Jan Tesarik

High-magnification ICSI overcomes paternal effect resistant to conventional ICSI

Previous studies have shown that repeated intracytoplasmic sperm injection (ICSI) failures can be caused by a paternal effect. Other studies have suggested that ICSI results are compromised if morphologically abnormal spermatozoa are injected into oocytes. This study was undertaken to evaluate the usefulness of a high-magnification optical system to select spermatozoa to be used for ICSI (high-magnification ICSI) in couples with repeated conventional ICSI failures. Couples with two or more previous conventional ICSI failures underwent an additional conventional ICSI attempt, followed by a high-magnification ICSI attempt. The outcomes of the two sequential attempts were compared. In 72 of these patients, sperm DNA integrity was assessed. In the whole group of 125 couples with repeated ICSI failures, high-magnification ICSI improved clinical outcomes (pregnancy, implantation, delivery and birth rates) without affecting biological outcomes (fertilization and cleavage rates, embryo morphology). The improvement of clinical ICSI outcomes was evident both in patients with an elevated degree of sperm DNA fragmentation and in those with normal sperm DNA status. It is concluded that high-magnification ICSI improves clinical outcomes in couples with previous repeated conventional ICSI failures.

Key elements of a highly efficient intracytoplasmic sperm injection technique: Ca2 + fluxes and oocyte cytoplasmic dislocation*

OBJECTIVE To analyze the mechanism by which modifications of the intracytoplasmic sperm injection (ICSI) technique influence success rates. DESIGN Prospective clinical study supplemented with an experimental analysis of Ca2+ fluxes provoked by the injection procedure. SETTING Private hospital and public research center. PATIENTS Patients treated by IVF and ICSI. INTERVENTIONS Intracytoplasmic sperm injection. MAIN OUTCOME MEASURES Fertilization and pregnancy rates and intracellular free Ca2+ concentration. RESULTS The inclusion of vigorous aspiration of oocyte cytoplasm improved outcomes of ICSI. In a series of 100 consecutive cases treated with this technique, the fertilization and pregnancy rates were 87% of total metaphase II oocytes injected and 52% of total treatment cycles, respectively. Enhanced Ca2+ influx into the injected oocytes and dislocation of the oocyte cytoplasm, including the development of a focus of persistent Ca2+ discharge around the injected sperm head, were the main characteristics of this highly successful technique. CONCLUSIONS Vigorous aspiration of oocyte cytoplasm may facilitate fertilization after ICSI by increasing the oocyte Ca2+ load at the time of injection, by establishing a more intimate contact of the injected sperm head with oocyte intracellular Ca2+ stores, or by a conjunction of these mechanisms.

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.

Comparison of Ca2+ responses in human oocytes fertilized by subzonal insemination and by intracytoplasmic sperm injection

OBJECTIVE To evaluate the consequences of bypassing the normal interaction between the sperm and oocyte surfaces for the form of Ca2+ responses developing in oocytes at fertilization. DESIGN Oocytes were fertilized by subzonal insemination (SUZI) (maintaining the normal interaction between cell surfaces of both gametes) or by direct intracytoplasmic sperm injection, and changes in intracellular free Ca2+ concentration were evaluated by confocal laser scanning microscopy after loading oocytes with a fluorescent Ca2+ indicator. SETTING Private hospital, public research center, and university-based laboratory. PATIENTS, PARTICIPANTS Patients participating in an assisted reproduction program. INTERVENTIONS In vitro fertilization, SUZI, intracytoplasmic sperm injection. MAIN OUTCOME MEASURES Changes in intracellular free Ca2+ concentration. RESULTS All oocytes fertilized after SUZI showed an oscillatory Ca2+ response introduced by a short initial phase with faster Ca2+ oscillations. In contrast, oocytes fertilized after intracytoplasmic sperm injection did not show a similar change in the oscillation rhythm. In both cases, Ca2+ increases were propagated throughout the ooplasm in a wave-like manner. CONCLUSIONS The results show that there is a relationship between gamete surface contact and the form of Ca2+ fluxes accompanying fertilization. When the contact between gamete surfaces is skipped by direct sperm injection to the ooplasm, a delayed, truncated Ca2+ response is produced which, however, can maintain the form of Ca2+ waves and Ca2+ oscillations typical of normal fertilization.

Pregnancy and birth after transfer of embryos that developed from single- nucleated zygotes obtained by injection of round spermatids into oocytes

OBJECTIVE To use injection of spermatids into oocytes as a mode of infertility treatment in cases in which spermatozoa are not available. DESIGN Prospective clinical evaluation and case report. SETTING In Vitro Fertilization Unit, Herzliya Medical Centers, Herzliya-on-Sea, Israel. PATIENT(S) Thirteen couples with male factor infertility in which the male partner lacked spermatozoa in the ejaculate or testicular biopsy samples. INTERVENTION(S) Round spermatid injection and elongated spermatid injection into oocytes. MAIN OUTCOME MEASURE(S) Evaluation of the rate of two-pronucleated and single-nucleated zygote development. RESULT(S) The rate of two-pronucleated zygote development after round spermatid injection and elongated spermatid injection was relatively low (27% and 36%, respectively). Single-nucleated zygotes develop more frequently after round spermatid injection and elongated spermatid injection (35% and 17%, respectively) than after intracytoplasmic sperm injection with mature spermatozoa. A normal pregnancy and childbirth resulted from the transfer of 4 cleaving embryos, each of which developed from a single-nucleated zygote in a round spermatid injection treatment cycle with ejaculated spermatids. CONCLUSION(S) Embryos derived from single-nucleated zygotes after spermatid conception can be viable and give rise to an ongoing clinical pregnancy and childbirth.

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.