David E. Battaglia

Unique checkpoints during the first cell cycle of fertilization after intracytoplasmic sperm injection in rhesus monkeys.

Intracytoplasmic sperm injection has begun an era of considerable improvements in treating male infertility. Despite its success, questions remain about the dangers of transmitting traits responsible for male infertility, sex and autosomal chromosome aberrations and possible mental, physical and reproductive abnormalities. We report here the first births of rhesus monkeys produced by intracytoplasmic sperm injection at rates greater or equal to those reported by clinics. Essential assumptions about this process are flawed, as shown by results with the preclinical, nonhuman primate model and with clinically discarded specimens. Dynamic imaging demonstrated the variable position of the second meiotic spindle in relation to the first polar body; consequently, microinjection targeting is imprecise and potentially lethal. Intracytoplasmic sperm injection resulted in abnormal sperm decondensation, with the unusual retention of vesicle-associated membrane protein and the perinuclear theca, and the exclusion of the nuclear mitotic apparatus from the decondensing sperm nuclear apex. Male pronuclear remodeling in the injected oocytes was required before replication of either parental genome, indicating a unique G1-to-S transition checkpoint during zygotic interphase (the first cell cycle). These irregularities indicate that the intracytoplasmic sperm injection itself might lead to the observed increased chromosome anomalies.

Failure of oocyte activation after intracytoplasmic sperm injection using round-headed sperm.

OBJECTIVE To examine the outcome of intracytoplasmic sperm injection (ICSI) with round-headed sperm (globozoospermia). DESIGN Retrospective analysis. SETTING In vitro fertilization laboratory with extensive ICSI experience. PATIENT(S) A patient couple with infertility because of globozoospermia seeking ICSI treatment. MAIN OUTCOME MEASURE(S) Fertilization, cleavage, and pregnancy rates. INTERVENTION(S) Intracytoplasmic sperm injection and calcium ionophore. RESULT(S) This couple experienced only 7% fertilization after ICSI in their first cycle. Treatment of the unfertilized oocytes with calcium ionophore 20 hours after ICSI-induced fertilization and cleavage of 70% of the oocytes. Embryo quality was fair to good. On the second cycle, 8 of the injected oocytes were treated with ionophore immediately after ICSI and the remaining 20 oocytes were untreated. Normal fertilization was achieved in 75% of the treated and 10% of the untreated oocytes. Treatment of these unfertilized oocytes with ionophore 20 hours after ICSI resulted in fertilization in 73%. Pregnancy was not achieved after either ICSI cycle. Ultrastructural analysis indicated multiple structural abnormalities in the sperm. CONCLUSION(S) These results indicate that the round-headed sperm from this patient were incapable of oocyte activation after ICSI. This may be the reason for the frequent ICSI fertilization failure seen with this condition. Current ICSI procedures may not always overcome the infertility associated with globozoospermia, and further study of the etiology of this condition is needed.

Follicular development, ovulation, and corpus luteum formation in cryopreserved human ovarian tissue after xenotransplantation

OBJECTIVE To assess the competency of human frozen/thawed ovarian follicles matured in xenografts to form functioning corpora luteae after human chorionic gonadotropin (hCG) administration. DESIGN Prospective controlled animal study. SETTING University research laboratory. PATIENT(S) Three women (19, 28, and 36 years) who underwent oophorectomy. ANIMAL(S) Nineteen female severe combined immunodeficient (SCID) mice. INTERVENTION(S) Cryopreserved human ovarian tissues were grafted into the s.c. space of bilaterally oophorectomized SCID mice. All the animals were stimulated with pregnant mares serum gonadotropin (PMSG) for 4 weeks starting from 16 weeks after transplantation. Twelve animals were injected with hCG at the end of gonadotropin stimulation. MAIN OUTCOME MEASURE(S) [1] The rate of grafts with growing follicles, with antral follicles, and/or with corpora luteae. [2] The histologic assessment of follicles and corpora luteae. [3] The serum progesterone and estradiol level in animals with corpus luteum in the grafts. RESULT(S) [1] The rate of grafts with growing follicles and with corpora luteae was 33% to 100%, and 28% to 50%, respectively. [2] Corpora luteae in xenografts were all morphologically normal. [3] The progesterone levels were all above 3.0 ng/mL. CONCLUSION(S) This study showed that the cryopreserved human ovarian follicles can be matured to a stage at which they can form functioning corpora luteae in the host animal.

The future of human ovarian cryopreservation and transplantation: fertility and beyond.

OBJECTIVE To review the current progress in ovarian cryopreservation and transplantation and to discuss the obstacles with the clinical application of this technique. DESIGN The literature on ovarian cryopreservation and transplantation was reviewed to facilitate understanding and predict future directions. The studies related to this topic were identified through MEDLINE and other bibliographic databases, focusing on the most recent developments. CONCLUSION(S) The experimental evidence for low-temperature storage of ovarian tissue is encouraging. Although restoration of fertility with cryopreserved ovarian grafts has been successful in various animals, there are uncertainties about the optimum use of stored ovarian tissue in humans. Autotransplantation appears to be promising, but the potential risk of transmitting malignant cells in women with cancer is of great concern. The maturation of primordial follicles with xenotransplantation is encouraging, but the efficacy and the safety of this method need further investigation. Furthermore, the quality of oocytes that have been matured in a host animal is unknown. The development of in vitro culture systems for oocyte maturation is still in its infancy. There are many issues to be resolved in ovarian transplantation before the full clinical use of this emerging technique. Most of all, there is an urgent need to optimize the freeze/thaw procedure and to find the means to protect grafts from ischemia-reperfusion injury. Nevertheless, ovarian transplantation should prove to be clinically useful for women at risk for premature ovarian failure.

Inhibin and reproductive aging in women

The monotropic FSH rise is the sentinel endocrine event that first indicates a woman is approaching the end of her reproductive potential. While a deficiency in inhibin has long been postulated as the immediate cause of the monotropic FSH rise, this has only recently been demonstrated to actually occur. It is our current hypothesis that when the number of preantral follicles in both ovaries drop below a threshold, then there is a subtle decrease in inhibin B which leads to the monotropic FSH rise which, in turn, accelerates follicular depletion and the attainment of the menopause.

The Gonadotropin Secretion Pattern in Normal Women of Advanced Reproductive Age in Relation to the Monotropic FSH Rise

Women of advanced reproductive age are known to demonstrate subtle FSH elevations (monotropic FSH rise) while still retaining ovulatory function. Te purpose of this study was to investigate the hypothesis that the physiologic basis for the monotropic FSH rise is an alteration in the secretion pattern of the GnRH pulse generator. The subjects were 11 normal women age 40–45 years who underwent 24 hours of frequent blood sampling in the follicular (EF) and/or midluteal (ML) phases of spontaneous menstrual cycles. The controls were 11 normal women age 20–25 years. Tlie respective gonadotropin secretion patterns were analyzed for LH pulse frequency, mean LH and FSH levels, and LH pulse amplitude. Time were no differences between the groups for estradiol (E2) and progesterone when the respective cycle phases were compared. The 24-hour mean FSH level was significantly increased in the older women in both the EF and ML phases. There were no differences between the groups in either cycle phase for LH pulse frequency, LH pulse amplitude, and mean LH levels. The results lend no support to the hypothesis that a slowing or other alteration of the GnRH pulse generator is the basis for the monotropic FSH rise in older ovulatory women. Other possibilities include the dynamics of E2 secretion or changes in FSH-modulating peptides (ie, inhibin) in these women.

Localization and developmental fate of ovoperoxidase and proteoliaisin, two proteins involved in fertilization envelope assembly

Fertilization of the sea urchin egg leads to the assembly of an extracellular matrix, the fertilization envelope. Ovoperoxidase, the enzyme implicated in hardening the fertilization envelope, is inserted into the assembling structure via a Ca2+-dependent interaction with the protein proteoliasin (P. Weidman and B. M. Shapiro, 1987, J. Cell Biol. 105, 561-567). In the present report, polyclonal antisera were raised to ovoperoxidase and proteoliasin (purified from eggs of Strongylocentrotus purpuratus) and characterized by Western blot analysis and an enzyme-linked immunoabsorbent assay (ELISA). By indirect immunofluorescence microscopy all cortical granules of unfertilized eggs, as well as the fertilization envelope, contained both proteoliasin and ovoperoxidase. At the ultrastructural level both proteins are localized to the electron-dense spiral lamellae of the cortical granules. Western blot analysis revealed that ovoperoxidase and proteoliasin persist in early embryos until hatching, but are absent from later developmental stages. Homogenates of eggs of several other echinoderm species (Strongylocentrotus droebachiensis, Strongylocentrotus franciscanus, Pisaster ochraceus, Dendraster excentricus, and Lytechinus pictus) also contain proteins antigenically similar to ovoperoxidase and proteoliaisin, indicating that many echinoderms utilize a similar strategy for assembly of the fertilization envelope. The results underline the need for postsecretory controls in the extracellular matrix modifications that accompany the cortical reaction.

Circulating levels of growth hormone, insulin-like growth factor-I and growth hormone binding protein in normal women of advanced reproductive age

OBJECTIVE Women experience an age‐related decline in fertility despite regular ovulatory cycles and normal production of ovarian steroids. Growth hormone and IGF‐I are both reported to decline with age, and there is evidence that both hormones promote intraovarian actions of gonadotrophins. The purpose of this study was to characterize circulating levels of GH and IGF‐I in normal, older reproductive age women with ovulatory cycles.

The anterior pituitary response to a gonadotropin-releasing hormone challenge test in normal older reproductive-age women

OBJECTIVE To assess the pituitary responsiveness to GnRH stimulation of premenopausal women relative to age. DESIGN Older and younger reproductive-age women underwent the GnRH stimulation test in the early follicular phase of the menstrual cycle. SETTING Female subjects in an academic research environment. PATIENTS Women aged 21 to 44 years consisting of normal volunteers and infertility patients. INTERVENTIONS Gonadotropin-releasing hormone was administered intravenously between days 2 and 4 of the menstrual cycle. Blood samples were collected from -20 minutes before to 120 minutes after administration. MAIN OUTCOME MEASURE Luteinizing hormone, FSH, inhibin, and E2 levels. RESULTS No significant difference in baseline values existed between older and younger women with regard to LH, inhibin, and E2, but basal FSH levels were higher in older women. A significantly diminished percent of LH and percent FSH change above baseline occurred 30 minutes after GnRH administration in the older women compared with younger women. No change in inhibin or E2 levels could be detected during the sampling period. CONCLUSIONS The present study demonstrates marked attenuation of the acute pituitary LH response (sensitivity) to GnRH stimulation in older women when compared with a younger cohort.

Ovarian Follicle Recruitment and Secretory Capacity in Women of Advanced Reproductive Age

The final common pathway in reproductive aging in women is attrition of the initial endowment of ovarian follicles, predominantly through atresia. Mathematical models based on morphometric studies have been used to hypothesize that follicular depletion is bi-exponential, with an accelerated loss occurring after about age 38 (1). Among individual women, the age of menopause and the length of the reproductive life span (both ultimately dependent on follicle depletion) are quite variable, yet the controlling factors are unknown. There is an intriguing temporal relationship between the onset of the accelerated phase of follicle atresia, decline in fecundity, and the monotropic rise in follicle stimulation hormone (FSH). In addition, older reproductive-age women exhibit an earlier onset of the intercycle rise in FSH associated with earlier recruitment and ovulation of the dominant follicle. The relationship, if any, between these phenomena remains speculative. This chapter on studies of endocrine and ovarian changes in older reproductiveage women will attempt to describe what is known about dominant follicle recruitment and function in women of advanced reproductive age.