FERTILITY AND STERILITY Vol. 55, No.6, June 1991

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1 Assisted technology roductive FERTILITY AND STERILITY Vol. 55, No.6, June 1991 Copyright ~ 1991 The American Fertility Society Printed on acid-free paper in U.S.A. Possible role of pure human follicle-stimulating hormone in the treatment of severe male-factor infertility by assisted reproduction: preliminary report* Anibal A. Acosta, M.D. t Sergio Oehninger, M.D. Hakan Ertunc, M.D. Christine Philput, Ph.D. The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia Objective: Experimental clinical trial assessing the potential of systemic follicle-stimulating hormone (FSH) to improve sperm fertilizing ability in in vitro fertilization (IVF). Design: Retrospective clinical evaluation of severe male factor patients failing fertilization in IVF or showing severe sperm defects. Setting: Academic tertiary clinical care unit. Patients, Participants: Fourteen patients (41 cycles) who failed IVF, 22 patients (32 cycles) with severe quantitative and qualitative semen abnormalities indicating poor fertilization. Interventions: Treatment: FSH 150 U 1M three times a week for 3 months. Main Outcome Measure(s): Pretreatment and post-treatment evaluation of endocrine profile, basic semen analysis, and fertilization and pregnancy rates. Hypothesis: FSH treatment may improve spermatogenesis quality by its multiple actions on the Sertoli-gamete cell compartment without interfering with testicular hormonogenic function. Results: No significant changes were observed in the endocrine profile or in semen parameters; individual cases showed improvements in sperm concentration and motility. Significant increase in fertilization rate of preovulatory oocytes was demonstrated; seven term pregnancies were achieved. Conclusions: A multicenter randomized, double-blind trial with crossover is needed to demonstrate the benefit of systemic FSH administration and if this effect is FSH exclusive. Fertil Steril 55:1150, 1991 Male-factor infertility patients with severe impairment of sperm parameters often do not achieve fertilization in the in vitro fertilization (IVF) system. Basic semen analysis, separation of the sperm motile fraction (swim-up), and tests of sperm function help clinicians to predict sperm inability to fertilize. 1 Different techniques of assisted fertilization (micromanipulation) have been proposed to overcome this Received October 1, 1990; revised and accepted February 15, * Supported in part by Serono Laboratories, Randolph, Mas sachusetts. t Reprint requests: Anibal A. Acosta, M.D., 825 Fairfax Avenue, Department of Obstetrics and Gynecology, Norfolk, Virginia problem, but the success rate is still very low, and they have a high degree of complexity.2 No efforts have been made in assisted reproduction to establish whether systemic treatment can improve sperm parameters, quantitatively or qualitatively, or sperm fertilizing performance in these severe male-factor infertility cases. Usually, conventional empirical treatments (antiestrogens [E], human menopausal gonadotropin [hmg], human chorionic gonadotropin [hcg], aromatase inhibitors) had been used clinically in many of these patients before their referral to assisted reproduction programs, in an attempt to improve testicular gametogenic function. The effect(s) on sperm, if any, have been quite variable and unpredictable. Properly controlled studies have failed to demonstrate real 1150 Acosta et al. FSH, IVF, male factor Fertility and Sterility

2 beneficial effects of these therapies in severely oligospermic men. 3,4 Most patients in this situation have a normal or slightly abnormal hypothalamic-pituitary-leydig cell axis as judged by basal serum levels of luteinizing hormone (LH), total testosterone (T), and T /LH ratio. 5 Spermatogenesis in the presence of normal T levels requires follicle-stimulating hormone (FSH) to reach full quantitative and qualitative development 6 and is totally related to the quality of the spermatogenic cell line and the Sertoli cell functions, most of which are FSH-dependent.7 Theoretically, treatment of this type of patients with pure FSH may provide adequate endocrine stimulation to the Sertoli cell to enhance its FSHdependent functions and to support spermatogenesis without interfering with Leydig cell physiology and without locally increasing E levels, which may have a deleterious effect on the initial stages of spermatogenesis. 8 Here, an experimental clinical trial was performed to obtain preliminary information on the effects of pure FSH treatment on endocrine and semen parameters and to assess the potential of this therapy to improve sperm fertilizing ability in assisted reproduction (IVF and embryo transfer [ET]). Two groups of severe male-factor patients were evaluated: patients with a history of failed fertilization in previous IVF attempts and cases with poor sperm parameters, indicating that fertilization would not occur. The results obtained need to be validated by a prospective, randomized, placebo-controlled, doubleblind trial with crossover. Whenever an expensive treatment modality like IVF and ET is used and only a small number of cases may be amenable to this kind of treatment, it is likely that only a multicentric, cooperative effort will allow performance of such an experimental design in an efficient manner and in a short time. This paper intends also to stimulate an interest in the subject to see if such a cooperative effort is possible. MATERIALS AND METHODS The Norfolk Program defines the male factor in infertility by the presence of any of these abnormalities: sperm density < 20 X 10 6 sperm/ml (severe at <5 X 10 6 /ml); progressive motility < 40% (severe at <10%); morphology of <14% norma! forms (severe at <4%), using strict criteria 9 ; total recovered motile fraction after swim-up of <10 X 10 6 sperm (severe at 1.5 X 10 6 ); adenosine triphosphate postswim-up < 40 pmol per 10 6 sperm, and sperm pen- etration assay (SPA) of <10% of the oocytes exposed. 1 The less severe parameters define a male factor in the clinical setting; the severe ones are those that have been demonstrated to cause problems in our embryology laboratory during IVF therapy, and therefore they are used to define the male factor in assisted reproduction. This study evolved in two phases. In the initial one, pure FSH systemic treatment (Metrodin; Serono Laboratories, Randolph, MA) was used in cases in which sperm were unable to fertilize normal preovulatory oocytes in previous IVF attempts (secondary treatment group). Fourteen patients and a total of 41 cycles were in this category. Patients attempted IVF again subsequently to FSH therapy, thereby allowing a controlled evaluation (before-after) of results. When the quantitative and qualitative semen criteria were established by critical assessment of that group of patients, the clinician was able to predict failure of fertilization; it was considered unethical at that point to put these patients through the IVF program only to demonstrate that fertilization would not occur. Thus, such patients were treated primarily with pure FSH, followed by IVF therapy (primary treatment group). Criteria used to put these patients on treatment and learned through the previous experience were: (1) a poor prognosis morphology pattern «4% normal forms) and severe impairment of the other parameters (i.e., sperm concentration < 5 X 10 6 /ml and/or motility < 10%); and (2) a consistent recovered motile fraction of <1.5 X 10 6, regardless of the other parameters. 1 Twenty-two patients and 32 treatment cycles were in this category. These two populations were retrospectively evaluated and compared in terms of basal endocrine values (LH by radioimmunoassay [RIA] and bioassay, prolactin [PRL], estradiol [E2], T, T/LH, and E2/ T ratios) and of basal semen analysis values (concentration, motility, and morphology), trying to determine if the two groups were similar. No significant differences among the two groups were found in any one of the parameters analyzed. The dose of pure FSH selected and used in this study was 150 IV delivered by intramuscular (1M) injection three times a week, for a period of at least 3 months. In the absence of clinical information about previous experience with the use of pure FSH in the treatment of male infertility, we selected a similar dose to that of hmg used in previous clinical trials.lo Patients included in the protocol had a careful review of all previous records including medical and Vol. 55, No.6, June 1991 Acosta et al. FSH, IVF, male factor 1151

3 surgical treatments and a complete physical examination. A battery of endocrine tests consisting of serum FSH-RIA (I-FSH) (FSH-Quant; Leeco Diagnostics, Inc., Southfield, MI), LH-RIA (I-LH) (LH-Quant; Leeco Diagnostics, Inc.), PRL-RIA (Diagnostic Products, Inc., Los Angeles, CA), T-RIA (ICN Biomedical Inc., Carson City, CA), and E2 by RIA (Pantex, Santa Monica, CA) was obtained before treatment started (baseline levels) and during the 2nd, 4th, and 8th weeks oftreatment. The intraassay and interassay coefficients of variation for each hormone were as follows: FSH, 4.8% and 9.0%; LH, 7.1% and 10.6%; T, 7.5% and 14.9%; E2, 5.8% and 5.7%; and PRL, 5.5% and 15.8%, respectively. Bioactive FSH (B-FSH) levels in serum samples were measured using the rat granulosa cell bioassayy The reference preparations (World Health Organization First International Reference Preparation 83/575 and Metrodin) were obtained from Serono Laboratories, Norwell, MA. For the FSH bioassay, granulosa cells obtained from immature diethylstilbestrol (DES)-treated, Sprague-Dawley rats were cultured in 16-mm 24-well culture plates. Each well contained 6 X 104 viable cells in 0.5 ml McCoy's 5a medium (Gibco, Grand Island, NY) supplemented with 2 mm/l glutamine, 100 V /ml penicillin, 100 JLg/mL streptomycin sulfate, 10-6 M methyltrienolone (New England Nuclear, Boston, MA), 10-6 M 19-0H-androstenedione (Sigma Chemical Co., St. Louis, MO), 10-7 M DES, mm I-methyl-3-isobutyl xanthine (Sigma), 1 ng/ml insulin (26.8 V /mg; Lilly Research Laboratories, Indianapolis, IN), and 30 ng/ml hcg (CR-121; 13,450 IV/mg; Center for Population Research, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD). Each sample was pretreated with polyethylene glycol and assayed in triplicate. Results were analyzed with a program that used a weighted logit-log regression analysis.11 Bioactive luteinizing hormone (B-LH) levels in serum samples were measured using a modification of the method described by Van Damme et al.i2 Follicle-stimulating hormone and LH bioactivities were run in a single assay. Radioimmunoassayjbioassay ratios were calculated, recognizing the limitations of its validity.13 All hormone determinations were done in pooled sera from three blood specimens taken 20 minutes apart. During treatment, blood was drawn on the days medication was not administered. Thirteen normal fertile men who had fathered a child within 2 years before this study (donors) were used as a control group for endocrine parameters. Semen samples were obtained simultaneously with the blood samples in most patients and in all of them before treatment was started and after its completion. Semen analysis included sperm density, motility, mean velocity, mean linearity, motility index, and total motile sperm fraction determined by computerized semen analysis (CellSoft; Labsoft Division ofcryo Resources, Ltd., New York, NY); volume, ph, and morphology evaluation were estimated by previously reported methods.14 The IVF techniques used in Norfolk have been already published in detail.i5 In summary, the stimulation protocol on the female partner consisted of administration of hmg and FSH alone or in combination, used in a step-down fashion. The patient's response was followed and monitored by daily serum E21evels and transvaginal pelvic ultrasound for follicle number and size. Evaluation of terminal E2 response patterns (indication of the degree of maturity of the follicles and a reflection of the adequacy of the terminal gonadotropin stimulation) was performed according to established guidelines. 16 An ovulatory-triggering dose of hcg, 10,000 IV, was given when the leading follicles reached 16 mm in diameter. Oocyte retrieval was performed 34 to 36 hours after administration of hcg using the transvaginal ultrasonically-guided technique. The oocytes obtained were evaluated and classified according to the Norfolk criteria,17 and insemination was performed at different intervals after retrieval according to guidelines published elsewhere.is To control for oocyte quality and maturational stage, only mature, preovulatory oocytes (metaphase I and II at aspiration) were considered in the study. The semen specimens delivered at the time of insemination were carefully evaluated and compared with the ones provided after 3 months of treatment with FSH. The sperm concentration at insemination was routinely increased in the male factor population from 5 X 10 4 sperm/ml per oocyte in 3 ml of insemination medium used when dealing with normal males to 1 X 105 to 5 X 105 sperm/ml per oocyte, the majority being inseminated with the latter concentration.19 When the number of sperm available was not enough to follow this protocol, the retrieved oocytes were put together in the well of a microtiter dish (0.3 to 1.0 ml) and inseminated with the entire number of sperm recovered after swimup separation. The inseminated oocytes were examined 16 hours thereafter, and if two pronuclei were not visualized, a new sperm sample was requested and reinsemination performed. Investigation of pronuclear for Acosta et al. FSH, IVF, male factor Fertility and Sterility

4 I!, i mation continued, and either delayed fertilization20 or lack of fertilization was diagnosed. If normal fertilization occurred, pre-embryos were transferred to the uterine cavity at 42 to 44 hours after insemination, usually between the two- and four-cell stages. Luteal phase of the stimulated cycles was routinely supplemented with progesterone (P). Twentyfive mg of P in oil were administered 1M starting on the day of transfer and continuing for 5 days. The dose was then increased to 50 mg/d until the period began or until hcg results were obtained. If the hcg determination was positive, P was continued for 7 to 10 days; after that, the patient received 17-hydroxy P caproate (Carter-Glogau Laboratory, Inc., Glendale, AZ) 250 mg/wk 1M; this was continued until the 14th week of pregnancy. The patient usually returned to her hometown either in the United States or abroad and continued follow-up with her private obstetrician in close communication with the Norfolk team. The outcome of pregnancy, (not pregnant, delivery, or miscarriage) was recorded and used for compilation of results. This research protocol and a consent form for the FSH treatment of the male factor were submitted and approved by the Institutional Review Board of the Eastern Virginia Medical School. An investigative new drug application (IND) was filed with the Food and Drug Administration for the use of pure FSH in the treatment of severe male-factor infertility. Statistical evaluation was performed using Student's t-test, paired t-test, Fisher's exact test, Duncan's multiple range test, and Pearson's correlation coefficient as appropriate. Results are presented as means ± SD. RESULTS Patients included in the protocol were classified according to basal serum FSH levels in three different categories: (1) normal basal FSH values (5 to 15 mlu /ml; within 3 SD of the mean for the control group)5; (2) mildly elevated basal FSH values (16 to 25 miu/ml); and (3) elevated basal FSH values (>25 miu/ml). Within each of these categories, the populations were subdivided into primary and secondary treatment subgroups, as previously described. The distribution of patients and cycles in each one of the groups and subgroups is shown in Table 1. The ovarian stimulation protocols used were a combination of FSH, hmg, two ampules FSH, four ampules FSH, six ampules FSH, and two ampules hmg. The great majority of the patients were Table 1 Groups and Subgroups According to Basal FSH Levels and Primary or Secondary Treatment No. of No. of No. of cycles No. of cycles Group patients cycles before FSH onfsh 5to 15 Primary Secondary to 25 Primary Secondary >25 Primary Total treated with the FSH, HMG protocol representing 20.9% of the primary subgroup, and 30.6% of the secondary subgroup in patient with normal basal FSH levels, 44.4% and 11.1%, respectively, in the primary and secondary subgroups in patients with moderately elevated gonadotropins, and 50% in the patients with elevated gonadotropins. The two FSH protocol was used in 11.2% ofthe primary group, in 16.1 % of the secondary group in patients with normal basal gonadotropin levels, and in 50% of patients with clearly elevated gonadotropins. A four FSH protocol was used in 3.2% of the patients in the primary subgroup and 9.6% of the patients in the secondary subgroup in the normal gonadotropin groups, and in 11 % of the patients in the secondary subgroup, patients with moderately elevated gonadotropins. The other modalities of stimulation were used in a very small number of patients. In our experience, all of these ovarian stimulation protocols have demonstrated provision of preovulatory oocytes with identical fertilization rate.21 The serum terminal E2 pattern distribution was similar in all the groups and subgroups with clear predominance of the A pattern (constant increase of E2 levels up to retrieval). Basal RIA LH levels showed a trend to increase as basal RIA FSH levels increased in the different groups (14.7 ± 9.8; 16.5 ± 6.7; 26.4 ± 9.05 mlu /ml), but this difference was not statistically significant. Prolactin did not show significant differences in the three categories of basal RIA FSH levels (7.2 ± 3.2; 8.3 ± 2.6; 6.3 ± 0.3 ng/ml). Basal RIA T levels increased also as RIA LH levels increased in groups 1,2, and 3 (5.5 ± 2.1; 6.5 ± 1.1; 7.0 ± 0.6 ng/ml) but did not differ significantly. Basal RIA E2 levels were similar!n the three groups (23.8 ± 14.9; 25.9 ± 20.8; 26.7 ± 10.2 pg/ml). Basal RIA T /LH ratios decreased as basal RIA FSH levels increased in the three main groups as an Vol. 55, No.6, June 1991 Acosta et al. FSH, IVF, male factor 1153

5 Table 2 Fertilization Rate and Pregnancy Outcome Before and After FSH Therapy and After FSH Treatment No. of No. of No. of patients cycles transfers Results Fertilization rate preovulatory oocytes Pregnancy rate Secondary group (before FSH) Secondary group (after FSH) Primary treatment group (after FSH) Total "P < 4 X 10-6 % 12 (abnormal) (9/74)" 0 49 (normal) (27/55)" 2 (14.2/cycle; 25.0/transfer)b 45.7 (normal) (75/164) 5 (15.6/cycle; 27.7/transfer)b 46.5 (102/219) 7 (14.5/cycle; 21.8/transfer)b b Numbers in parentheses are percents. expression of a greater LH than T increase (0.4 ± 0.2; 0.4 ± 0.1; 0.2 ± 0.2) and the basal E2/T ratios showed a similar trend (4.5 ± 2.2; 3.8 ± 2.4; 3.8 ± 1.8). However, these trends were not statistically significant. Basal sperm concentration values in the group with normal FSH levels were significantly higher (55.3 ± 43.9 X 10 6 /ml) than in the groups with elevated ones (6.3 ± 3.7 and 6.4 ± 7.7 X 10 6 /ml) (not significant). Sperm motility did not show significant differences in the three groups (31.2% ± 13.1%; 21.5% ± 14.3%, and 25.0% ± 29.6%). Normal morphology showed a tendency to decrease as basal FSH levels increased (4.19% ± 4.4%; 2.0% ± 1.2%, and 1.7% ± 1.4%) (not significant). A longitudinal evaluation was performed in each one of the groups at 2, 4, and 8 weeks of FSH treatment. No significant changes were observed for FSH, LH, and PRL RIA levels. Testosterone showed a significant reduction (P < 0.05) only in group 3 (7.0 ± 0.6 versus 4.7 ± 0.5 ng/ml) after 8 weeks of therapy, and T/LH and E2/T ratios showed no significant changes. The overall population was then studied before and after FSH treatment (3 months). Radioimmunoassay LH levels did not change significantly. Bioactive LH levels did not show any significant changes (8.57 ± 1.80 mlu/ml versus 8.49 ± 1.99 mlu /ml) and were not significantly different from those of controls (9.33 ± 1.35 miu/ml). The bio FSH/immuno-FSH (B/I) LH ratio was not significantly different in any of the groups. The RIA-FSH levels were significantly higher in the post-treatment group (11.98 ± 7.78 versus ± 9.74 P < 0.05). Bioactive FSH levels did increase after treatment (22.6 mlu/ml versus 25.1 miu/ml), but the difference did not reach statistical significance. The B/I ratios were 1.9 and 1.6, much lower than those of controls.3,s Sperm concentration, motility, and morphology did not change significantly in anyone ofthe groups and subgroups when basal and post-treatment specimen results were compared. Sperm concentration at insemination was very constant following the protocol that Norfolk uses in the male-factor patients.14,19 A substantial change was noticed in the secondary treatment group in terms of fertilization rates per oocyte (Table 2). Before treatment, only 9 of cytes fertilized (12%), and all of these fertilizations were abnormal (delayed fertilization after primary insemination or reinsemination).20 After treatment, the fertilization rate increased to 49.0% (P < 4 X 10-6 ). In the primary treatment group, the fertilization rate after FSH therapy was very similar to the previous one: 45.7% confirming the similarities of both treatment groups and the reliability of the criteria used to select the patients. As a direct result of the improvement in the fertilization rate, the pregnancy rate improved dramatically in the secondary treatment group reaching 14.2%/cycle and 25.0%/transfer. Corresponding rates for the primary treatment group were 15.6% and 27.7%. It is interesting to note that all seven pregnancies achieved went to term, resulting in the delivery of healthy infants. Six pregnancies occurred in male patients with a serum basal FSH level of 5 to 15 mlu /ml and one in a patient with FSH > 26 mlu /ml. The same endocrine and semen variables were compared before and after FSH tre!ltment in the group that obtained fertilization within the secondary category. Once again, no significant differences were found in any variable Acosta et al. FSH, IVF, male factor Fertility and Sterility

6 I The pregnant and nonpregnant groups were compared in terms of basal FSH, LH, PRL, E2, T, TjLH, and E2jT ratios. No significant differences were found. No differences were found either when the same values post-treatment were used for comparison. Basal and post-treatment sperm parameters showed no significant differences between these two groups. The mean number of pre-embryos transferred (derived from preovulatory oocytes) in the pregnant group was almost three times higher than in the nonpregnant group (2.8 versus 1.0) as an expression of the improved fertilization rate. DISCUSSION Assisted reproduction is the ultimate diagnostic test and therapeutic effort for male infertility due to severe sperm disturbances. When failure of fertilization is demonstrated, only assisted fertilization procedures can overcome the problem. The efficiency of these methods is extremely low and, when using sperm with severe abnormalities, may be even lower. An effort is therefore justified to try to find an alternative to these treatment procedures. The criteria outlined in the Norfolk program to determine and predict the fertilizing ability of the sperm seem to be effective and adequate as established by clinical and laboratory work 1 ; the similarities between the secondary and primary groups of patients selected using those criteria in the present clinical trial give further confirmation as to their efficacy. The dose of pure FSH selected in our study was chosen on a completely empirical basis because no information was available in the literature on the use of pure FSH in the treatment of the male factor in assisted reproduction in the human; nor were data in the nonhuman primate available at the time this study was initiated. The dose used was selected on the basis of previous clinical trials using hmg.lo Work in the nonhuman primate that appeared more recently22 suggests the need for a higher dose to obtain effects that can be detected on peripheral endocrine levels, on spermatogenic function through a semen analysis, or on microscopic investigation of testicular biopsies. The basal FSH levels in these patients clearly indicate different degrees of testicular tubular deficiencies, but they do not seem to be predictive of the final outcome because pregnancies were obtained even in patients with clearly elevated FSH levels (>25 mivjml). Basal LH and T levels increased in parallel to the basal FSH levels, although these changes did not reach statistical significance. The T jlh ratio decreased, perhaps as an expression of a subtle, subclinical deficiency of the Leydig cell compartment, which is compensated by an increase in the LH output. Similar findings were reported by Giagulli and Vermeulen 5, who interpreted these changes also as an expression of mild, compensated Leydig cell deficiency. After treatment, only RIA FSH levels showed a significant increase. The determination of B-FSH and B-LH and their respective BjI ratios do not seem to offer any advantage in the evaluation of the problem. This is in agreement with conclusions reached by other authors.23 An interesting finding was that the number of sperm with normal morphology diagnosed using strict criteria tended to decrease as basal FSH levels increased. This would be further indication that abnormal sperm morphology is associated with impaired Sertoli cell function. The significant increase in fertilization rates obtained in these patients in spite of previous failure and poor semen characteristics seem to indicate an effect of pure FSH mainly in sperm function (quality), and the viability and normality of the pregnancies achieved seem to confirm this reasoning. We have continued using this protocol after the review of these series of patients was completed, with identical results. A cooperative effort and a prospective clinical experimental design with the characteristics already described is now mandatory to validate the present preliminary data. In view of our results, we believe that there is a group of infertile male patients with inability to fertilize oocytes in the human in vitro system, represented mainly by severe abnormal morphology or a combination of abnormal parameters as previously defined, who can be helped by systemic treatment with pure FSH before IVFjET. The dose of pure FSH used does not seem to be enough to modify the main endocrine indicators or the classical semen parameters in the majority of the cases. Nevertheless, the fertilization rate of preovulatory oocytes and the pregnancy rate were substantially improved. There are indications in the subhuman primate that increasing the dose of pure FSH to 4.6 IV jkg per day administered on a daily basis, instead of 2.5 IV jkg per day three times a week as we have used, the peripheral FSH levels and the classical semen parameters can be increased.22 A readjustment of the dose may therefore be necessary inthe future if the treatment proves effective. Failure of fertilization in those cases with severe teratozoospermia is associated with impaired rec- Vol. 55, No.6, June 1991 Acosta et al. FSH, IVF, male factor 1155

7 ognition and binding of the sperm to the zona pellucida as demonstrated by the use of the hemizona assay24; in addition, the SPA using the hamster-zona free system is also impaired in some teratozoospermic patients showing good sperm binding to the zona in the human in vitro system.24,25 This reinforces the need for new markers of sperm physiological capabilities that can predict fertilization in vitro and that can be used to monitor the results of treatment. Acknowledgments. The authors thank Serono Laboratories, Randolph, Massachusetts, for providing Metrodin; Kristine D. Dahl, Ph.D., Research Assistant Professor, Department of Medicine and Physiology, University of Washington, for performing the FSH bioassays; Robert Williams, Ph.D., Associate Scientific Director, The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Viriginia; Sue Sieg, M.S., and Susan Kendall, B.S., for the LH bioassays; and all the members of the In Vitro Fertilization, Embryology Laboratory, and Andrology Laboratory teams for their cooperation in this study. In addition, we are grateful to Ms. Pauline M. Clynes for her editorial assistance, and to Ms. Martha Wilson for graphical contributions. REFERENCES 1. Acosta AA, Oehninger S, Morshedi M, Swanson RJ, Scott R, Irianni F: Assisted reproduction in the diagnosis and treatment of male factor. Obstet Gynecol Surv 44:1, Ng S-C, Bongso A, Sathananthan H, Ratnam SS: Micromanipulation: its relevance to human in vitro fertilization. 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J In Vitro Fert Embryo Transfer 5:249, van Alpen MMA, van de Kant HJG, de Rooij DG: Folliclestimulating hormone stimulates spermatogenesis in the adult monkey. Endocrinology 123:1449, Jockenhovel F, Khan SA, Nieschlag E: Diagnostic value of bioactive FSH in male infertility. Acta Endocrinol (Copenh) 121:802, Franken DR, Oehninger S, Burkman LJ, Coddington CC, Kruger TF, Rosenwaks Z, Acosta AA, Hodgen GD: The hemizona assay (HZA): a predictor of human sperm fertilizing potential in in vitro fertilization (IVF) treatment. J In Vitro Fert Embryo Transfer 6:44, Kruger TF, Swanson RJ, Hamilton M, Simmons KF, Acosta AA, Matta JF, Oehninger S, Morshedi M: Abnormal sperm morphology and other sperm parameters related to the outcome of the hamster oocyte human sperm sperm penetration assay. Int J Androlll:107, Acosta et ai. FSH, IVF, male factor Fertility and Sterility