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In Vitro Assessment of Some Sperm Function Following Exposure To Hermanny et al. Reproductive Biology and Endocrinology 2012, 10:8 http://www.rbej.com/content/10/1/8 RESEARCH Open Access In vitro assessment of some sperm function following exposure to levonorgestrel in human fallopian tubes Alexia Hermanny1, M Valeria Bahamondes1, Francisco Fazano1, Nadia M Marchi1, Maria Elena Ortiz2, Maria Heloisa RR Genghini1, Horacio B Croxatto3 and Luis Bahamondes1* Abstract Background: The mechanism of action of levonorgestrel (LNG) as emergency contraception (EC) remains a subject of debate and its effect on sperm function has been only partially explained. The aim of this study was to assess whether LNG at a similar dose to those found in serum following oral intake for EC could affect spermatozoa when exposed to human fallopian tubes in vitro. Methods: Fifteen mini-laparotomies were performed, the side on which ovulation occurred was recorded, and both tubes were removed and perfused with a suspension containing 1 × 10(6) motile spermatozoa, with or without LNG. Following 4-hour incubation, the tubes were sectioned to separate the isthmus and the ampulla. Each segment was flushed and the material was evaluated to quantify the number of motile sperm, the number of spermatozoa adhering to the oviductal epithelium and the acrosome reaction (AR) rate. Results: The addition of LNG did not significantly alter the number of recovered motile spermatozoa either at the isthmus or at the ampulla, nor did it have any effect on the number of recovered spermatozoa adhered to the human tubal epithelium. Furthermore, LNG did not affect the AR rate. No significant differences were found even when the side on which ovulation occurred was taken into account. Conclusions: In a similar dose to that observed in serum following oral intake for EC, LNG had no effect on the number of motile spermatozoa recovered from the human fallopian tubes in vitro, on their adhesion to the tubal epithelium, distribution or AR rate. The possible effect of LNG as EC on sperm function remains poorly understood. Keywords: Emergency contraception, Levonorgestrel, Acrosome reaction, Fallopian tubes, Human spermatozoa Background to sexual intercourse and on the phase of the menstrual Levonorgestrel (LNG) is a progestin used in emergency cycle in which it is taken [3,4]. contraception (EC). The currently recommended LNG One of the proposed mechanisms of action concerns dose consists of two 0.75 mg pills taken 12 h apart or the effect of LNG on spermatozoa and their functions one dose of 1.5 mg up to 72 h after unprotected sexual [5-8]. However, this effect is still poorly understood. intercourse [1,2]. Despite the disseminated use of LNG Progesterone (P) promotes changes in aspects of sperm as EC worldwide, its mechanism of action remains function related to fertilization such as capacitation, under debate and may involve several mechanisms that sperm hyperactivation and acrosome reaction (AR) are dependent on the time of administration in relation [9-15]. Although LNG exerts a weak agonistic effect on P sperm receptors, high LNG concentrations (200-800 ng/mL) have been shown to be capable of inducing AR in human spermatozoa in vitro [8]. However, when the * Correspondence: [email protected] 1Human Reproduction Unit, Department of Obstetrics and Gynecology, spermatozoa were exposed to LNG concentrations simi- School of Medicine, University of Campinas (UNICAMP), 13084-971 lar to those observed in serum after intake for EC [16], Campinas, SP, Brazil no effect was observed [17]. Additionally, when human Full list of author information is available at the end of the article © 2012 Hermanny et al; BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Hermanny et al. Reproductive Biology and Endocrinology 2012, 10:8 Page 2 of 9 http://www.rbej.com/content/10/1/8 spermatozoa were treated in vitro with LNG at doses of having used any other form of hormone therapy, and 1 ng/mL, 10 ng/mL and 100 ng/mL, representing lower, not having been breast feeding or pregnant in the 6 similar and higher levels than those observed in serum months preceding the study. after LNG intake for EC, motility was found to be impaired with the 10 ng/mL dose; however, no effect Experiments was observed on the AR [7]. Otherwise, administration Women were instructed to use condoms as their of 1.5 mg of LNG to sterilized women at different method of contraception from the first day of the cycle moments following coitus failed to have any effect on until the day of surgery in order to avoid unplanned the quality of cervical mucus, sperm penetration to the pregnancy. Follicular development was monitored daily uterine cavity or AR [18]. in all participants from the 8th day of the menstrual After ejaculation, mammalian spermatozoa are unable cycle by ultrasound with a 5.0 MHz vaginal probe (Jus- to fertilize the oocyte and this capacity is subsequently tavision 400, Toshiba, Toshigi-Ken, Japan). When the acquired as a consequence of a series of physiological dominant follicle reached a diameter of 14-17 mm, sur- and functional changes called capacitation. Sperm capa- gical sterilization was scheduled for the following day. citation occurs during sperm migration within the All in vitro experiments were performed prior to ovula- female genital tract [19]; however, it has been noted that tion to avoid the effect of the P milieu on AR and any adhesion of spermatozoa to the tubal epithelium consti- possible confounding effect of P on sperm adhesion to tutes an important condition for in vivo fertilization in the tubal epithelium. To confirm the follicular phase of several species [20]. The interaction between spermato- the menstrual cycle, a blood sample was drawn on the zoa and the endosalpinx offers some protection to sper- day of surgery and the serum was separated and stored matozoa and in vitro sperm motility has been found to at -20°C until analysis. Serum P levels < 3 ng/mL were be longer when spermatozoa are incubated with tubal considered as confirmation of the follicular phase of the epithelium [21]. Spermatozoa undergo two forms of cycle [24]. P levels were measured in duplicate using a change in preparation for fertilization: capacitation and commercial electrochemiluminescence immunoassay hyperactivation. Capacitation includes changes at the (Roche Diagnostics GmbH, Mannheim, Germany) with plasma membrane including the protein layer and sev- a measurement range of 0.030-60.0 ng/ml, an inter- eral lipids that prepare the cells for the AR and fertiliza- assay coefficient of variation (CV) of 2.4% and an intra- tion [22]. Those spermatozoa that complete AR assay CV of 2.7%. precociously are unable to penetrate the zona pellucida All the women were anesthetized and mini-laparot- (ZP) because they lose enzymatic acrosome content omy was then performed. Both fallopian tubes were [10,23]. removed gently from the proximal portion. The side on The effect of LNG as EC on sperm function is still which the dominant follicle was present was identified poorly understood and to the best of our knowledge, and recorded. The tubes were placed in two separate this effect has not been tested at the site of fertilization. Petri dishes with HEPES-buffered modified Human Therefore, the objective of this study was to assess Tubal Fluid medium (HTF-HEPES; Irvine Scientific, whether LNG, at a similar dose to those observed in Santa Ana, CA, USA) and transferred immediately to serum following oral intake for EC, could affect sperma- the laboratory. The excess tissue (mesosalpinx) was tozoa exposed to human fallopian tubes under in vitro removed. conditions. Semen samples Methods Fifteen semen samples were obtained from healthy The study was conducted at the Human Reproduction donors with normal sperm according to the analysis cri- Unit, Department of Obstetrics and Gynecology, School teria defined in the World Health Organization manual of Medicine, University of Campinas (UNICAMP), Cam- [25]. Semen was collected by masturbation in sterile pinas, Brazil. All the women and semen donors gave plastic jars after 3 days of sexual abstinence. After eva- their written informed consent and the study protocol luation, the sample was separated into two tubes of was approved by the institution’s internal review board. equal volume. Motile spermatozoa of each fraction were Women of 25-41 years of age were invited to participate selected by a swim-up technique in which 1 mL of in the study. The admission criteria consisted of: women HTF-HEPES medium supplemented with 35 mg/mL of who had requested surgical sterilization and for whom bovine serum albumin (BSA), 1 nM of estradiol (E2) and the abdominal route of surgery was indicated, with regu- 25 nM of LNG (0.1% solution obtained from a stock lar menstrual periods (25-35 day intervals), no known solution containing 25 μM of LNG in ethanol) were tubal diseases, not having used hormonal or intrauterine used in one tube, while the same final concentration of contraceptives during the cycle of the experiment, not ethanol present in the LNG-treated sample was added Hermanny et al. Reproductive Biology and Endocrinology 2012, 10:8 Page 3 of 9 http://www.rbej.com/content/10/1/8 to the other tube. After 1 h of incubation, the superna- tubes and centrifuged and the pellets were also diluted tant with the motile spermatozoa was carefully removed in 100 μL of PBS for further evaluation of the number and an aliquot was placed into a Makler counting cham- of motile sperm and AR rate. ber (Sefi Medical Instruments, Haifa, Israel), warmed to The samples obtained following flushing with Triton 37°C and examined under a microscope.
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