Effect of volumetric mixtures of peritoneal and follicular fluid from the same woman on sperm motility and acrosomal reactivity in vitro*

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1 FERTILITY AND STERILITY Copyright 1992 The Amerian Fertility Soiety Printed on aid-free paper in U.S.A. Effet of volumetri mixtures of peritoneal and folliular fluid from the same woman on sperm motility and arosomal reativity in vitro* Alberto Revelli, M.D.t:j: Gianni Soldati, Ph.D.t Jiirg Stamm, M.D.t Maro Massobrio, M.D.:j: Edda Topfer-Petersen, Ph.D. Maro Balema, Ph.D.tll "La Carita" Hospital. Loarno, Switzerland, University of Turin, Turin, Italy, and University of Munih, Munih, Germany Objetive: To study the effet of peritoneal (PF) and folliular fluids (FF from the same woman) as well as of given volumetri ombinations thereof on sperm motility and aero somal reativity. Design: Prospetive. Peritoneal fluid and FF were inubated separately or in given volumetri ombinations (PF/FF = 1/,75/25,5/5,25/75,/1; vol/vol) with swim-up sperm suspensions. Setting: In vitro fertilization and general infertility lini and laboratories. Patients, Partiipants: Women partiipants of the gamete intrafallopian transfer program (motility study, n = 2; arosomal reation study, n = 14). Sperm donors of the artifiial insemination program and men with given sperm parameters. Interventions: Hormonal stimulation. Laparosopy. Main Outome Measures: Progressive veloity and perentage of motile gametes measured with multiple-exposure photography. Arosomal reativity measured by an immunofluoresent tehnique. Results: Folliular fluid always influened progressive motility and also sustained the number of motile gametes, as funtion of time, better than PF or the PF /FF mixtures (P <.5). The arosomal reativity of sperm inubated in the various PF /FF ombinations was low; after 5 hours only the FF -sperm suspensions showed a signifiant enhanement of arosomally reated gametes. Conlusion: At ovulation, FF transmits positive (motility- and arosomal reativity-enhaning) signals to sperm, whereas PF may transmit positive, neutral, or negative signals (noise signals). The volumetri ombination of FF and PF in the tubal environment, whih may differ from yle to yle and from woman to woman, ould therefore result in synergi (or antagonisti) effets on the sperm fertility potential. Fertil Steril 1992;57:654-6 Key Words: Peritoneal fluid, folliular fluid, human spermatozoa, sperm motility, arosome reation Reeived May 17, 1991; revised and aepted November 2, * Supported by grants / from the Swiss National Foundation for Sientifi Researh, Bern, Switzerland, and by grant TO 114/1-2 from the Deutshe Forshungsgemeinshaft, Bonn, Germany. t Andrology Laboratory, Gyneologial Endorinology Unit, "La Carita" Hospital. :j: Institute of Gyneology and Obstetris, Chair D, Mauriziano "Umberto I" Hospital, University of Turin. Department of Dermatology, Andrology Unit, University of Munih. II Reprint requests: Maro Balerna, Ph.D., Andrology Laboratory, Gyneologial Endorinology Unit, "La Carita" Hospital, 66 Loarno, Switzerland. 654 Revelli et al. PF/FF mixtures and sperm motility An inreasing bulk of evidene indiates that the fluids sereted by the female genital trat may influene spermatozoa during the migration toward the ooyte and the subsequent interations between the gametes. Beause they an be easily obtained during assisted reprodution proedures, peritoneal fluids (PFs) and folliular fluids (FFs) have been the most studied in the last years (1-4). However, despite the growing interest on the interation of these fluids with sperm, the mehanisms and the physiologial impliations involved are still poorly understood. To further ompliate matters, the in vitro studies made hitherto have produed

2 heterogeneous and often onfliting results. Some investigators observed, e.g., that PF an adversely affet sperm motility (5, 6), whereas others did not find any toxi effet of this fluid on spermatozoa (7). Moreover, although the inorporation of FF in assisted reprodution protools has laimed to improve the linial results (8, 9), the in vitro studies exploring the effet of FF on spermatozoa have generated onfliting reports. Some authors observed inhibitory effets on FF on sperm motility (1, 11), whereas others reported stimulating effets on both sperm motility and arosomal reation (12-15). These partly ontraditory findings are probably explained by the biologial variability of sperm, PF, and FF and by the different experimental approahes employed to test the interations between spermatozoa and these fluids. Using objetive motility measurements, we previously reported on the in vitro effet of 8% PFand 8% FF -ontaining buffer on sperm motility (2, 12). In those studies, we observed that although FF generally indued a positive effet on sperm veloity, PF was able to maintain, inrease, or inhibit sperm motility as funtion of time. Beause FF mixes with PF at ovulation and beause this mixture may onstitute the final fertilization milieu or ontribute to it, we deided to study in vitro the effets indued by different volumetri mixtures of PFs and FFs on both sperm motility and arosome reation. The data presented herein show that, depending on their respetive onentration in the final inubation milieu, PFs and FFs from a given woman indue harateristi effets on the motility parameters and on the arosomal reation of a partiular sperm sample. Patients MATERIALS AND METHODS Twenty women with a mean age of 34.8 ± 3.7 years (range: 3 to 42) and omplaining of infertility lasting from 5 ± 2.3 years (range: 1 to 1) were entered in this study. They were lassified in our gamete intrafallopian transfer (GIFT) program as male-related infertility (n = 11), endometriosis (n = 3), or unexplained infertility ases (n = 6). Superovulation was indued with the administration of daily intramusular injetions of 15 U of human menopausal gonadotropin (hmg, Pergonal 75; Serono, Aubonne, Switzerland) starting from day 3 of the yle. From day 7, folliular growth was monitored daily with ultrasound measurements and serum estradiol determinations. On the day the leading follile reahed 18 mm, hmg injetions were stopped, and 1, U of human horioni gonadotrophin (hcg, Profasi HP; Serono) were administered. Laparosopy was sheduled 36 hours after hcg injetion. Peritoneal fluids and FFs were obtained during laparosopy, proessed, and stored as previously desribed (2, 12). Sperm Preparation Semen samples reeived in our enter for routine semen analysis or donors partiipating in our artifiial insemination by donor program were used in this study. The ejaulates, obtained after 3 to 5 days of abstinene, were allowed to liquefy for about 3 minutes in sterile glass ontainers at room temperature (RT). Only sperm samples having the following harateristis were employed: >7 X 1 6 sperm/ml; >4% progressive motility at 3 minutes, and >3% normal forms (16). Motile sperm were olleted in B 2-Mtmezo ulture medium (Bio Merieux, Mary L'Etoile, Frane) using the swimup tehnique without prior sperm washing (1 hour, 37 C in a 5% CO 2 atmosphere). If neessary, the sperm onentration was adjusted to 15 X 1 6 sperm/ml by entrifugation at 6 X g for 1 minutes at RT and aspiration of enough medium to obtain the desired onentration. In Vitro Interation of Sperm and Volumetri Mixtures of PF IFF Twenty-five miroliters of the motile-rih sperm suspension were mixed in a sterile Eppendorf tube (Sarsted, Sevelen, Switzerland) with 1 JlL of B2- Menezo medium (ontrol) or with 1 JlL ofthe orresponding PF /FF mixture (PF /FF volumetri ratios: 1/, 75/25, 5/5, 25/75, /1; this gives perent onentrations of PF /FF = 8%/%, 6%/ 2%,4%/4%,2%/6%, and %/8% in the final sperm-fluid suspensions). Three objetive assessments of progressive motility (veloity straight linear [VSL]) were performed for eah PF/FF-sperm suspension by means of the multiple-exposure photography tehnique at time (t) =, 2.5, and 5 hours after mixing as previously desribed (2). Aliquots for the motility assessments were taken from the same sperm suspension at the designated times. During the entire duration of the test, the suspensions were kept in an inubator at 37 C and 5% CO 2 Arosome Reation Assessment For 11 semen samples, aliquots of the ontroland PF /FF -sperm suspensions were obtained at the Revelli et al. PFjFF mixtures and sperm motility 655

3 A B Progresslll9 I19loi ty (um/s) 36 Motile sperm ( % ) 9 t t 2.5 hours t 5 hours t t 2.5 hours t 5 hours th perentile t t 3 75 ntt t tt t 5th perentile 28 7 ~t ( median) th perentile 24 6 ttrf tit t 22 55, , , , ContrOl medium; 2-6 PF/FF mixtures 1 Control medium; 2-6 PF/FF mixtures Figure 1 Effet of ontrol medium (1), (2), three PF/FF volumetri mixtures (3 to 5) and (6) on the progressive motility (A) and perent of motile sperm (B) of 2 sperm samples. Spermatozoa obtained by swim-up were inubated as desribed under Materials and Methods. The plots show the quartiles (diagram) of the 2 progressive veloities (in!lm/s, A) and of the 2 perentages of motile gametes (B) as funtion oftime and of the inubation medium. Only sperm inubated with FF had higher veloities at all times (bar 6 versus other bars in A; P <.5). The PF/FF mixtures indued higher perentages of motile ells with respet to the ontrol medium (ompare bars 2 to 6 with. bar 1 at all times in B; P <.5). A progressive redution of motile spermatozoa as funtion of time was observed in all inubations exept for the inubation with 8% FF (B, bar 6). t ri designated inubation periods, and smears were prepared for the arosome reation assessment. The smears were stained for the presene/absene of their outer arosomal membrane with a polylonal antibody raised in rabbits against boar spermatozoa and ross-reating with human outer arosomal antigens (17). Briefly, the smears were allowed to air dry at RT for 3 minutes, fixed in methanol, and inubated for 3 minutes in phosphate-buffered saline (PBS) supplemented with.5% bovine serum albumin (ph 7.5). The smears were then exposed to 5 ML of a 1:1 dilution of rabbit anti-outer arosomal antigen serum in PBS and inubated for 1 hour in a moist hamber at 37 C. After three washings with PBS (3 times for 1 minutes), 8 ML of fluoresent goat anti-rabbit immunoglobulins (diluted 1:8 in PBS) were pipetted onto the smears. These were again plaed in a moist hamber and inubated for 1 hour at 37 C. The smears were then washed (3 X 1 minutes) in PBS and mounted with a 1:9 glyerol/pbs solution. Using a fluoresent mirosope (Zeiss, Jena, Germany), 1 spermatozoa were lassified as having reated (absene of fluoresene in the arosomal area) or non-reated (fluoresent arosomal area). The effiieny of this immunofluoresent staining tehnique was ontrolled by performing the same proedure on spermatozoa inubated in B 2- Menezo and exposed to a freezethaw yle. Using this proedure, at least 6% of the spermatozoa were found to have lost their outer arosomal membrane. Statistial Analysis The normal distribution of the data was heked by the Lilliefors test at the 5% level (18). In view of the skewness of the distributions, between -group omparisons were performed by the Kruskal-Wallis (unpaired samples) or the Friedman test (paired samples). The two-sample Komolgorov-Smirnov test was used to ompare sperm veloity distribution lasses. RESULTS The effets of a sperm survival medium or of different volumetri ratios of PF and FF on the medians of the progressive sperm veloities and on the perentage of motile sperm as funtion of time are shown in Figure la and B. As asertained by the Kruskal-Wallis statisti, spermatozoa inubated with 8% FF showed signifiantly higher median veloities with respet to all other inubations at t = (Fig. la, olumn 6; n = 2). Interestingly, at longer inubation times, an inrease in the volu- 656 Revelli et al. PF/FF mixtures and sperm motility

4 metri ratio of FF in the PF IFF mixtures was not aompanied by a onomitant inrease of veloity. However, and in all instanes, the effet of 8% FF was found to be higher than that indued by all other PF IFF ombinations (these effets were signifiant for all mixtures exept for the 6% 12% PF IFF mixtures at 2.5 and 5 hours; Figure la, olumns 3 and 6; Kruskal-Wallis test). Furthermore, the progressive veloity showed a redution at t = 2.5 hours in all PF IFF mixtures, followed by an inrease at t = 5 hours. Conerning the perentage of motile ells, a signifiant differene was observed between the ontrols and all PF IFF mixtures (Figure 1B; ompare bar 1 with bars 2 to 6 at all times). With the. exeption of the inubations with 8% FF, the perentage of motile ells progressively delined in all PF IFF mixtures as funtion of time. Considering the effets after 5 hours of inubation of PF IFF mixtures on the median of progressive veloity of eah of the 2 semen samples tested, we observed the following patterns: (1) 1 ases in whih the minimal stimulation was observed with 8% PF and 4%/4% PF IFF mixtures (a typial ase is given in Fig. 2A); (2) 7 ases with a single minimum of the median of sperm veloity in the 4%/4% PF/FF inubation mixture (a typial ase is illustrated in Fig. 2B); (3) 2 ases in whih there was a progressive inrease of the motility from 8% PF to 8% FF (not shown); and (4) 1 ase in whih all PF IFF mixtures exerted similar effets (not shown). However, onerning patterns 1 and 2 above, no signifiant differenes ould be asertained by applying the Friedman test to the progressive motility distributions of the different mixtures. Furthermore, these patterns were apparently neither related to the type of semen used (beause the same sperm was able to show different patterns when inubated with the fluids from another woman; data not shown) nor to the type of pathology (e.g., endometriosis or unexplained infertility) affeting the women from whom PF and FF were obtained (the low number of pathologial ases studied does not, however, permit a definite statement). As far as the arosomal reation is onerned, only a low perentage of spermatozoa was found to have reated even after 5 hours of inubation (Fig. 3). The perentage of spontaneous arosomal loss (ontrol medium inubations: bar 1 in Fig. 3) was found to be signifiantly lower than that of the 8% FF inubations at all times (row 6 in Fig. 3). In ontrast, the reativity indued by 8% FF was sig- U 31 Q) '-. E ;: J [/) G :s D 29 E Z' 27 Qj > E Q; 26 Q ' '" : 24 U 28 Q) "- -.J [/) 2: 26 Q "'5 D 25.;::: U; Z' Qj 23 > E Q; Q 22 (fj ' D '" Q) :2 A ', Inubation medium ( 1 = ontrol: 2-6 = PF/FF mixtures) 21 2 B Inubation medium ( 1 = ontrol: 2-6 = PF/FF mixtures) Figure 2 Effets of ulture medium (ontrol) or PF IFF mixtures prepared with the fluids of two women on the progressive motility of the sperm of two men. The single points are the medians of the sperm veloity distributions as registered after 5 hours of in vitro inubation in the respetive media. Patterns of this type were observed in 1 (pattern a) and 7 (pattern b) ases, respetively. nifiantly higher than those indued by 8% PF and by the PF IFF mixtures only at t = 5 hours. DISCUSSION Along their way to the fertilization site, spermatozoa enounter various fluids sereted by the female genital trat. In vitro studies performed during the Revelli et al. PF/FF mixtures and sperm motility 657

5 Arosomal reallons ( % ) 14r r ~ _, t " t"25hours t"5hours 12 * U-~~~-L4-~~~-L-L~~1~1~_L_W " Control medium; 2-6 ; PF/FF mixtures Figure 3 Perentage of arosomally reated spermatozoa in B 2-Mimezo medium (ontrol) and in the various PF/FF mixtures as funtion of time. The bars represent the values of the quartiles for the 11 data points (see diagram in Fig. 1). The rate of arosomal loss in tlte ontrol medium was lower than that in the 8% FF inubations at all times (P <.5). In ontrast, the reativity indued by 8% FF was higher than that indued by 8% PF and the PF/FF mixtures only at t = 5 hours (P <.5). last few years, in partiular with human PF and FF, have shown that suh fluids are probably involved in the modulation of some fundamental sperm funtion, suh as motility (2, 12, 15) and arosome reation (19, 2). However, despite the potential impliations of suh studies toward the understanding of some of the basi mehanisms of ouple infertility (or of natural ontraeption), our knowledge of the effets indued by these fluids on sperm is still limited. Three main fators may explain this situation: (1) the in vitro experiments performed so far often involved different experimental approahes (this issue was partiularly disussed in two previous papers [2, 12]); (2) the intrinsi intrasample and intersampie biologial variability of sperm and female genital trat fluids; and (3) the lak of efforts made to approah in vitro the omplex situation found in vivo, where FF, PF, and the fluid sereted by the fallopian tubes probably mix after the rupture of the follile, forming what ould be defined as the fertilization milieu at the fertilization site. In the present study, we tried to ope with at least some of these diffiulties. Our approah inluded: (1) a mild proedure to separate sperm from seminal plasma after its fluidifiation; (2) a paired exposure of a given sperm to five volumetri ratios of PF and FF from the same hormonally stimulated woman; and (3) a kineti analysis of the effets indued by these fluids on sperm motility and arosome reation up to 5 hours of inubation. The exposure of spermatozoa to 8% PF, 8% FF, and to three other volumetri ombinations of these fluids (PF/FF ratios = 2%/6%, 4%/4%, and 6%/2%, final onentrations) was performed beause one does not know a priori where fertilization might take plae and to what kind of medium the gametes are exposed when they meet and begin to fuse. As already observed by other workers (13-15) and by ourselves (12), inubation up to 5 hours of sperm with 8% FF never resulted in a loss of sperm veloity or in a derease in the perentage of motile spermatozoa as funtion of time. Furthermore, as previously reported (12), a positive effet on sperm motility was noted in many ases almost immediately after mixing sperm with FF, indiating that FF ontains motility-promoting fators able to rapidly interat with spermatozoa. Two reent reports have pointed to the fat that FFs, espeially those obtained from stimulated yles (21), ontain high onentrations of agents (in partiular steroids) able to promote sperm motility (13). The influene of these substanes ould also help to explain linial observations, suh as the improvement in the suess rate observed in intrauterine insemination and G 1FT proedures by treatment of spermatozoa with FF (8, 9) (see, however, Tuker et al. [22]). In ontrast, as far as PF is onerned, some workers reported inhibitory effets on sperm motility (5, 6), whereas others did not (7). A previous study by our group found that approximately only one third of the PFs from infertile subjets was able to inrease the veloity distribution and the proportion of motile sperm after 5 hours of inubation, and that, furthermore, PFs from different women affeted a given sperm in a different (and peuliar) way (2). In the present study, when ompared with the ontrol medium, PF did not indue a generalized ativating effet on sperm veloity. Although in some ases a limited effet in this diretion was noted, it appeared only after at least 2.5 hours of inubation. Moreover, PF did not preserve the proportion of motile spermatozoa as a funtion of time. We, therefore, hypothesize that PF laks or ontains an insuffiient amount of substanes able to ativate and/or sustain 658 Revelli et al. PFIFF mixtures and sperm motility

6 sperm motility and that in some ases it might even ontain sperm-immobilizing fators. With the experimental data obtained in our previous studies, we expeted to observe a progressive, dose-dependent enhanement of sperm motility by adding inreasing amounts of FF to the final inubation mixture. Surprisingly, exept for two ases, this was not observed. The two major patterns that were found to relate sperm motility to the relative amount of PF and FF in the inubation medium (Fig. 2A and B) indiated that PF alone (or then PF and PF/FF at about a 1:1 volumetri ratio) were able to redue the linear omponent of progressive motility and that what distinguished the two patterns was in fat the behavior of spermatozoa in pure PF. Beause no statistial differene was found to exist by examining the progressive veloity distributions in the different media for the 2 ases examined, this finding must be onsidered as a trend. Two hypotheses ould explain suh findings. First, PF alone or in ombination with inreasing amounts of FF ould have led to the formation of milieus able to affet sperm veloities by virtue of their partiular biohemial omposition (e.g., the level of free FF steroids [13] may have dereased as a result of binding with PF substanes). Alternatively, the observed patterns ould have been indued by a hange of the sperm motility type (transition from progressive to hyperativated motility). Beause the multiexposure photography tehnique used herein only allows estimation of the initial and final point of sperm displaement during 1 seond (VSL) and beause hyperativated motility is known to be aompanied by a derease of the linear omponent of motility (whiplash or figure-eight pattern [23, 24]), suh a hange would have esaped our observation and would have resulted in a derease of progressive veloity. In addition, the transition to a hyperativated state ould also help to explain the generalized derease of sperm veloities at t = 2.5 hours followed by an inrease at t = 5 hours. This ould have been beause of the reovery of the linear progressive omponent of motility. Only automated objetive sperm veloity assessments made with omputerized devies will provide further insight into this issue. Another important point in this study was the observation that 8% PF (and also the PF /FF mixtures) from various women affeted differently the motility of a given sperm, whereas the inubation with 8% FF always improved its motility. The results obtained with the 8% PF and 8% FF inubations onfirmed the results previously reported (2,12), whereas the behavior of the PF /FF mixtures was rather unexpeted. Overall, the results obtained in this and in the previous studies seem to indiate that, at ovulation, it is the FF that transmits positive (motility-enhaning) biohemial signals to spermatozoa (13), whereas the PF may transmit positive, neutral, or negative signals (noise signals). The omposition of FF and PF, as well as their volumetri ombination in the tubal environment, whih may differ from yle to yle, from woman to woman, and also be dependent on the presene of a given pathology (e.g., endometriosis, unexplained infertility ases), ould result in synergi or then antagonisti effets in terms of sperm motility. To further ompliate matters, these effets ould also be sperm-dependent. The synergi ombination of two positive signals (from PF and FF) may indue a maximal stimulation of sperm motility. In other ases, the two signals may antagonize or even result in a negative effet (the role of PF in endometriosis and/or unexplained infertility ases remains to be eluidated). However, as disussed above, suh a stimulation would not neessarily be refleted in the enhanement of the linear (progressive) omponent of sperm motility but ould failitate spermatozoa in reahing the hyperativated state. Further studies are needed to onfirm this hypothesis. Conerning the extent of arosomal reations, and in agreement with other reports (1, 14, 19, 2), an onset of the arosomal reation was observed uniquely after 5 hours of inubation and only in those ases in whih sperm were inubated with the highest onentration of FF (8%). It appears, therefore, that only FF ontains a suffiient onentration of the fators able to rapidly indue an arosomal reation. At the same time, our results onfirm the findings of other workers (1, 19), proposing long inubation times to obtain high perentages of arosomally reated sperm. Aknowledgment. The authors gratefully aknowledge the skillful help of the laboratory staff in Loarno. REFERENCES 1. Syrop CH, Halme J. 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