Carmen Mendoza

Luteinizing hormone affects uterine receptivity independently of ovarian function

Previous studies have suggested that LH, in addition to its well-known effects on the ovary, may exert direct effects on the uterus. This study evaluated the effects of mid-cycle administration of human chorionic gonadotrophin (HCG), which signals through the LH receptor, on endometrial thickness and uterine receptivity in two groups of women lacking ovarian activity and receiving embryos from an oocyte donation programme. Patients in one group still had ovulatory cycles, but their ovarian function was suppressed by pituitary down-regulation with a gonadotrophin-releasing hormone (GnRH) agonist in the embryo transfer cycle, resulting in low endogenous LH concentrations. Patients in the other group were menopausal women whose pituitary function was not down-regulated in the embryo transfer cycle and whose endogenous LH concentrations were thus high. Patients in each of the two groups were randomized into two subgroups. Patients in one subgroup were given 5000 IU of HCG 2 days before oocyte recovery in the corresponding donor. Patients in the other subgroup received placebo at the same time. Oocytes from each donor were randomly distributed between one patient from the HCG subgroup and one patient from the placebo subgroup in each patient group. Endometrial growth and secretory transformation were stimulated by sequential treatment with oestradiol valerate and progesterone. In women with low endogenous LH receiving placebo, endometrial thickness stopped increasing at the beginning of secretory transformation. Mid-cycle HCG administration resulted in a continuous increase in endometrial thickness through this period, improved the implantation rate after embryo transfer in these women (30.6 versus 20.7%) and augmented the number of multiple pregnancies. No similar stagnation of endometrial thickness and no effects of mid-cycle HCG administration on endometrial thickness, the implantation rate and the number of multiple pregnancies were found in women with high endogenous LH. It is concluded that endometrial maturation is disturbed in women with low endogenous LH but can be rescued by mid-cycle stimulation of LH receptor with exogenous HCG in the absence of ovarian activity.

Selective expression of a progesterone receptor on the human sperm surface

Objective To visualize progesterone (P) binding sites on the sperm surface, examine the relationship between hormone binding and hormone action (acrosome reaction), and determine the size of the hormone-responsive sperm subpopulation. Design Kinetic analysis of P binding was combined with the assessment of the hormone effect using a fluorescent acrosomal marker. Setting Private hospital, medical research center, and a university-based andrological laboratory. Patients, Participants Sperm samples were from healthy volunteers with normal spermiogram values. Interventions None. Main Outcome Measures Progesterone binding was analyzed by fluorescence microscopy and flow cytometry using P coupled to fluorescein isothiocyanate-labeled bovine serum albumin. Tetramethylrhodamine isothiocyanate-labeled Pisum sativum agglutinin was used as acrosomal marker in double-labeling experiments. Results After in vitro capacitation, only few spermatozoa (approximately 10%) were able to bind P to the cell surface, but most of these cells subsequently generated the acrosome reaction in response to hormone binding. Conclusions The expression of P receptor sites on the human sperm surface is a major factor controlling the P-induced acrosome reaction. Further studies are warranted to explore if defective expression of the receptor can compromise fertility.

Use of a modified intracytoplasmic sperm injection technique to overcome sperm-borne and oocyte-borne oocyte activation failures

OBJECTIVE To examine whether sperm-borne and oocyte-borne oocyte activation failures can be overcome by mechanical means that entail modifying the ICSI technique. DESIGN Case report series. SETTING Private clinics. PATIENT(S) Six infertile couples undergoing ICSI. INTERVENTION(S) Standard ICSI and modified ICSI based on mechanical manipulation that facilitated entry of calcium into the oocyte. MAIN OUTCOME MEASURE(S) Fertilization rate and pregnancy outcome. RESULT(S) In three cases of sperm-borne and three cases of oocyte-borne oocyte activation deficiencies, the modified ICSI technique enabled normal fertilization and development of embryos with good morphology. In terms of fertilization, the efficacy of modified ICSI was similar to use of a calcium ionophore, without producing extensive embryo fragmentation during postfertilization development. Term pregnancies resulting in the birth of normal children were achieved with the modified ICSI technique in five cases. CONCLUSION(S) Sperm-borne and oocyte-borne oocyte activation failures can be overcome by modifying the ICSI technique. The modification obviates the need to use insufficiently tested and potentially harmful drugs.

Effects of phosphodiesterase inhibitors caffeine and pentoxifylline on spontaneous and stimulus-induced acrosome reactions in human sperm.

OBJECTIVE To determine whether the phosphodiesterase inhibitors caffeine and pentoxifylline influence the acrosome reaction in the conditions in which they are currently used as sperm movement enhancers. DESIGN The frequency of acrosome reaction occurring spontaneously in capacitating media or induced by physiological (follicular fluid [FF]) and artificial (ionophore A23187) stimuli was compared in the presence and absence of the phosphodiesterase inhibitors. SETTING Private hospital and research laboratory. PATIENTS, PARTICIPANTS Patients undergoing routine semen examination before in vitro fertilization (no pathology detected) and healthy sperm donors. INTERVENTIONS None. MAIN OUTCOME MEASURE Percentage of acrosome-reacted sperm determined with the use of fluorescein-labeled Pisum sativum agglutinin as acrosomal stain. RESULTS Caffeine alone augmented the frequency of acrosome reaction, but this effect was not observed with pentoxifylline alone. However, pentoxifylline increased sperm responsiveness to the acrosome reaction-inducing stimuli, FF and ionophore A23187. CONCLUSIONS The promotion of spontaneous acrosome reaction may counteract the benefits from application of caffeine as motility stimulant. On the other hand, the sensitization to physiological acrosome reaction stimuli is expected to contribute to the improvement of sperm fertilizing ability by pentoxifylline and make this drug a potential candidate for the treatment of acrosome reaction anomalies.

Sperm nuclear DNA damage: update on the mechanism, diagnosis and treatment

Previous studies have shown that repeated intracytoplasmic sperm injection failures can be associated with sperm DNA damage. This paper reviews the current understanding of the mechanism of sperm DNA damage, discusses different diagnostic methods and their threshold values to discriminate between good- and poor-prognosis patients, and outlines the currently available treatment options. A rational approach to the interpretation of sperm DNA fragmentation data and to the choice of the optimal treatment method is suggested.

Fertilizable oocytes reconstructed from patient's somatic cell nuclei and donor ooplasts

The only assisted reproduction treatment now available for women with ovarian failure or irreparable oocyte defects is oocyte donation. However, some women experience psychological barriers to the recourse to donor oocytes, related to the lack of contribution of their proper genes to the progeny. A pilot study in humans suggests that this problem may be overcome by the development of techniques for haploidization of somatic cell nuclei, allowing the formation of new oocytes bearing the complete nuclear genome of the patient. Somatic cell nuclei were obtained from cumulus cells of a patient who failed to produce fertilizable oocytes and were transferred into enucleated oocytes (ooplasts) from a donor. Out of six ooplasts injected with the somatic cell nuclei and fertilized with spermatozoa from the patients husband, signs of haploidization were detected in three oocytes, two of which subsequently started embryonic development and were cryopreserved for eventual future transfer to the genetic mother. These data show that human oocytes can be used for both reprogramming and haploidization of somatic cell nuclei, allowing reconstruction of genetically own oocytes for patients without, or with seriously disturbed, ovarian function.

Sperm treatment with pentoxifylline improves the fertilizing ability in patients with acrosome reaction insufficiency

Objective To test whether pentoxifylline, a drug previously shown to sensitize spermatozoa from normal samples to the action of acrosome reaction stimuli, can be used to improve the fertilizing ability of spermatozoa from patients with acrosome reaction insufficiency. Design Prospective analysis of pentoxifylline effects on the acrosome reaction and zona-free egg penetration; retrospective comparison of IVF results with and without the use of pentoxifylline. Setting Private hospital, public research center, and university-based laboratory. Patients In vitro fertilization patients selected on the basis of a previous acrosome reaction test. Interventions None in the experimental part; IVF-ET in the clinical part of this study. Main Outcome Measures Frequency of the acrosome reaction, rate of penetration of zona-free eggs, normal fertilization in IVF attempts. Results Pentoxifylline improves the acrosome reaction scores and zona-free egg penetration rates in patients with acrosome reaction insufficiency. Preliminary clinical experience shows an improvement of IVF results in these patients. Conclusions In vitro pentoxifylline treatment of spermatozoa to be used in IVF improves the sperm fertilizing ability in patients with acrosome reaction insufficiency. However, the effect of pentoxifylline on the acrosome reaction should be tested individually in each patient before the application of the drug in this new indication.

Progesterone action through aggregation of a receptor on the sperm plasma membrane

Rapid steroid effects, reported in several cell types, have pointed out the possibility of non‐genomic mechanisms of action, presumably on cell surface receptors. Here we analyzed the effects of antibody‐mediated aggregation of a novel type of progesterone receptor on the plasma membrane of human sperm cells. We report that aggregation of hormone‐receptor complexes induces Ca2+ influx and a Ca2+‐dependent exocytotic event in this system. These data suggest a possible mechanism for rapid steroid‐induced events. Further research is warranted to examine if a similar mechanism is involved in rapid steroid effects in other cell types.

Expression of D-mannose binding sites on human spermatozoa: comparison of fertile donors and infertile patients

OBJECTIVE The hypothesis that defective expression of D-mannose binding sites (presumptive elements of the sperm-zona pellucida binding mechanism) is related to male infertility was tested. DESIGN Experiments were performed on sperm samples from two groups of men classified, respectively, as fertile and infertile, based on their reproductive history. SETTING The study was carried out in an andrologic laboratory of a University Hospital. PATIENTS, PARTICIPANTS Fertile men were healthy sperm donors; infertile men were patients with fertility problems. INTERVENTIONS D-mannose binding sites were visualized by fluorescence microscopy using a mannosylated neoglycoprotein probe. MAIN OUTCOME MEASURES The hypothesis as reported in the objective section was formulated before data collection and was not modified thereafter. RESULTS Sperm from fertile men displayed a characteristic pattern of changes in the expression of D-mannose binding sites during in vitro capacitation. This pattern was altered in sperm from infertile men. CONCLUSIONS If the relationship between defective expression of D-mannose binding sites and decreased sperm fertilizing ability is validated by parallel tests of sperm-zona binding, it may be used for development of chemical tests replacing the current ones using human zonae pellucidae.

Fast acrosome reaction measure: a highly sensitive method for evaluating stimulus-induced acrosome reaction

OBJECTIVE To test a new method for evaluation of stimulus-induced acrosome reaction (AR). To determine whether this method, based on the definition of a specific staining pattern of recently reacted spermatozoa, brings an advantage of increased sensitivity as compared with a standard procedure. DESIGN The hypothesis that a specific staining pattern with fluorescein-labeled Pisum sativum agglutinin corresponds to recently acrosome-reacted spermatozoa was tested by analyzing the frequency of this pattern at different time points after addition of ionophore A23187, an artificial AR inducer. The AR results obtained with a new method based on this relationship were compared with those obtained with a standard method of AR evaluation. SETTING Private hospital, public research center, and a university-based research laboratory. PARTICIPANTS Healthy sperm donors. MAIN OUTCOME MEASURE Changes in the frequency of specific sperm-staining patterns. RESULTS A specific sperm-staining pattern with P. sativum agglutinin was shown to be associated with a recently occurred AR, whereas the absence of staining was typical of reactions having occurred a longer time ago. This phenomenon was used to define a formula for fast AR measure after stimulus addition. An increased sensitivity provided by this formula as compared with a standard evaluation was demonstrated. CONCLUSIONS Fast AR measure is a simple, easy-to-perform method for AR evaluation. It is particularly suitable for testing the effects of rapidly acting stimuli.