples were collected and spermatozoa was selected by a direct ‘swim-up’ technique according to the WHO laboratory manual (Mortimer, 1994a,b). Then, the concentration of sample was counted by the improved Neubauer haemocytometer and adjusted to 5 9 106 ml using sperm preparation medium (Origio company, Malov, Denmark). Finally, the sample was placed at 37 °C for 3 h. Sperm morphology was assessed by modified papanicolaou staining according to WHO criteria (Organization, 2010). At least 200 spermatozoa were assessed using strict criteria (Menkveld & Kruger, 1995). Progesterone-induced AR The sperm samples described above were divided into two groups, with a final volume of 125 ll in each group: one as the progesterone group with the addition of 0.5 ll progesterone solution (2.5 mg progesterone was dissolved in 1 ml anhydrous ethanol) to induce AR and the other one as the control group with the addition of 0.5 ll anhydrous ethanol. Both of the two groups were inculcated at 37 °C for 1 h. After that, sperm samples were washed with Earle’s balanced salt solution (EBSS) buffer for three times and the concentration was adjusted to 4 9 106 ml 1. Then, 50 ll reacted semen solution was lied in the semen ring on the glass slide, which was then dried up at 70 °C for 30 min, added with vinyl alcohol on the semen ring and incubated at 4 °C overnight away from light. Immunohistochemistry and assessment of AR Fluorescein isothiocyanate-labelled pisum sativum agglutinin (FITC-PSA; Sigma, St Louis, Mo., USA) is widely used for assessing the AR (Ho et al., 1994; Porter et al., 2003). First, 2 mg FITC-PSA was added in 1 ml phosphate-buffered saline (PBS) buffer to prepare the stain solution. Second, 2 ll stain solution was added to each sperm ring, and 50 ll PBS solution was added to spread over the smears. After 1 h of dyeing, smears were washed with distilled water for three times, then sealed and fixed with mounting medium. The sperm smears were viewed at 9400 magnification under oil immersion using a fluorescence microscope (Nikon, Tokyo, Japan) at 450–490 nm excitation. A © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
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minimum of 200 spermatozoa were examined and categorised as follows: 1 Acrosome intact (AI): 50–75% of the spermatozoa head is brightly and uniformly fluorescing (Fig. 1). 2 Acrosome reacted (AR): Only a tiny band of fluorescence was localised at the equatorial segment or no fluoresce in the acrosome region (Fig. 1). Detection of fertilisation rate The fertilisation rate was calculated as the number of oocytes fertilised divided by the number of retrieved oocytes. The normal fertilisation rate (2PN) and the abnormal fertilisation rate (3PN) were both detected. Statistical analysis Results were expressed as means standard deviation (SD) or standard error of mean (SEM). Data were analysed by SPSS 16.0 (Chicago, IL, USA). The results of both the progesterone and control groups in the progesteroneinduced AR follow the non-normal distribution, and the comparison between the two groups was performed with the nonparametric test and P < 0.05 indicates statistically significance. Correlation analysis was performed with the Spearman correlation analysis test. P < 0.01 in a double side test was considered statistically significant. Results The basic characteristics of 26 couples with UI and undergoing IVF treatment are shown in Table 1.
Relationship of AR and IVF rate
Table 1 Basic characteristics of 26 couples Parameters
Value (mean SD)
Time of infertility (year) Female age (year) Retrieved oocytes Male age (year) Semen density Semen live rate (%) a% b% Normal sperm morphology rate (%) Fertilised oocytes Number of high-quality embryos Number of multipronuclear zygotes
5.25 31.23 10.50 33.35 38.26 67.56 17.80 15.74 8.65 7.65 5.92 0.77
2.88 3.43 4.70 4.91 21.138 11.26 6.196 3.468 7.52 4.60 3.95 0.95
Statistical analysis showed that male (r = 0.152, P = 0.459) and female age (r = 0.183, P = 0.37) had no effect on fertilisation rate. Although the AR rate of progesterone group was significantly higher than that of control group (15.6 5.88% versus 9.66 5.77%, P < 0.05, data not shown), AR rate of progesterone group had no correlation with the fertilisation rate, rate of good-quality embryo development, number of 3PN zygotes, rate of 3PN zygotes production or 3PN development embryo rate in the progesterone group (Table 2). The normal sperm morphology rate was divided into six intervals based on its value. The parameters of number of 3PN zygotes, rate of 3PN zygotes production (3PN zygotes/retrieved eggs) and rate of 3PN embryo development (3PN zygotes/retrieved eggs) at each interval were calculated and shown in Table 3. Using Spearman correlation analysis, we found that no significant correlation between the normal sperm morphology rate and the number of 3PN zygotes, rate of 3PN zygotes production or rate of 3PN embryo development (Table 4). Discussion
Fig. 1 View of spermatozoa stained with fluorescein isothiocyanatelabelled pisum sativum agglutinin (FITC-PSA) at 9400 magnification under oil immersion using a fluorescence microscope at 450–490 nm excitation. Spermatozoa were classified as either acrosome intact or acrosome reacted. AI, Acrosome intact; AR, acrosome reacted.
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
Progesterone has been widely used for induction of AR in vitro (Osman et al., 1989; Saaranen et al., 1993), and progesterone-induced AR rate has been reported to be correlated with fertilisation rate in vitro (Calvo et al., 1994a). In this study, AR rate in the progesterone group was higher than control group, but AR rate of progesterone group was not significantly correlated with fertilisation rate or rate of high-quality embryo development. Besides, we also observed that sperm normal morphology was not significantly related with amount of 3PN zygotes, rate of 3PN zygotes production, rate of 3PN embryo development, fertilisation rate or the progesterone-induced AR rate. In this study, progesterone can specifically induce sperm AR, but no simple correlation between AR and the 3
Relationship of AR and IVF rate
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Table 2 Spearman correlation analysis between age, AR rate and fertilisation rate-related parameters in the progesterone group Fertilisation rate of IVF (%) Male age r P Female age r P AR rate r P
Good-quality embryo development rate (%)
Number of 3PN zygotes (n)
Rate 3PN zygotes production (%)
Rate of 3PN embryo development (%)
0.152 0.459
0.301 0.135
0.132 0.519
0.069 0.738
0.075 0.715
0.183 0.370
0.037 0.858
0.13 0.948
0.031 0.879
0.006 0.978
0.053 0.798
0.055 0.789
0.140 0.494
0.174 0.395
0.156 0.446
1 ≤ r ≤ 1; P < 0.05 indicates significant difference.
Table 3 Number of 3PN zygotes, rate of 3PN zygotes production or rate of 3PN embryo development at each interval of normal sperm morphology rate Normal morphology sperm rate (%)
Number of 3PN zygotes
4–5 5–6 6–7 7–8 8–9 >9
1.00 0.17 1.17 1.33 1.00 0.50
1.00 0.41 1.17 1.15 1.41 0.84
Rate of 3PN zygotes production 0.12 0.01 0.08 0.08 0.11 0.05
0.13 0.02 0.09 0.07 0.16 0.08
Rate of 3PN embryo development 0.13 0.01 0.10 0.11 0.25 0.07
0.14 0.03 0.10 0.09 0.35 0.11
Table 4 Spearman correlation analysis test between normal sperm morphology rate and fertilisation rate-related parameters
Fertilisation rate
P4-induced AR rate
Number of 3PN zygotes (n)
Normal sperm morphology rate interval r 0.184 0.214 0.029 P 0.369 0.293 0.957
Rate of 3PN zygotes production (%)
0.200 0.704
Rate of 3PN embryo development (%)
0.406 0.425
1 ≤ r ≤ 1; P < 0.01 indicates significant difference.
fertilisation rate in vitro was found, which was not inconsistent with previous studies (Krausz et al., 1995, 1996). In addition to progesterone, there is a variety of hormones involved in AR regulation. By the nuclear receptors and the cytoplasm receptors, estradiol has been found to be able to react in a variety of cells including sperm (Hess et al., 1997; Baldi et al., 2009). Angiotensin II in bovine and human can induce AR through the expression of AT1 receptor in sperm (Gur et al., 1998; K€ ohn et al., 1998). Atrial natriuretic peptide (ANP), a hormone produced by myocardial cell, has been 4
repeatedly found in mammalian reproductive system organisation, such as fallopian tube (Zhang et al., 2006) and follicular fluid (Anderson et al., 1994). There is evidence indicating that ANP can induce AR for many kinds of mammals, including humans (Anderson et al., 1994, 1995). Besides, prolactin (Hashimoto et al., 1988), relaxin (Miah et al., 2006), insulin (Lampiao & Du Plessis, 2008), catecholamine, epinephrine and norepinephrine in high concentration in the mammalian oviduct (Way et al., 2001) have been shown to be able to associate with mammalian AR. So, we think that fertilisation is a complex and delicate process, affected and constrained by multiple factors, such as the sperm function, internal environment and endocrine environment. Thus, it is difficult to use single factor to induce the sperm AR in in vitro experiments to simulate the in vivo process. Our research showed that, among the 26 couples with UI, 12 couples had zygotes with 3PN, accounting for 46.15% (12/26). In addition, there are 20 3PN fertilised oocytes, which accounts for 7.33% of the total number of eggs (data not shown). The 3PN incidence of 7.33% is close to that reported elsewhere (Porter et al., 2003). There is no clear correlation between sperm morphology and 3PN in this study. Similarly, our results showed that normal sperm morphology was not correlated with amount of 3PN zygotes and 3PN rate of embryo development. It has been reported that use of frozen semen was significantly correlated with the risk of recurrence comparing with patients having no tripronucleate zygotes in IVF (Plachot & Crozet, 1992). Polyspermic fertilisation has been reported to be related with sperm concentration (Wolf et al., 1984). In this study, we firstly report the correlation between sperm morphology and 3PN fertilisation rate. In conclusion, an assumption that progesteroneinduced AR rate could alternatively be used to diagnose fertilisation rate with greatly increased feasibility was proposed and evaluated in this study by analysing a sample © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
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consist of 26 couples of UI undertaking the treatment of IVF. However, no statistically significant correlation was found either between the progesterone-induced AR rate and the fertilisation rate in vitro, or between normal sperm morphology and the amount of 3PN zygotes, refuting the proposed assumption. Thus, progesterone rate may not indicate the fertilisation rate in vitro, and the sperm morphology may not reflect the 3PN fertilisation rate. However, correlation between progesteroneinduced AR and fertilisation rate and between normal sperm morphology and 3PN fertilisation rate need to be investigated by analysing a larger patients population. Acknowledgement This study was supported by Shanghai Municipal Bureau of Health Projects (Bureaus). References Anderson RA, Feathergill KA, Drisdel RC, Rawlins RG, Mack SR, Zaneveld LJ (1994) Atrial natriuretic peptide (ANP) as a stimulus of the human acrosome reaction and a component of ovarian follicular fluid: correlation of follicular ANP content with in vitro fertilization outcome. J Androl 15:61–70. Anderson RA, Feathergill KA, Rawlins RG, Mack SR, Zaneveld LJ (1995) Atrial natriuretic peptide: a chemoattractant of human spermatozoa by a guanylate cyclase-dependent pathway. Mol Reprod Dev 40:371–378. Baldi E, Luconi M, Muratori M, Marchiani S, Tamburrino L, Forti G (2009) Nongenomic activation of spermatozoa by steroid hormones: facts and fictions. Mol Cell Endocrinol 308:39–46. Blackmore P, Beebe S, Danforth D, Alexander N (1990) Progesterone and 17 alpha-hydroxyprogesterone. Novel stimulators of calcium influx in human sperm. J Biol Chem 265:1376–1380. Calvo L, Vantman D, Banks S, Tezon J, Koukoulis G, Dennison L, Sherins R (1989) Follicular fluid-induced acrosome reaction distinguishes a subgroup of men with unexplained infertility not identified by semen analysis. Fertil Steril 52:1048. Calvo L, Dennison-Lagos L, Banks SM, Dorfmann A, Thorsell LP, Bustillo M, Schulman JD, Sherins RJ (1994a) Andrology: acrosome reaction inducibility predicts fertilization success at in-vitro fertilization. Hum Reprod 9:1880–1886. Calvo L, Dennison-Lagos L, Banks SM, Sherins RJ (1994b) Andrology: characterization and frequency distribution of sperm acrosome reaction among normal and infertile men. Hum Reprod 9:1875–1879. Flaherty SP, Dianna P, Swann NJ, Matthews CD (1995a) Aetiology of failed and abnormal fertilization after
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
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intracytoplasmic sperm injection. Hum Reprod 10:2623–2629. Flaherty SP, Payne D, Swann NJ, Matthews CD (1995b) Assessment of fertilization failure and abnormal fertilization after intracytoplasmic sperm injection (ICSI). Reprod Fertil Dev 7:197–210. Flaherty SP, Payne D, Matthews CD (1998) Fertilization failures and abnormal fertilization after intracytoplasmic sperm injection. Hum Reprod 13:155–164. Gnoth C, Godehardt D, Godehardt E, Frank-Herrmann P, Freundl G (2003) Time to pregnancy: results of the German prospective study and impact on the management of infertility. Hum Reprod 18:1959–1966. Gur Y, Breitbart H, Lax Y, Rubinstein S, Zamir N (1998) Angiotensin II induces acrosomal exocytosis in bovine spermatozoa. Am J Physiol-Endocrinol Metabol 275:E87–E93. Harper CV, Barratt CL, Publicover SJ (2004) Stimulation of human spermatozoa with progesterone gradients to simulate approach to the oocyte. Induction of [Ca2+]i oscillations and cyclical transitions in flagellar beating. J Biol Chem 279:46315–46325. Hashimoto H, Izumi S, Mori C, Nakane P, Hoshino K (1988) Immunohistochemical observation of prolactin receptor in rat epididymal spermatozoa by backscattered electron imaging. In: Prolactin Gene Family and Its Receptors Molecular Biology to Clinical Problems, Excerpta Medica International Congress Series. Hoshino K (ed.). Elsevier, Amsterdam, pp. 341–344. Hess RA, Bunick D, Lee K-H, Bahr J, Taylor JA, Korach KS, Lubahn DB (1997) A role for oestrogens in the male reproductive system. Nature 390:509–512. Ho P-C, Yeung W, Chan Y-F, So W, Chan S (1994) Factors affecting the incidence of polyploidy in a human in vitro fertilization program. Int J Fertil Menopausal Stud 39:14–19. Karimzadeh MA, Ayazi Rozbahani M, Tabibnejad N (2009) Endometrial local injury improves the pregnancy rate among recurrent implantation failure patients undergoing in vitro fertilisation/intra cytoplasmic sperm injection: a randomised clinical trial. Aust N Z J Obstet Gynaecol 49:677–680. Kim YJ, Ku SY, Jee BC, Suh CS, Kim SH, Choi YM, Kim JG, Moon SY (2011) Increased responsiveness may be associated with tripronuclear embryos in both conventional in vitro fertilization and intracytoplasmic sperm injection cycles using gonadotropin-releasing hormone agonist long protocols: a self-matched observational study. J Obstet Gynecol Res 37:37–44. K€ ohn FM, M€ uller C, Drescher D, Neukamm C, Mulla KE, Henkel R, H€agele W, Hinsch E, Habenicht U, Schill WB (1998) Effect of angiotensin converting enzyme (ACE) and angiotensins on human sperm functions. Andrologia 30:207–215. Krausz C, Bonaccorsi L, Luconi M, Fuzzi B, Criscuoli L, Pellegrini S, Forti G, Baldi E (1995) Intracellular calcium increase and acrosome reaction in response to progesterone
5
Relationship of AR and IVF rate
in human spermatozoa are correlated with in-vitro fertilization. Hum Reprod 10:120–124. Krausz C, Bonaccorsi L, Maggio P, Luconi M, Criscuoli L, Fuzzi B, Pellegrini S, Forti G, Baldi E (1996) Andrology: two functional assays of sperm responsiveness to progesterone and their predictive values in in-vitro fertilization. Hum Reprod 11:1661–1667. Lampiao F, Du Plessis SS (2008) Insulin and leptin enhance human sperm motility, acrosome reaction and nitric oxide production. Asian J Androl 10:799–807. Lishko PV, Botchkina IL, Kirichok Y (2011) Progesterone activates the principal Ca2+ channel of human sperm. Nature 471:387–391. Liu DY, Baker HG (1994) Andrology: disordered acrosome reaction of spermatozoa bound to the zona pellucida: a newly discovered sperm defect causing infertility with reduced sperm-zona pellucida penetration and reduced fertilization in vitro. Hum Reprod 9:1694–1700. Menkveld R, Kruger T (1995) Advantages of strict (Tygerberg) criteria for evaluation of sperm morphology. Int J Androl 18:36. Miah A, Tareq K, Hamano K, Kohsaka T, Tsujii H (2006) Effect of relaxin on acrosome reaction and utilization of glucose in boar spermatozoa. J Reprod Dev 52:773–779. Morales P, Llanos M, Gutierrez G, Kohen P, Vigil P, Vantman D (1992) The acrosome reaction-inducing activity of individual human follicular fluid samples is highly variable and is related to the steroid content. Hum Reprod 7:646– 651. Mortimer D (1994a) Laboratory standards in routine clinical andrology. Reprod Med Rev 3:97–111. Mortimer D (1994b) Practical Laboratory Andrology. Oxford University Press, New York. Nagy Z, Staessen C, Liu J, Joris H, Devroey P, Van Steirteghem A (1995) Prospective, auto-controlled study on reinsemination of failed-fertilized oocytes by intracytoplasmic sperm injection. Fertil Steril 64:1130–1135. Osman RA, Andria ML, Jones AD, Meizel S (1989) Steroid induced exocytosis: the human sperm acrosome reaction. Biochem Biophys Res Commun 160:828–833.
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T. Jiang et al.
Plachot M, Crozet N (1992) Fertilization abnormalities in human in-vitro fertilization. Hum Reprod 7:89–94. Porter R, Han T, Tucker MJ, Graham J, Liebermann J, Sills ES (2003) Estimation of second polar body retention rate after conventional insemination and intracytoplasmic sperm injection: in vitro observations from more than 5000 human oocytes. J Assist Reprod Genet 20:371–376. Publicover S, Harper CV, Barratt C (2007) [Ca2+] i signalling in sperm-making the most of what you’ve got. Nat Cell Biol 9:235–242. Saaranen M, Calvo L, Dennison L, Banks S, Bustillo M, Dorfmann A, Goldstein M, Thorsell L, Schulman J, Sherins R (1993) Andrology: acrosome reaction inducing activity in follicular fluid correlates with progesterone concentration but not with oocyte maturity or fertilizability. Hum Reprod 8:1448–1454. Way A, Barbato G, Killian G (2001) Identification of norepinephrine in bovine oviductal fluid by high performance liquid chromatography. Life Sci 70:567–576. Wolf D, Byrd W, Dandekar P, Quigley M (1984) Sperm concentration and the fertilization of human eggs in vitro. Biol Reprod 31:837–848. World Health Organization (2010) WHO Laboratory Manual for the Examination and Processing of Human Semen. Cambridge University Press, Cambridge. Yovich J, Edirisinghe W, Yovich J (1994) Use of the acrosome reaction to ionophore challenge test in managing patients in an assisted reproduction program: a prospective, double-blind, randomized controlled study. Fertil Steril 61:902. Zhang M, Hong H, Zhou B, Jin S, Wang C, Fu M, Wang S, Xia G (2006) The expression of atrial natriuretic peptide in the oviduct and its functions in pig spermatozoa. J Endocrinol 189:493–507. Zhu D, Zhang J, Cao S, Zhang J, Heng BC, Huang M, Ling X, Duan T, Tong GQ (2011) Vitrified-warmed blastocyst transfer cycles yield higher pregnancy and implantation rates compared with fresh blastocyst transfer cycles-time for a new embryo transfer strategy? Fertil Steril 95: 1691–1695.
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