Corifollitropin alfa for poor responders patients, a prospective randomized study.

BACKGROUND: Poor ovarian response remains one of the biggest challenges for reproductive endocrinologists. The introduction of corifollitropin alpha (CFA) offered an alternative option to other gonadotropins for its longer half-life, its more rapid achievement of the threshold and higher FSH levels. We compared two different protocols with CFA, a long agonist and a short antagonist, and a no-CFA protocol. METHODS: Patients enrolled fulfilled at least two of the followings: AFC < 5, AMH < 1,1 ng/ml, less than three oocytes in a previous cycle, age > 40 years. Ovarian stimulation with an antagonist protocol was performed either with 300 UI rFSH and 150 UI rLH or 300UI HMG. In the long agonist group, after pituitary suppression with triptorelin, CFA was given the 1-2th day of cycle and 300 UI rFSH and 150 UI rLH the 5th day. In the short antagonist group CFA was given the 1-2th day of cycle and 300 UI rFSH and 150 UI rLH the 5th day. The primary objective was the effect on the number of oocytes and MII oocytes. Secondary objective were pregnancy rates, ongoing pregnancies and ongoing pregnancies per intention to treat. RESULTS: The use of CFA resulted in a shorter lenght of stimulation and a lower number of suspended treatments. Both the CFA protocols were significantly different from the no-CFA group in the number of retrieved oocytes (p < 0,05), with a non-significant difference in favour of the long agonist protocol. Both CFA groups yielded higher pregnancy rates, especially the long protocol, due to the higher number of oocytes retrieved (p < 0,05), as implantation rates did not differ. The cumulative pregnancy rate was also different, due to the higher number of cryopreserved blastocysts (p < 0,02). CONCLUSIONS: The long agonist protocol with the addition of rFSH and rLH showed the best results in all the parameters. A short antagonist protocol with CFA was less effective, but not significantly, although provided better results compared to the no-CFA group. We suggest that a long agonist protocol with CFA and recombinant gonadotropins might be a valuable option for poor responders. TRIAL REGISTRATION: The study was approved by the local Ethics Committee (EudraCT2015-002817-31).

Objective evaluation of the viability of cryopreserved oocytes.

Recent studies of fundamental cryobiology, empirical observations and more systematic clinical experiences have generated a renewed interest in oocyte cryopreservation. Poor survival rate has long been the limiting factor which has prevented widespread adoption of oocyte storage. Slow-cooling and vitrification protocols developed in the last few years have apparently solved this problem, ensuring high recovery of viable oocytes from liquid nitrogen storage. However, the definition of oocyte viability appears rather vague. In fact, post-storage survival as assessed on morphological criteria, indicated by the absence of overt cell degeneration, is not necessarily synonymous with viability. Despite its sensitivity to low temperatures, the meiotic spindle can be preserved after cryopreservation and its constitution after thawing can be monitored non-invasively through polarized light microscopy. Assessment of oocyte cryopreservation via clinical parameters is a daunting task. Most studies are small and difficult to interpret because of confounding factors, such as age, patient selection and quality and strategy of use of the cryopreserved material. Some progress has been made, however, as suggested by recent experiences in which the implantation efficiency of embryos produced from thawed oocytes approaches that reported using cryopreserved embryos directly.

Oocyte cryopreservation: clinical outcome of slow-cooling protocols differing in sucrose concentration.

Oocyte cryopreservation represents an important option for management of female fertility, avoiding the ethical concerns associated with embryo storage. This retrospective study evaluated the clinical outcome of two alternative slow freezing protocols involving different sucrose concentrations. From January 2004 to March 2006, spare oocytes from selected couples undergoing IVF or intracytoplasmic sperm injection were frozen using a slow-cooling protocol and thawed at a later stage. Patients were divided into two groups: group A (n = 65), whose oocytes were frozen with propane-1,2-diol (PrOH) and 0.1 mol/l sucrose; and group B (n = 66) whose oocytes were frozen with 0.3 mol/l sucrose. A total of 543 oocytes were thawed in group A and 601 in group B, achieving a survival rate of 24.3 and 71.2% respectively. Whilst fertilization rate (53.5 and 80.4% respectively) was higher in group B, enhanced results for group A were achieved over all (implantation rate per transferred embryos 12.2 versus 5.7%; pregnancy rate per transfer 16.7 versus 9.5%). Normal births and ongoing pregnancies have occurred in both groups. Although in slow-cooling methods higher sucrose concentration in the freezing mixture allows higher post-thaw survival and fertilization rates, overall this did not coincide with an improved clinical outcome.

The use of Matrigel at low concentration enhances in vitro blastocyst formation and hatching in a mouse embryo model.

OBJECTIVE: To determine the effect of Matrigel at a low concentration on the growth of mouse embryos in culture. DESIGN: Randomized case-control study of mouse embryos. SETTING: An academic research environment. ANIMALS: Mouse embryos. INTERVENTION(S): Embryos were cultured in Quinn's or Celbio's human tubal fluid (HTF) enriched with 1.5% bovine serum albumin and 0.8% liquid Matrigel. Each HTF was compared with the same medium devoid of Matrigel. Afterward, Quinn's and Celbio's HTF, both containing Matrigel, were compared directly. Embryos were cultured in four-well dishes, and their morphology and viability were assessed at 96 hours. MAIN OUTCOME MEASURE(S): Level of interleukin-1alpha in media collected at the end of culture. RESULT(S): In both types of HTF, the presence of Matrigel allowed a larger number of embryos to reach the blastocyst stage and to hatch; blastocyst morphology also was improved. These positive effects were enhanced in Quinn's HTF: embryos cultured in its Matrigel-enriched version secreted a higher level of interleukin-1alpha than those in Celbio's HTF plus Matrigel and also showed a better morphology. CONCLUSION(S): In the mouse embryo model, Matrigel improves culture conditions in terms of both embryo viability and morphology, and these effects are enhanced in Quinn's HTF.

Evidence that a functional fertilin-like ADAM plays a role in human sperm-oolemmal interactions.

Fertilin is a protein initially identified in guinea pig spermatozoa; it is the prototype of a larger family of conserved, proteins designated as a disintegrin and a metalloproteinase (ADAM). These heterodimers which consist of alpha and beta subunits, containing metalloproteinase-like and disintegrin-like domains, appear to play a role in mammalian fertilization. Peptides derived from the disintegrin domains of two ADAMs, fertilin and cyritestin, interfere with gamete adhesion and sperm-egg membrane fusion in non-human species. It has been suggested that fertilin-beta binds to an oolemmal integrin, and it is proposed that the tripeptide FEE (Phe-Glu-Glu) is the integrin recognition sequence in human fertilin-beta. We evaluated whether fertilin beta plays a role in human fertilization by studying the effects of a linear octapeptide containing the FEE sequence, SFEECDLP, and a scrambled octapeptide with the same amino acids, SFPCEDEL, on the incorporation of human spermatozoa by human zona-free eggs. The effects of G4120, a potent RGD-containing (Arg-Gly-Asp) thioether-bridged cyclic peptide which blocks both fibronectin and vitronectin receptors, and the relationship between FEE- and RGD-receptor interactions on sperm-egg interactions were also studied. The FEE-containing peptide, but not the scrampled peptide, inhibited sperm adhesion to oocytes and their penetration, over the range 1-5 microM. The inhibition induced by SFEECDLP was reversible and occurred only in the presence of peptide itself. The G4120 peptide exhibited 10-fold less inhibitory effects on sperm adhesion and penetration than did SFEECDLP. When combined, SFEECDLP and G4120 exhibited strong inhibition of both adhesion and penetration at concentrations that individually had been ineffective, suggesting co-operation between the two receptor-ligand interactions during fertilization. We propose that a fertilin-like molecule is functionally active on human spermatozoa and that its interaction with an oolemmal integrin receptor plays a role in fertilization in humans.

An investigation of the latency period between sperm oolemmal adhesion and oocyte penetration.

In clinical studies of the ability of capacitated human sperm to penetrate zona-free hamster eggs, we have previously observed that the ratio of oolemmal adherent to penetrating sperm varied between men. Sperm incorporation did not occur immediately following gamete adhesion and not all adherent sperm penetrated the egg. To further investigate this phenomenon, comparisons were made of the kinetics of gamete adhesion, membrane fusion, and sperm incorporation of capacitated mouse and human spermatozoa by zona-free hamster eggs and of mouse sperm by zona-free mouse and hamster eggs. Eggs were inseminated with either capacitated human or mouse sperm or combinations of both, washed out of sperm suspension after initial gamete adherence, and further incubated in sperm-free medium. Gamete membrane fusion was judged by dye transfer of Hoechst 33342 and sperm entry of the cortical ooplasm by observation of expanded sperm heads within acridine orange stained eggs. Oolemmal adherent mouse and human sperm fused with and penetrated zona-free hamster eggs at different times whether eggs were inseminated in parallel or with combinations of sperm of both species. Oolemmal adherent mouse sperm penetrated zona-free hamster eggs prior to their penetration of zona-free mouse eggs. Ultrastructural studies of zona-free human eggs inseminated with human sperm confirmed prior observations with hamster eggs that only acrosome-reacted human sperm adhere to the oolemma. These results have lead us to postulate that sperm entry into the egg may occur through a "zipper" mechanism involving the ligation of local gamete receptors similar to the incorporation of target particles by phagocytes and suggest that not all oolemmal adherent human sperm are capable of being incorporated although they have undergone an acrosome reaction.

Premature ovarian failure: steroid synthesis and autoimmunity.

The androgen biosynthesis and autoimmunity of 25 patients with premature ovarian failure (POF) and 18 control subjects with normal cycles were examined. Serum levels of dehydroepiandrosterone sulfate (DHEAS), 17-hydroxyprogesterone (17-OHP), androstenedione, and testosterone were analyzed in POF patients with or without organ-specific autoimmunity, and the results compared with those of women with normal ovarian function. The comparative analysis of DHEAS, 17-OHP, androstenedione and testosterone showed that POF patients had significantly lower values than normal women (DHEAS, androstenedione and testosterone p < 0.01, 17-OHP p < 0.05). Furthermore, we found one or more organ-specific autoantibodies in 11 patients with POF (44%), while only one woman in the control group showed autoimmunity (antithyroid microsome) (5.5%). Only one patient had both anti-ovarian and anti-adrenal antibodies (4%). The comparison of androgen levels in POF patients with or without autoimmunity revealed a statistically significant reduction of DHEAS levels in POF patients with organ-specific autoimmunity (p < 0.01). These data reveal the reduction in androgen synthesis in POF patients, particularly in those with organ-specific autoimmunity.

Detection using antisperm monoclonal antibodies of shared epitopes expressed by human spermatozoa and oocytes.

OBJECTIVE: To determine whether human spermatozoa and oocytes share common antigenic epitopes, supporting the hypothesis that their cross-linking by antisperm antibodies present in the clinical sera of infertile couples could promote sperm adhesion to the oolemma. DESIGN: Human and hamster eggs were studied for the presence of antigens recognized by a panel of World Health Organization Task Force monoclonal antibodies (mAbs) originally raised against human spermatozoa. A new technique was devised, using frozen sections of paraformaldehyde-fixed individual human and hamster eggs, to screen rapidly antisperm mAbs for egg reactivity. Living zona-free human and hamster eggs then were exposed to Covaspheres (Duke Scientific, Palo Alto, CA) coupled with these mAbs to document the presence of reactive epitopes on the oolemma. SETTING: Academic research environment. MAIN OUTCOME MEASURE(S): Indirect immunofluorescence and Covasphere rosetting. RESULT(S): Eleven of 37 antisperm mAbs tested reacted with fixed hamster eggs and 10 reacted with human eggs. Five of 6 mAbs reactive with both fixed eggs also reacted with the oolemma of living, zona-free eggs. CONCLUSION(S): Common antigenic epitopes, some of which are shared with somatic tissues, exist on the oolemma of human eggs and on the plasma membrane of human spermatozoa.

Immunogenicity and contraceptive potential of a human zona pellucida 3 peptide vaccine.

Immunization with zona pellucida 3 (ZP3) glycoprotein induces infertility in primates and is a target antigen for a contraceptive vaccine. However, loss of ovarian function is a long-term side effect. A possible mechanism is autoimmune ovarian disease induced by ZP3-specific autoreactive T cells, demonstrated in mice immunized with a murine ZP3 peptide in complete Freund's adjuvant. Indeed, a murine contraceptive vaccine that elicits antibodies to zona pellucida (ZP) without concomitant pathogenic T-cell activation has been achieved by a chimeric peptide (CP) consisting of a native ZP3 B-cell epitope and a foreign helper T-cell peptide. Herein, we evaluate the CP strategy in primate for human ZP3 (hZP3) vaccine development. A CP was constructed that consisted of a known helper T-cell epitope from the malarial circumsporozoite protein and a native B-cell epitope of hZP3. The human CP elicited antibodies to ZP3 in macaques without a measurable T-cell response to the hZP3 peptide. The serum antibodies reacted with macaque and human ZP and significantly inhibited human sperm binding to oocytes in vitro. Moreover, the CP elicited antibodies to human ZP in mice that lack murine major histocompatibility complex (MHC) class II molecules but express transgenic human HLA-DR3, -DQ6, or DQ8 molecules. Therefore, this study 1) provides evidence to support the feasibility of the CP strategy in hZP3 vaccine development and 2) describes a novel approach for evaluating the influence of polymorphic human MHC on vaccine immunogenicity without human immunization.

In controlled ovarian hyperstimulation, steroid production, oocyte retrieval, and pregnancy rate correlate with gene expression of vascular endothelial growth factor.

PURPOSE: Whether the gene expression of vascular endothelial growth factor (VEGF) in human granulosa cells is a predictor of fertilization was evaluated in patients participating in an in vitro fertilization program. METHODS: Fifty patients with normal ovaries who were participating in an in vitro fertilization program at the University of Milan, San Raffaele Scientific Institute, were included in the study. We correlated E2 and P serum levels on the day of oocyte collection, the number of follicles, oocytes collected, and fertilized, and pregnancies with mRNA for VEGF of luteinizing granulosa cells obtained at the time of oocyte retrieval. RESULTS: Comparing E2 and P serum levels, the number of follicles, oocytes collected and fertilized, and pregnancies with gene expression for VEGF, we found a positive correlation. E2 and P serum levels were higher in patients with increased VEGF (P < 0.01). Furthermore, there were more follicles, oocytes collected and fertilized, and pregnancies in patients with maximum expression of VEGF, and the difference was statistically significant (P < 0.05). CONCLUSIONS: Our results suggest that VEGF may be important for vascular development during follicular growth and luteal differentiation, oocyte maturation, and fertilization.

Is vitronectin the velcro that binds the gametes together?

Evidence has been presented that the adhesion of human spermatozoa to the oolemma is mediated by integrins recognizing the Arg-Gly-Asp sequence (RGD). Fibronectin and vitronectin, glycoproteins that contain functional RGD sequences, are both present on human spermatozoa, and integrins that recognize these ligands have been detected on spermatozoa and eggs. In this work, we studied the effects of oligopeptides specifically designed to block fibronectin or vitronectin receptors on the interaction of human spermatozoa with zona-free hamster oocytes. GRGDdSP, a peptide blocking cell attachment to fibronectin, was without effect, while GdRGDSP, which blocks both fibronectin and vitronectin receptors, significantly inhibited the binding of human spermatozoa to the oolemma of zona-free hamster eggs, in a concentration-dependent manner, over a range 1-100 microM. As these experiments suggested that a vitronectin receptor plays a role in sperm-oolemmal adhesion, we performed a series of experiments studying the effects of exogenous vitronectin, when added to spermatozoa and oocytes, on gamete interactions. Sperm-oolemmal adherence, as well as sperm aggregation, was promoted by vitronectin, over range of 2.2 nM to 1 microM, but only in the presence of calcium ions. We propose that vitronectin released during the sperm acrosome reaction is recognized by both gametes and plays a role in their adhesion.

Use of growth hormone-releasing factor in ovulation induction in poor responders.

OBJECTIVE: To evaluate the effect of growth hormone-releasing factor (GRF), given in addition to follicle-stimulating hormone (FSH), after pituitary down-regulation, on follicular development in poor responders. GRF was added in a prospective, randomized manner to an existing stimulation protocol. STUDY DESIGN: Forty-two women, demonstrated to be poor responders in previous attempts at induction of ovulation, were included in the study. Follicular stimulation with FSH was started after pituitary downregulation obtained using gonadotropin-releasing hormone agonist (GnRH-a). GRF, 1,000 mg/day, was given in addition to FSH to 22 patients randomly chosen until human chorionic gonadotropin administration. RESULTS: The number of ampules of FSH needed to obtain follicular growth was significantly reduced in the group of women who received GRF. Also, the number of follicles obtained was higher and the days of treatment generally reduced. However, a greatly beneficial effect for some women was observed, while a second subgroup did not have any. No differences were observed in follicular steroid and insulin growth factor 1 (IGF-1) between GRF patients and controls or in serum IGF-1 between the two subgroups of patients who received GRF. CONCLUSION: In vivo administration of GRF with FSH after pituitary down-regulation may be beneficial for some poor responders, although prognostic criteria could not be established. However, the use of GRF does not seem to influence the chance of obtaining pregnancy; it remains low in these patients.

Success in inducing ovulation in a case of premature ovarian failure using growth hormone-releasing hormone.

Ovulation was obtained in a 29-year-old woman affected by premature ovarian failure who had previously failed to respond to two attempts performed administering human menopausal gonadotropin or follicle-stimulating hormone after the spontaneous gonadotropin production was suppressed using a gonadotropin-releasing hormone analog (buserelin). Induction of ovulation succeeded when 1000 mg/day growth hormone-releasing hormone was added to the induction scheme. Five mature follicles were obtained after 27 days therapy and the serum level of 17 beta-estradiol was 975 pg/ml (195 pg/ml per follicle) at the time of human chorionic gonadotropin administration.

P-selectin is expressed on the oolemma of human and hamster oocytes following sperm adhesion and is also detected on the equatorial region of acrosome-reacted human spermatozoa.

Selectins are a family of adhesive molecules, involved in the interactions between leukocytes and endothelium and in platelet adhesion. P-selectin, one of the members of this family, is stored in alpha-granules and dense granules of platelets as well as in Weibel-Palade bodies of endothelial cells, and it is rapidly redistributed to the cell surface after activation. It recognizes carbohydrate structures as ligands, in particular sialyl-Lewis(x), which is part of the CD15 antigen. In this work we studied P-selectin expression on gametes. While zona-free human and hamster oocytes did not react with a monoclonal antibody directed against P-selectin, oocytes from both species displayed a reactivity with this antibody following their contact with human spermatozoa, as demonstrated both by covasphere binding and indirect immunofluorescence. Artificial activation of zona-intact human oocytes by means of the calcium lonophore A23187 induced the expression on the oolemma of a moiety reacting with anti-P-selectin antibody as well. P-selectin also appeared to be expressed on the sperm surface following the acrosome reaction, as demonstrated by a flow cytometric study of reactivity of spermatozoa with the anti-P-selectin antibody, using the expression of CD46 as a marker of the acrosome reaction. The localization of the P-selectin moiety on the equatorial region of the plasma membrane of acrosome reacted spermatozoa was confirmed by transmission electron microscopy using immunogold labelling. We suggest that P-selectin might be involved in gamete interactions.

The expression of alpha v, alpha 5, beta 1, and beta 3 integrin chains on ejaculated human spermatozoa varies with their functional state.

Evidence has been presented suggesting the involvement of integrins and their ligands in mammalian fertilization. In this study we asked whether the alpha 5, alpha v, beta 1 and beta 3 integrin chains, which form receptors for fibronectin and vitronectin, are present on human spermatozoa. Fresh ejaculate spermatozoa and capacitated spermatozoa, before and after a calcium ionophore (A23187)-induced acrosome reaction, were either fixed and their reaction with anti-integrin monoclonal antibodies detected by immunoperoxidase staining or studied without fixation, using cytofluorimetric scanning. Expression of specific integrin chains varied with the functional state of spermatozoa. The alpha 5 chain was not detected on fresh living spermatozoa, but was present on capacitated spermatozoa, whether fixed or living. The pattern of beta 1 expression on living spermatozoa paralleled that of alpha 5. No further increase in the expression of either alpha 5 or beta 1 was observed following an ionophore-promoted acrosome reaction. In contrast, alpha v was detected on neither fresh, living ejaculate spermatozoa, nor following capacitation (< 10% reactive). The percentage of alpha v positive cells increased substantially following ionophore exposure. Expression of beta 3 was similar to alpha v, and the percentage of cells displaying beta 3 correlated with the proportion of spermatozoa that had undergone an acrosome reaction, following ionophore exposure. These results indicate that the expression of integrins on spermatozoa is dynamic, varying with their functional state and that integrin receptors for fibronectin (alpha 5 beta 1) become apparent on the spermatozoan surface during capacitation and vitronectin (alpha v beta 3) following the acrosome reaction.

In vitro production of human anti-sperm antibodies: effect of an oligoclonal antibody (F6) on sperm-egg interaction.

A method has been developed to establish lines of transformed lymphocytes able to produce in vitro the same anti-sperm antibodies as those naturally occurring in immuno-infertile individuals. We utilized lymphocytes from a male donor whose serum contained anti-sperm antibodies of the IgG class up to the dilution 1:10,000, as detected by means of immunobead binding. T lymphocytes were separated from B lymphocytes using magnetic beads coated with anti-T antibody. B lymphocytes were then placed at a concentration of 5 x 10(6)/ml in a 96-well plate, stimulated with phytohaemagglutinin (PHA) and transformed with Epstein-Barr virus. After a few days, only transformed cells continued growing and these were collected. The supernatant was tested for production of anti-sperm antibodies and those transformed lymphocytes shown to be synthesising antibodies directed against the sperm head and the tail were cloned. We obtained a clone of cells producing antibodies of the IgG1 class directed against the head of the spermatozoon. This oligoclonal antibody (F6) recognized a 58-kDa band from a lysate of sperm membranes and was able to reduce the penetration of zona-free hamster oocytes by capacitated spermatozoa.

Production of mouse monoclonal antibodies directed against the oolemma of human and hamster oocytes by intra-splenic injection of oocytes.

PROBLEM: To develop an additional approach for the study of oolemmal surface moieties involved in gamete interactions, we decided to obtain monoclonal antibodies by intrasplenic injection of human and hamster oocytes in Balb/c mice. METHOD: Two Balb/c males were injected three times intrasplenically at 15-day intervalS with approximately 40 zona-free hamster and 3-5 zona-free human oocytes. After the third injection, spleen cells were fused and hybridomas developed. We used a novel screening system based upon the use of sections of frozen human and hamster eggs, tested by means of indirect immunofluorescence. The antibodies that we produced were evaluated for their ability to interfere with the zona-free hamster eggs penetration by human spermatozoa. The B2B5 antibody was also developed as ascitic fluid and further characterized. RESULTS: Seven antibodies reactive with hamster oocytes were produced. Six of them also reacted with human oolemmas. The binding was confined to the oolemma, and no staining of the zona nor the cytoplasm was present. One of these antibodies reduced the penetration of zona-free hamster eggs by human spermatozoa. This antibody, B2B5, an IgM kappa, was confirmed to interact with the oolemma by means of indirect immunofluorescence of fresh eggs and Covasphere binding. B2B5 did not react with other human or hamster tissues except capacitated human spermatozoa. The reactivity with the oolemma of hamster oocytes was not lost after egg penetration by human sperm. CONCLUSIONS: Intrasplenic immunization using zona-free human and hamster oocytes allows the production of anti-oolemma antibodies. A system of screening based upon the use of sections of frozen eggs also allows an easy and quick scoring of many supernatants. B2B5 monoclonal anti-oolemma antibody deserves further studies in that is able to interfere with fertilization and its antigen appears to be confined to the gametes surface.

Vitronectin is an intrinsic protein of human spermatozoa released during the acrosome reaction.

Evidence has been presented that oolemmal integrins and their ligands on spermatozoa may play a role in gamete interactions leading to fertilization. We previously demonstrated that vitronectin (Vn) could be extracted from fresh human spermatozoa and detected in Western blots, and Vn was observed on the surface of living, capacitated sperm by indirect immunofluorescence. In the present experiments, messenger RNA encoding Vn was detected in human testis poly (A+) RNA using Northern analysis, and Vn was localized within the acrosomal region of ejaculated sperm by immunoperoxidase and immunofluorescence staining. During the acrosome reaction, induced in capacitated spermatozoa by lonomycin, Vn was released into the medium in a calcium-dependent manner. Vn appears to be a specific product of intratesticular spermatozoa that is secreted during the acrosome reaction. These findings suggest that Vn is positioned to play a strategic role in gamete interactions leading to fertilization.

Effects of the coculture with human endometrial cells on the function of spermatozoa from subfertile men.

OBJECTIVE: To evaluate the effects of a coculture with human endometrial cells on the function of spermatozoa from samples obtained from infertile couples. DESIGN: In a prospective study, human spermatozoa selected by swim-up from fresh samples were cultured on human endometrial feeder layers. Thereafter, their viability, motility, acrosome integrity, and ability to penetrate zona-free hamster oocytes were evaluated. Spermatozoa from the same samples incubated under the same conditions but in the absence of endometrial cells, as well as in the medium previously spent for cell culture, were used as controls. SETTING: Andrology Laboratory of the Infertility Center of San Raffaele Hospital. PATIENTS: Spermatozoa were obtained from 17 infertile men attending the Infertility Center at our hospital. RESULTS: Spermatozoa incubated in the presence of endometrial cell feeder layers did not differ from controls with regard to their viability or motility. Conversely, the percent spontaneous acrosome reactions after 18 hours of incubation was significantly higher for spermatozoa cocultured (19.7 +/- 2.2 versus 11.2 +/- 1.9; mean +/- SE). The mean number of spermatozoa penetrating hamster oocytes was also significantly improved (1.24 +/- 0.3 versus 0.68 +/- 0.24). This effect did not seem to be solely due to the secretion of soluble factors by endometrial cells in the medium, in that spermatozoa incubated in the medium spent for endometrial cell culture had a similar acrosome reaction percentage but a lower rate of hamster egg penetration. CONCLUSIONS: The coculture with human endometrial cells appeared to be beneficial for improving the sperm function. This effect partially may be due to the secretion of steroids in the medium, which increases the quota of spontaneous acrosome reaction and in part due to the direct contact of cells with spermatozoa, maybe for the detoxification of the medium or the release of trophic factors. Coculture might be a promising approach to preparing spermatozoa for assisted fertilization in cases of subfertile males.