Seminal leukocytes and clinical outcomes with donor sperm insemination

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1 Seminal leukocytes and clinical outcomes with donor sperm insemination Virginie Barraud-Lange, M.D., Ph.D., a,b Jean-Christophe Pont, Pharm.D., a Khaled Pocate, M.D., a Jean Marie Kunstmann, M.D., a Celine Chalas-Boissonas, M.D., Ph.D., a Beatrice Ducot, M.D., c and Jean Philippe Wolf, M.D., Ph.D. a,b a Service d Histologie-Embryologie-Biologie de la Reproduction, CECOS, H^opital Cochin, AP-HP, Universite Paris Descartes, Paris; b Inserm 1016, Physiology and Physiopathology of Reproduction, Paris; and c Inserm, CESP Center for Research in Epidemiology and Population Health, U1018, Epidemiology of Reproduction and Child Development Team, F-94276, Universite Paris Sud 11, Le Kremlin-Bic^etre, France Objective: To report the level of leukocytospermia in fertile donors semen. Surprisingly, seminal leukocytes protect fertilization properties of sperm and are associated with normal or improved assisted reproductive technology outcomes in infertility patients. This raises the question of whether leukocytospermia exists in fertile men as well. We report a study of sperm donors who, by law in France, have to be of proven fertility. Design: Retrospective analysis. Setting: University laboratory. Patient(s): One hundred fifty-five donors were selected for cryobanking. Results of their sperm analyses were compared with those from 10,242 infertile men. Intervention(s): None. Main Outcome Measure(s): The men s first ejaculate was studied by cytologic analysis to determine the round cell and polymorphonuclear cell (PMN) contents. A total of 3,875 donor sperm inseminations (DSIs) were performed, and their outcomes were analyzed over an 8-year period. Result(s): PMN is more elevated in semen from infertility patients than in semen from fertile donors, but some donors (6.5%) had high leukocytospermia (R10 6 /ml). The post-dsi pregnancy rate was increased when round cells were present (P<.02) but not with higher PMN concentrations. Furthermore, high leukocytospermia was associated with an increased post-dsi miscarriage rate. Conclusion(s): In fertile donors, as in infertility patients, high leukocytospermia (>10 6 /ml) is associated with a normal pregnancy rate but an increased percentage of early pregnancy loss. (Fertil Steril Ò 2011;96: Ó2011 by American Society for Reproductive Medicine.) Key Words: Leukocytospermia, donor sperm insemination, IUI, sperm bank The significance of leukocytospermia and its relevance to male infertility are still debatable. We have already reported that for an infertility patient, moderate leukocytospermia is associated with better sperm parameters, assisted reproduction technologies (ART) outcome, and live birth rate. We also showed that even in the presence of leukocytospermia >10 6 /ml, and contrary to what was expected, leukocytes protect sperm function and are associated with an increased clinical pregnancy rate in ART compared with rates for infertility patients without leukocytospermia (1). We have also showed that moderate leukospermia appears to be physiologic and constitutes a favorable factor for sperm quality and functions (2), in contrast to high leukocytospermia, which is linked to prostatitis. In addition, elevated leukocytospermia does not hinder any sperm fertilization functions (1). This was not anticipated and raises several questions: What should the normal Received May 11, 2011; revised July 15, 2011; accepted August 16, 2011; published online October 7, V.B.-L. has nothing to disclose. J.-C.P. has nothing to disclose. K.P. has nothing to disclose. J.M.K. has nothing to disclose. C.C.-B. has nothing to disclose. B.D. has nothing to disclose. J.P.W. has nothing to disclose. Reprint requests: Jean Philippe Wolf, M.D., Ph.D., Service d Histologie-Embryologie-Biologie de la Reproduction, H^opital Cochin, AP-HP, Universite Paris Descartes, 123, Bd. Port Royal Paris, Universite Paris Descartes, France ( jean-philippe. wolf@cch.aphp.fr). level of seminal leukocytes be considered to be? What is the level of leukospermia in normal fertile men? Can a man be fertile despite high leukocytospermia? It is difficult to answer such questions, because fertile men do not come to the infertility clinic except as sperm donors. By law in France, these men have to be of proven fertility (i.e., already fathers). Their sperm is analyzed before cryopreservation and used later in donor sperm insemination (DSI). The sperm can be used for inseminating several women until ten babies result from the same donor. Therefore, it is possible not only to analyze the semen leukocytes in their sperm, but it is also possible to correlate the level of seminal leukocytes of the ejaculate with the outcome of the associated DSI. In the present study, the total leukocyte content was assessed, and more specifically the polymorphonuclear leukocytes (PMNs) by cytologic analysis in the laboratory of the Centre pour l Etude et la Conservation des Ovocytes et du Sperme (CECOS; Center for the Study of Oocyte and Sperm Preservation) of Paris Cochin. POPULATION AND METHODS We analyzed the semen from donors who came to the sperm bank program in the CECOS in Cochin Hospital, Paris, France, over an 8-year period. Two hundred ninety-eight men were candidates for sperm donation, of whom only 155 were selected for the program. To be eligible, donors must have good sperm parameters and the sperm must have good survival rates after cryopreservation and thawing. In addition, the men should not have 1320 Fertility and Sterility â Vol. 96, No. 6, December /$36.00 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 TABLE 1 Comparison of semen characteristics between infertility patients and fertile sperm donors. Infertility patients Donors P value No. of couples 10, Sperm volume 3.5 (1.7) 3.9 (1.6).0009 Sperm count 65.4 (68.1) (69.1).0001 Total sperm number (232.4) (252.4).0001 Round cells 4.7 (7.6) 6.3 (12.3).01 PMN leukocytes 2.1 (16.5) 0.8 (4.9).31 Motility a 13.3 (8.9) 22.3 (10.6).0001 Motility b 19.2 (7.7) 24.9 (8.6).0001 Motility a þ b 32.5 (12.8) 47.1 (9.7).0001 Motility c 6.6 (3.3) 5.3 (1.6).0001 Vitality 63.2 (17.6) 67.2 (12.3).007 Normal forms 21.4 (13.9) 34.5 (14.4).0001 Note: Values are mean (SD). PMN ¼ polymorphonuclear. any genetic problems in their families or personally. Sperm parameters were assessed for each ejaculate, but sperm cytology and PMN determination were evaluated only for the first ejaculate provided by these donors (n ¼ 155), with the use of cytologic analysis (3). Seventy percent of infertility patients used donor sperm because of azoospermia (obstructive azoospermia 2.6%, nonobstructive 67.4%), 24.3% because of oligoasthenoteratospermia (14.2% severe oligospermia, 5.6% moderate oligospermia), and 4.5% because of asthenoteratospermia. Three percent cited familial genetic disorders and 2.3% had other reasons. When DSI is planned, straws are delivered to the center where the patients were examined. The biologist selects the most appropriate technique to prepare the sperm. If the sperm parameters are very good, which is usually the case, a PureSperm gradient (90% 45%) is performed and the pellet is then washed with Ferticult (Fertipro). If the sperm parameters are not good enough, the straw contents are just washed with Ferticult medium before insemination. Eight hundred ninety-seven DSIs were performed with the first ejaculates of the donors. The total number of DSIs performed between 2000 and 2008 was 3,875. Round cell counts were determined on every ejaculate. The results from donor ejaculates were compared with semen analyses performed for 10,242 male partners of couples attending the Cochin infertility clinic for initial investigation and for whom leukocytospermia was analyzed using the same techniques for cytologic determination used for donors. Among the male partners, 265 had normal semen parameters according to the 1993 World Health Organization (WHO) criteria (3) used for this study; 826 azoospermic patients were not included in the study. Sperm Analysis Sperm analyses were performed according to WHO 1993 guidelines (3). Semen was collected by masturbation within the laboratory facilities after 3 5 days of sexual abstinence and allowed to liquefy for half an hour. Sperm counts were then determined with the use of a Malassez slide by counting the number of spermatozoa present in the two grids or a minimum of 200 spermatozoa in each. Double determinations were performed and were considered to be satisfactory when the difference between the two chambers was <10%. Otherwise, the determination was repeated. Final concentration was obtained by multiplying the result according to the dilution of the sperm FIGURE 1 Distribution of the ratio of round cells (RC) to sperm count (Spz) in (A) patients (n ¼ 277) and (B) fertile sperm donors (n ¼ 10,242). The y axis is limited to 30 in both figures. Fertility and Sterility â 1321

3 TABLE 2 Pregnancy rate and outcomes after donor sperm insemination (DSI) according to the concentration of round cells in the semen. Round cells 0 <10 6 R10 6 P value Total DSI (n) 444 1,980 1,451 3,875 Pregnancy, n (%) 55 (12.4) 357 (18.0) 244 (16.8) < (16.9) Deliveries, n (%) 44 (80.0) 289 (81.4) 200 (83.0) (81.9) Abortion, n (%) 10 (18.2) 55 (15.5) 36 (14.9) (15.5) Ectopic, n (%) 1 (1.8) 9 (2.5) 2 (0.8).3 12 (1.8) Therapeutic termination, n (%) 0 2 (0.6) 3 (1.2).5 5 (0.8) Total pregnancy outcome, n suspension. All round cells present within the two chambers of the Malassez slide were counted, and the concentration was determined according to the suspension dilution. Sperm motility was assessed by examination of a 20- ml drop of homogenized sperm suspension under a cover slip at 37 Cat a high power field. Sperm morphology was determined by analysis of a Schorr-stained smear from the first ejaculate only. A minimum of 100 spermatozoa were analyzed for the determination. PMN Determination PMNs were determined according to the Schorr technique (3). Briefly, two smears were done using 20-mL drops of semen. They were air-dried, fixed with alcohol, stained with Harris dye (hematoxylin) and with the Schorr dye (Merck), and analyzed under a bright-field microscope using a 100 immersion optic (1,000 magnification). One hundred spermatozoa were counted, and the PMNs were evaluated as a percentage of the sperm cells. The final PMN concentration was obtained by multiplying the sperm concentration by this percentage. Statistical Analysis Statistical analysis was done using analysis of variance, chi-square test, or Fisher exact test. To take into account the role of several variables on fertilization rates and pregnancy and child birth outcomes, logistic regressions were performed and included ejaculate as a random-effect variable and women s age and the year of the DSI as covariables. RESULTS Sperm parameters of infertility patients and fertile donors are compared in Table 1. Fertile donors had a higher mean number of round cells in their semen than infertility patients ( vs , respectively; P<.004), but the ratio of round cells to sperm count was >5% in 29.4% of infertility patients compared with 5.2% of fertile sperm donors (P<.001; Fig. 1). The PMN concentration was >10 6 /ml in 14.1% and 6.5% of infertility patients and fertile sperm donors, respectively (P<.0001). Therefore, although leukocytospermia is higher in infertility patients than in fertile sperm donors, some fertile donors also have significant levels of leukocytospermia. Among 656 pregnancies that were obtained by DSI, only 651 outcomes were known (Table 2). The pregnancy rate rose from 12.4% when the donor sperm contained no round cells, to 18.0% and 16.8% with increasing concentrations of round cells, from <10 6 /ml to >10 6 /ml, respectively (P<.02). There were 533 deliveries with a delivery rate over to 80% regardless of the level of round cells (Table 2). Rates of miscarriage, extrauterine pregnancy, and therapeutic termination did not differ between the three groups (no round cells, <10 6 /ml, or >10 6 /ml; Table 2). Using a logistic regression analysis and taking into account the age of the women and the year of insemination, the chance of having a baby was multiplied by 1.41 (P¼.05) and 1.35 (P¼.10) when round cell concentrations were <10 6 /ml and >10 6 /ml, respectively, compared with no round cells. Considering the ratio of round TABLE 3 Pregnancy rate and pregnancy issues after donor sperm insemination (DSI) according to the concentration of polymorphonuclear (PMN) leukocytes in the first ejaculate from donors. PMN leukocytes 0 <10 6 /ml >10 6 /ml P value Total (%) DSI (n) Pregnancy, n (%) 131 (16.9) 4 (9.1) 11 (14.5) (16.3) Delivery, n (%) 108 (83.7) 4 (100) 7 (63.6).06 a 119 (82.6) Pregnancy loss, n (%) 17 (13.2) 0 (0) 4 (36.4) a (14.6) Ectopic pregnancy, n (%) 4 (3.1) 0 (0) 0 (0) 4 (2.8) Total pregnancy outcome, n a Comparison after merging of groups 0 and <10 6 /ml Barraud-Lange et al. Seminal PMN and donor sperm insemination Vol. 96, No. 6, December 2011

4 cells to sperm cells, the pregnancy rate rose from 12.4% to 17.5% and 17.1% (P<.02) for the group with no round cells to the group with a ratio <5% and the group with a ratio >5%, respectively. The early pregnancy loss rates were 18.2%, 14.9%, and 29.4%, respectively (P¼.2). The relationship between leukospermia and donor insemination outcomes was assessed with the first ejaculate provided by the donor (the only one for which PMN level was determined). Out of 897 associated DSIs, there were 146 pregnancies (16.3%; Table 3). No variation could be seen among the pregnancy rates between the different groups. But interestingly, there was a dramatic increase in the rate of pregnancy loss, from 13.2% for the group with no leukocytospermia to 36.4% when leukocytes were >10 6 /ml (P¼.07; Table 3) and a subsequent decrease in the delivery rate from 83.7% to 63.6%, respectively (P¼.06). These differences did not reach statistical significance, however, possibly because of the size the sample. The 76 DSIs performed in 55 recipients of this group were obtained from seven different donors, only two of them being associated with miscarriages in four women. Sperm parameters were compared according to the DSI outcome (Supplemental Table 1). Motility, vitality, and sperm morphology were significantly better in cases in which a pregnancy was obtained. However, logistic regression taking all theses items into account still showed a significant effect of the PMN concentration (Supplemental Table 1). DISCUSSION This is the first time that donor sperm quality has been related to the pregnancy outcomes of DSI in a study involving >100 fertile donors and run over an 8-year period. For the first time, we can correlate semen parameters with the pregnancy outcomes of DSI. Evaluation of PMN levels was made through cytologic evaluation run by well trained technicians with multiple quality-control tests performed every year. Among the fertile donors, there was a correlation between round cells and sperm count (r ¼ 0.29), showing that round cell count is correlated with sperm production. But interestingly, in infertility patients, the lower the sperm count, the higher the ratio of round cells to sperm cells, suggesting a desquamation of the seminal epithelium in cases of low spermatogenesis. In these cases, there is also a higher ratio of round cells to sperm cells than in fertile donors, suggesting that this increase may be linked to the pathology underlying their infertility. Indeed, when the ratio of round cells to sperm count is >5%, there is an increase in the miscarriage rate among pregnancies resulting from DSI. But when the sperm contains no round cells, significantly fewer clinical pregnancies result, suggesting that the optimum ratio of round cells to sperm cells would be between >0% and 5%. It appears that having some leukocytes is favorable for the fertilization ability of sperm (2), but a concentration >10 6 /ml could be detrimental for continued pregnancy (1). Some fertile sperm donors also have leukocytes in their semen and even elevated leukocytospermia >10 6 /ml. The leukospermia does not influence the pregnancy rate following DSI, but as in infertility patients, it is associated with increased pregnancy loss. Because the same donors sperm was used for different women with normal fertility, the male factor appears to be determinant in the early pregnancy loss, and the association with elevated leukocytospermia is reinforced for the DSI outcome. This increase in pregnancy loss is not statistically significant, however, because of the small sample. This is similar to what was found in infertility patients having in vitro fertilization (IVF) or intracytoplasmic sperm injection procedures to achieve pregnancy, except that in in vitro ART, leukocytospermia favored the clinical pregnancy rate (1). The relationship between PMNs and miscarriage may be due to impairment of DNA structure. Leukocytospermia is associated with increase reactive oxygen species (ROS) production by human spermatozoa (4) and an increase in DNA damage. Such stimulation of ROS production may be mediated via direct cell-cell contact or by soluble products released by leukocytes (4, 5). Similarly, a trend toward an increased miscarriage rate with elevated DNA fragmentation index has been reported with decreased IVF embryo transfer outcomes (6, 7). However, leukocytospermia has been reported to have at most a very weak effect on DNA integrity (8). DNA fragmentation is strongly positively correlated with intrinsic ROS production, whereas this correlation is weaker for extrinsic ROS production (9). Therefore, no correlation appears to exist between DNA fragmentation and the number of PMNs in semen. This suggests that the increased miscarriage rate may not be correlated to leukocytospermia per se. To answer the questions that were asked in the introduction, we conclude that the presence of round cells and leukocytospermia is normal in the semen as long as the ratio of round cells to sperm is <5%. In normal fertile donors, as in infertility patients, leukocytes protect fertilization ability of sperm, but high concentrations >10 6 /ml are associated with an increased rate of early pregnancy loss. However, they may not be directly responsible for it, as suggested by Henkel and Tomlinson (9, 10). Finally, a man can be fertile and have leukocytospermia, but it does not mean that fertility will remain if the reasons for the high leukocytospermia persist. Should leukocytospermia be treated? First, the level of spontaneous variation of leukocytospermia is very important, as seen in examinations performed 3 months apart (11). To answer this question, a therapeutic trial has to be run using antiinflammatory drugs against a placebo to avoid any bias. Such a study is currently ongoing in our department (Clinical Trial. Protocol ID: P081215). REFERENCES 1. Barraud-Lange V, Pont JC, Pocate K, Sifer C, Cedrin- Durnerin I, Fechtali B, et al. Seminal leukocytes are Good Samaritans for spermatozoa. Fertil Steril 2011;96: Ziyyat A, Barraud-Lange V, Sifer C, Ducot B, Wolf JP, Soufir JC. Paradoxical increase of sperm motility and seminal carnitine associated with moderate leukocytospermia in infertile patients. Fertil Steril 2008;90: World Health Organization. Schorr staining for sperm morphology evaluation. In: Auger J, Jouannet P, editors. WHO laboratory manual for the examination of human semen and sperm cervical mucus. 3rd ed. Paris: Edition Inserm; p Saleh RA, Agarwal A, Kandirali E, Sharma RK, Thomas AJ, Nada EA, et al. Leukocytospermia is associated with increased reactive oxygen species production by human spermatozoa. Fertil Steril 2002;78: Erenpreiss J, Hlevicka S, Zalkalns J, Erenpreisa J. Effect of leukocytospermia on sperm DNA integrity: a negative effect in abnormal semen samples. J Androl 2002;23: Lin MH, Kuo-Kuang LR, Li SH, Lu CH, Sun FJ, Hwu YM. Sperm chromatin structure assay parameters are not related to fertilization rates, embryo quality, and pregnancy rates in in vitro fertilization and intracytoplasmic sperm injection, but might be related to spontaneous abortion rates. Fertil Steril 2008;90: Frydman N, Prisant N, Hesters L, Frydman R, Tachdjian G, Cohen-Bacrie P, et al. Adequate ovarian follicular status does not prevent the decrease in pregnancy rates associated with high sperm DNA fragmentation. Fertil Steril 2008;89:92 7. Fertility and Sterility â 1323

5 8. Moskovtsev SI, Willis J, White J, Mullen JB. Leukocytospermia: relationship to sperm deoxyribonucleic acid integrity in patients evaluated for male factor infertility. Fertil Steril 2007;88: Henkel R, Kierspel E, Stalf T, Mehnert C, Menkveld R, Tinneberg HR, et al. Effect of reactive oxygen species produced by spermatozoa and leukocytes on sperm functions in nonleukocytospermic patients. Fertil Steril 2005;83: Tomlinson MJ, Barratt CL, Cooke ID. Prospective study of leukocytes and leukocyte subpopulations in semen suggests they are not a cause of male infertility. Fertil Steril 1993;60: Lackner JE, Lakovic E, Waldh or T, Schatzl G, Marberger M. Spontaneous variation of leukocytospermia in asymptomatic infertile males. Fertil Steril 2008;90: Barraud-Lange et al. Seminal PMN and donor sperm insemination Vol. 96, No. 6, December 2011

6 SUPPLEMENTAL TABLE 1 Comparison of sperm characteristics between success (pregnancy) and failure (no pregnancy) after donor sperm insemination (DSI). Sperm characteristic No pregnancy a Pregnancy a P value b P value c n 3, Sperm volume 4.3 (1.5) 4.2 (1.5) Sperm count (66.7) (64.1) Total sperm no (275.2) (291.2) Motility a 22.3 (10.2) 23.0 (10.0) Motility b 24.3 (7.9) 24.9 (8.4) Motility a þ b 46.6 (8.7) 47.9 (8.8) Motility c 5.2 (1.3) 5.0 (0.7) Vitality 67.2 (11.5) 68.5 (11.1) Normal forms 35.5 (14.4) 37.2 (13.7) a Values are mean (SD). b Comparison between pregnancy and no-pregnancy groups. c Test of relationship between round cells in the semen and success of DSI, adjusted for sperm characteristics. Fertility and Sterility â 1324.e1