'Why have women not returned to use their frozen oocytes?': a 5-year follow-up of women after planned oocyte cryopreservation.

RESEARCH QUESTION: What are the reproductive choices and retrospective reflections of women at least 4 years after planned oocyte cryopreservation (POC)? DESIGN: This was an internet survey, using the REDCap application, of women who underwent POC, at a single-centre university-affiliated IVF unit, 4-8 years before the survey. The questionnaire addressed reproductive choices and outcomes following POC. RESULTS: Seventy-nine women who underwent POC during 2011-2014 were invited to participate, and 70 (89%) responded. Mean age at cryopreservation was 37.1 ± 2.4 (range 30-41) years, mean age at study participation 42.6 ± 2.6 (range 35-48) years, and mean time from first cryopreservation cycle to study participation 5.5 ± 1.3 (range 4-8) years. The main retrospectively reported reason for POC was not wanting to become pregnant without a partner (59, 84%). During the follow-up period, 44 women (63%) attempted to conceive either naturally or by assisted reproductive technology using fresh or cryopreserved oocytes. Of those, 28 women achieved a live birth (64% of those who tried to conceive). Fourteen respondents (20% of all respondents) reported using their cryopreserved oocytes, and three (21%) achieved a birth using those oocytes. Fifteen women (34%) of those who tried to conceive used donor spermatozoa. CONCLUSIONS: The most common reasons for not using frozen oocytes were achieving pregnancy without frozen oocytes or preferring not to have a child without a partner. A considerable proportion of women who had POC and were not interested in being a single parent by choice eventually try to conceive using donor spermatozoa several years later.

Biopsy-induced inflammatory conditions improve endometrial receptivity: the mechanism of action.

A decade ago, we first reported that endometrial biopsy significantly improves the success of pregnancy in IVF patients with recurrent implantation failure, an observation that was later confirmed by others. Recently, we have demonstrated that this treatment elevated the levels of endometrial pro-inflammatory cytokines and increased the abundance of macrophages (Mac) and dendritic cells (DCs). We therefore hypothesised that the biopsy-related successful pregnancy is secondary to an inflammatory response, and aimed at deciphering its mechanism of action. Supporting our hypothesis, we found that the pro-inflammatory TNFα stimulated primary endometrial stromal cells to express cytokines that attracted monocytes and induced their differentiation into DCs. These monocyte-derived DCs stimulated endometrial epithelial cells to express the adhesive molecule SPP1 (osteopontin (OPN)) and its receptors ITGB3 and CD44, whereas MUC16, which interferes with adhesion, was downregulated. Other implantation-associated genes, such as CHST2, CCL4 (MIP1B) and GROA, were upregulated by monocyte-derived Mac. These findings suggest that uterine receptivity is mediated by the expression of molecules associated with inflammation. Such an inflammatory milieu is not generated in some IVF patients with recurrent implantation failure in the absence of local injury provoked by the biopsy treatment.

Endometrial inflammation and effect on implantation improvement and pregnancy outcome.

Implantation failure, which is presently the major barrier in human fertility, is attributed, in many cases, to the failure of the uterus to acquire receptivity. The transition into a receptive uterus includes cellular changes in the endometrium and the modulated expression of different cytokines, growth factors, transcription factors, and prostaglandins. These molecules partake in the generation of an inflammatory response followed by the recruitment of immune cells. These cells have shown to be involved in the maternal immune tolerance toward the implanted embryo as well as in the maternal-fetus interaction during pregnancy. Most of the accumulated evidence indicates that embryo implantation is associated with an active Th1 inflammatory response while a Th2-humoral inflammation is required for pregnancy maintenance. Yet, recent findings suggest that a Th1 inflammatory response is also necessary for the acquisition of uterine receptivity. This notion was originally suggested by reports from our and other clinical centers worldwide that IVF patients with repeated implantation failure subjected to endometrial biopsy exhibit a substantial improvement in their chances to conceive. These findings, followed by the demonstration of an elevated pro-inflammatory cytokine/chemokine expression, as well as an increased abundance of immune cells, in the endometrium of these patients, raised the idea that acquisition of uterine receptivity is closely associated with an inflammatory response. This review summarizes the molecular and biochemical evidence that confirm this notion and proposes a mechanism by which injury-induced inflammation improves uterine receptivity and the subsequent pregnancy outcome.

A fast signal-induced activation of Poly(ADP-ribose) polymerase: a novel downstream target of phospholipase c.

We present the first evidence for a fast activation of the nuclear protein poly(ADP-ribose) polymerase (PARP) by signals evoked in the cell membrane, constituting a novel mode of signaling to the cell nucleus. PARP, an abundant, highly conserved, chromatin-bound protein found only in eukaryotes, exclusively catalyzes polyADP-ribosylation of DNA-binding proteins, thereby modulating their activity. Activation of PARP, reportedly induced by formation of DNA breaks, is involved in DNA transcription, replication, and repair. Our findings demonstrate an alternative mechanism: a fast activation of PARP, evoked by inositol 1,4,5,-trisphosphate-Ca(2+) mobilization, that does not involve DNA breaks. These findings identify PARP as a novel downstream target of phospholipase C, and unveil a novel fast signal-induced modification of DNA-binding proteins by polyADP-ribosylation.

Ovulation-selective genes: the generation and characterization of an ovulatory-selective cDNA library.

Ovulation-selective/specific genes, that is, genes preferentially or exclusively expressed during the ovulatory process, have been the subject of growing interest. We report herein studies on the use of suppression subtractive hybridization (SSH) to construct a 'forward' ovulation-selective/specific cDNA library. In toto, 485 clones were sequenced and analyzed for homology to known genes with the basic local alignment tool (BLAST). Of those, 252 were determined to be nonredundant. Of these 252 nonredundant clones, 98 were analyzed by probing mouse preovulatory and postovulatory ovarian cDNA. Twenty-five clones (26%) failed to show any signal, and 43 cDNAs tested thus far display a true ovulation-selective/specific expression pattern. In this communication, we focus on one such ovulation-selective gene, the fatty acid elongase 1 (FAE-1) homolog, found to be localized to the inner periantral granulosa and to the cumulus granulosa cells of antral follicles. The FAE-1 gene is a beta-ketoacyl-CoA synthase belonging to the fatty acid elongase (ELO) family, which catalyzes the initial step of very long-chain fatty acid synthesis. All in all, the present study accomplished systematic identification of those hormonally regulated genes that are expressed in the ovary in an ovulation-selective/specific manner. These ovulation-selective/specific genes may have significant implications for the understanding of ovarian function in molecular terms and for the development of innovative strategies for both the promotion of fertility and its control.

Molecular control of meiosis.

The animal cell cycle consists of a round of chromosomal DNA replication in S-phase, followed by segregation of the replicated chromosomes into the daughter nuclei during M-phase. In most animal cells, gap phases termed G(1) and G(2) are introduced between the M- and S-phases, respectively. Meiosis is a particular example of cell division occurring in germ cells. This specialized cell cycle consists of two successive rounds of chromosome segregation that follow a round of DNA replication. Meiosis produces progeny cells with half as many chromosomes as their parents, thus making sexual reproduction possible. This review is concerned with the factors that have been implicated in the control of meiosis, although research in progress may reveal additional regulatory processes.

Epidermal growth factor induces maturation of rat follicle-enclosed oocytes.

Gonadotropin-induced differentiation of ovarian granulosa cells in culture is inhibited by epidermal growth factor (EGF). The present study was undertaken to test a possible inhibitory effect of EGF on LH-induced maturation of rat follicle-enclosed oocytes. We have found that EGF not only failed to affect LH action but served by itself as an inducer of maturation of follicle-enclosed oocytes. EGF action on the oocytes was dose and time dependent and could be prevented by (Bu)2 cAMP. The response of the oocytes was specific to EGF and could not be elicited by other growth factors such as nerve growth factor and insulin. The response to EGF was not limited to the large antral follicles, as oocytes enclosed by small antral follicles (less than 0.4 mm) were induced to mature by EGF as well. In addition, we have demonstrated that oocytes, induced to mature by EGF, are concomitantly uncoupled from the follicular cells. Based on these results we suggest that EGF may terminate the transfer of a follicular inhibitor to the oocyte. It is also possible, however, that EGF induces oocyte maturation by a mechanism independent of its effect on communication between the cellular components of the follicle.

Binding of human chorionic gonadotropin by rat cumuli oophori and granulosa cells: a comparative study.

Cumulus oocyte complexes isolated from PMS gonadotropin-primed immature rats bind [125I]hCG. Unlabeled hCG and ovine LH compete with the labeled hormone for binding sites, while rat FSH and ovine PRL are without effect. Since specific binding of [125I]hCG could not be detected in preparations of oocytes from which the cumuli had been removed, it appears that the binding exhibited by the complex can be attributed to the cumulus cells. The concentration dependence of binding is consistent with the presence of one population of high affinity (apparent Kd, 1.4 X 10(-10) M) binding sites (223 +/- 33 sites/cumulus cell). Rat granulosa cells bind hCG with equivalent apparent affinity and specificity but contain more sites (2060 +/- 180/cell) than cumulus cells.

Receptors for gonadotropin releasing hormone are present in rat oocytes.

Specific receptors for gonadotropin releasing hormone (GnRH) in the rat oocyte have been identified by using two independent methods. Light microscopic autoradiography, utilizing an iodinated biologically active photoaffinity derivative of GnRH, revealed specific binding of the neurohormone to rat oocytes. Furthermore, the presence of GnRH-receptor is also evident from indirect fluorescent immunocytochemistry that shows binding of GnRH-receptor antibodies to rat oocytes which is neither detected with non immune serum nor with antiserum depleted of GnRH-receptor antibodies. These antibodies to the GnRH-receptor, also bind to both cumulus and granulosa cells but not to rat basophilic leukemia cells. The presence of specific GnRH receptors on rat oocytes provides an experimental basis for understanding the molecular events involved in GnRH-induced oocyte maturation.

Effect of luteinizing hormone on respiration of the preovulatory cumulus oophorus of the rat.

The effect of luteinizing hormone (LH) on the respiration of isolated cumulus cell complexes (the oocyte surrounded by cumulus granulosa cells) obtained from immature Sprague-Dawley rats injected with 10 IU pregnant mare's serum gonadotropin on day 30 was investigated. The cell complexes were isolated from preovulatory follicles of rats killed at specific time intervals on the day preceding ovulation, i.e., on day 32. The samples were incubated in Eagle's tissue culture medium. Oxygen uptake was recorded either with the Cartesian diver technique or with a recently described microspectrophotometric technique using hemoglobin as an indicator. Exposure to exogenous bovine LH in vitro or in vivo or to endogenous LH, i.e., the preovulatory LH-surge, resulted in a marked decrease in respiratory activity of the cumulus cell complex, as revealed by both techniques. The cumuli exposed to LH showed an oxygen uptake of approximately 40-65% of the control cumuli. The results suggest that LH has a direct effect on this cell complex resulting in a decreased oxidative metabolism.

Induction of maturation in rat follicle-enclosed oocyte by forskolin.

The diterpene forskolin, which was found to be a potent and reversible activator of adenylate cyclase in intact tissues as well as in broken cell preparations, was employed to investigate the role of cAMP in the induction of oocyte maturation. We have found that forskolin can mimic the effect of LH on the ovarian follicle stimulating both cAMP accumulation and oocyte maturation. These findings suggest that LH-induced maturation in follicle-enclosed oocytes is a cAMP-mediated response.

Dissociation between the inhibitory and the stimulatory action of cAMP on maturation of rat oocytes.

The possible mediatory role of cAMP in the induction of oocyte maturation by luteinizing hormone (LH) is not yet clear since evidence for both inhibitory and stimulatory actions of the nucleotide on the oocyte has been provided. To elucidate the role of cAMP in regulation of oocyte meiosis we tried in the present study to dissociate between the inhibitory and stimulatory action of this nucleotide on oocyte maturation. To induce maturation, oocytes enclosed by their follicles were transiently exposed to either dibutyryl cAMP (dbcAMP) or to the phosphodiesterase inhibitor methylisobutylxanthine (MIX). Inhibition of maturation was obtained by the addition of the above agents to either follicle-enclosed oocytes incubated in the presence of LH or isolated cumulus-free oocytes that mature spontaneously in vitro. We found that inhibition of oocyte maturation is obtained by a relatively low dose of either dbcAMP or MIX while higher concentrations of these agents are required to induce oocyte maturation. Coupling of the oocyte to the cumulus cells, as expressed by the fraction of labeled uridine transferred from the cumulus cells to the oocyte following exposure of the follicle-enclosed cumulus-oocyte complex to MIX, was also determined. We found that uncoupling of the oocyte from the cumulus cells corresponded with the induction, but not inhibition of oocyte maturation, both by its concentration dependence and time-course. We suggest that cAMP has a dual role in regulation of oocyte maturation. Lower levels of the nucleotide act to maintain meiotic arrest, while elevated levels of cAMP mediate LH action to induce meiosis resumption.

Rat oocyte maturation: role of calcium in hormone action.

We studied the role of extracellular calcium (Ca0) in oocyte maturation and oocyte-cumulus cells interaction in rat follicles in vitro. Luteinizing hormone (LH) or a gonadotropin-releasing hormone analog (GnRHa) induced full maturation at [Ca0] = 1.3 mM. At [Ca0] = 0.6 mM, maturation induced by LH or GnRHa was inhibited by 65%. Chelatin of [Ca0] resulted in 45% maturation and neither hormone caused a further increase of maturation. [Ca0] = 20 mM enhanced the response to suboptimal concentrations of GnRHa but inhibited that to LH. Divalent cation ionophores caused [Ca0]-dependent maturation, which was fully inhibited by dibutyryl cAMP. Changes in [Ca0] also affected oocyte-cumulus interaction. At [Ca0] = 1.3 mM, either LH or GnRHa caused partial dispersion of the cumulus. Chelation of [Ca0] also resulted in an almost complete dispersion of the cumulus. The ionophores, however, caused maturation with the oocyte-cumulus complex preserved intact. Our data suggest that GnRHa may induce maturation via cAMP-sensitive calcium mobilization into the oocyte-cumulus-granulosa complex.

Rat oocytes induced to mature by epidermal growth factor are successfully fertilized.

Epidermal growth factor (EGF), which is a known mitogen, can also induce resumption of meiosis in the rat oocyte. The present study was designed in an attempt to elucidate whether oocytes, induced to mature by EGF in a follicle-enclosed oocyte culture, are fertilizable and can further develop into two-cell embryos. For further clarification of the effect of EGF on steroidogenesis in the ovarian follicle, progesterone concentrations were determined by radioimmunoassay. We found that oocytes matured by EGF (100 ng/ml) were successfully fertilized. Even though their rate of fertilization was relatively lower as compared to that of oocytes stimulated by luteinizing hormone (LH) both in vitro and in vivo (61%, 79%, and 83% respectively), once fertilized they exhibit an equal potential for further development (EGF: 48%, LH: 45%). On the other hand, EGF-induced progesterone production was very poor. These findings strongly support the idea that both mitogenesis and meiogenesis can be mediated by common signals. The results further suggest that progesterone production and oocyte maturation, in the rat, are independent events.

Fertilization and early development of rat oocytes induced to mature by forskolin.

Forskolin has been shown to successfully induce maturation of rat oocytes as assessed by morphological markers. The present study was designed in an attempt to elucidate whether oocytes, induced to mature by forskolin (10(-4) M, group A) in a follicle-enclosed oocyte culture, are fertilizable and can further develop into two-cell embryos. Oocytes exposed in vitro to either luteinizing hormone (LH, 5 micrograms/ml, group B) or a GnRH agonist analogue (10(-7) M, group C) as well as oocytes that underwent maturation in vivo (group D), served as positive controls. We found that similar rates of fertilization were obtained in the experimental and all of the above mentioned control groups (A = 78.9 +/- 4.2%, B = 77.9 +/- 3.1%, C = 77.5 +/- 5.5% and D = 84.7 +/- 2.7%). Cleavage rate of fertilized eggs from group A was significantly higher than that of eggs from groups B & C, and similar to that of eggs from group D (A = 63.1 +/- 6.7%, B = 37.8 +/- 4.9%, C = 50.0 +/- 4.1%, D = 67.8 +/- 4.1%). Using functional parameters we hereby demonstrate that forskolin and LH are at least equally potent in producing fertilizable eggs that have a high potential of development into two cell embryos. These results further support the idea that cAMP is a mediator of LH action in inducing oocyte maturation.

Dissociation between the direct stimulatory and inhibitory effects of a gonadotropin-releasing hormone analog on ovarian functions.

The paradoxical effects of gonadotropin-releasing hormone (GnRH) on the ovary have hitherto been believed to result from different regimens of administration; an acute treatment was shown to stimulate the ovary while chronic administration of the hormone inhibited LH-induced responses. In the present report we demonstrate that a single injection of a GnRH analog (D-Ala6)des-Gly10-GnRH-N-ethylamide (GnRHa, 2 micrograms/rat) is sufficient to obtain a significant inhibition (75%) of hCG-induced ovulation in PMSG-primed, either intact or hypophysectomized, immature rats. Inhibition of ovarian development, in terms of growth and ovulation, by multiple injections with GnRHa (2 micrograms/rat, twice daily for 3 days) could be obtained only upon administration of the hormone at early stages of follicular development, i.e. concomitantly with the PMSG injection. When administered after PMSG, GnRHa could not inhibit the ovary but rather induced ovulation by itself in the absence of hCG. A 12-24 h delay in initiation of GnRHa treatment triggered 65% of the rats to ovulate while a delay of 48 h resulted in 100% ovulation. Under both regimes of GnRHa administration, either the inhibitory or the stimulatory, the oocytes of the treated rats were induced to resume meiotic maturation. Since under the inhibitory regime ovulation did not occur, maturation was followed by a massive degeneration of the oocytes trapped within their follicles. These findings demonstrate that the follicular stage of development rather than the dose and/or duration of GnRHa administration determines whether GnRHa inhibits ovarian growth and ovulation, while the competence of the oocytes to respond to the GnRHa stimulus and mature is independent of hormonal priming.

Expression and modification of PKA and AKAPs during meiosis in rat oocytes.

Meiosis in oocytes is initiated during fetal life, arrested around birth and resumed after puberty. Meiotic arrest is controlled by a cAMP-dependent protein kinase (PKA)-mediated cAMP action. We examined oocytes for the presence and modulation of the regulatory (R) subunits of PKA and the A-kinase anchoring proteins (AKAPs) that target PKA to specific subcellular locations. We found that rat oocytes express the two regulatory subunit isoforms, RI and RII of PKA. Immunocytochemistry revealed that the regulatory subunits underwent cellular translocation upon resumption of meiosis. We also demonstrated the presence of a novel 140 kDa AKAP, AKAP140 that exhibited a retarded electrophoretic motility at reinitiation of meiosis. The mobility shift of AKAP140 was susceptible to alkaline phosphatase and prevented by inhibition of p34cdc2 kinase. We conclude that rat oocytes express AKAP140 that is phosphorylated during meiosis. AKAP140 phosphorylation is sensitive to p34cdc2 kinase inhibitors. We hypothesize that AKAP140 and its phosphorylation state may influence the translocation of the R subunits of PKA throughout resumption of meiosis.

Regulation of oocyte maturation. The role of cAMP.

We suggest that cAMP is a key molecule in regulation of oocyte maturation. In the small follicle, tonic levels of cAMP are continuously transferred to the oocytes to maintain meiotic arrest. In the preovulatory follicle, in response to LH, cAMP levels are elevated. These increased concentrations of the nucleotide affect the cumulus cells and interrupt communication in the cumulus-oocyte complex. Under these conditions the flow of cAMP to the oocyte declines, inhibition is relieved, and meiosis is resumed.

Serum bioactive and immunoreactive follicle-stimulating hormone in oligozoospermic and azoospermic men: application of a modified granulosa cell bioassay.

Serum follicle-stimulating hormone (FSH) levels measured by radioimmunoassay (RIA) usually correlate well with the rate of spermatogenesis. However, in certain cases this correlation does not exist. The purpose of this study was to establish a reliable bioassay of FSH for the andrological clinic. Follicle-stimulating hormone was measured by both standard RIA and bioassay in 98 men subgrouped into normospermic, oligospermic, and azoospermic. Bioactivity of FSH was determined using in vitro cultures of granulosa cells utilizing progesterone measurements for assessing FSH activity. Results of FSH levels obtained by both methods correlated well (r = 0.55, P less than 0.01) within themselves, and both correlated negatively and significantly with sperm concentration. The ratio between bioactivity and immunoreactivity of FSH did not correlate with sperm density. Thus, the decrease in sperm concentration and other sperm variables resulting from a germinal epithelial dysfunction was not mediated or associated with low biological activity of FSH. The application of this method can be of clinical value in cases where a discrepancy is found between serum RIA-FSH levels and sperm quality.

Experimental extension of the time interval between oocyte maturation and ovulation: effect on fertilization and first cleavage.

OBJECTIVE: To test the hypothesis that impaired fertility in human patients with high LH concentrations throughout the follicular phase of the menstrual cycle reflects premature maturation of their oocytes. DESIGN: Previous information that resumption of meiosis is induced by lower hCG concentrations than that required for stimulation of follicular rupture was confirmed and used for establishment of a rat animal model in which oocyte maturation and ovulation can be separated experimentally. In further experiments hypophysectomized, pregnant mare serum gonadotropin (PMSG)-primed, immature female rats injected with 1.1 IU of hCG, a dose found to induce maturation in 72.9% +/- 6% of the rats with no effect on ovulation, were administered with a second injection of an ovulatory dose (4 IU) of hCG, 24 hours later. The ovulated eggs were subjected to IVF. RESULTS: Fertilization and first cleavage in oocytes recovered from our experimental animal model were similar to that observed in control PMSG-primed, either hypophysectomized or intact rats, treated by a single injection of 4 IU of hCG. CONCLUSIONS: The extension of the time interval between oocyte maturation and ovulation in the rat does not result in a lower rate of fertilization or a reduced incidence of cleavage. However, an inferior developmental capacity of these embryos cannot be ruled out.