Premature progesterone rise in ART-cycles

Premature rise of progesterone during the late follicular phase in stimulated IVF cycles is a frequent event and its effect on the endometrial receptivity and on the ART (Assisted Reproductive Technique) – outcome has become a matter of intense debate and research. An emerging body of evidence demonstrates that premature progesterone rise does have a negative impact on the outcome of the ART-success. Until now, the exact cause of progesterone elevation is not fully clear, however lately published studies points to the fact, that premature progesterone elevation might be caused by enhanced FSH stimulation. The impact of elevated peripheral progesterone levels seems to be mainly on the endometrium and the window of implantation, leading to an asynchrony between the endometrium and the developing embryo. Hence, new data show additional an influence on the embryo quality. This review aims to summarize the up-to-date knowledge on the causes of premature progesterone rise during hormonal stimulation, on its influence on endometrial receptivity and embryo quality, on the impact on pregnancy and live birth rates as well as on the possible strategies to prevent this event or to deal with premature progesterone elevation in case it could not be avoided.     

The effects of metabolic stress on plasma progesterone in healthy volunteers and schizophrenic patients.

A number of preclinical studies suggest that progesterone may play an important role in the stress response, however, the effects of stress on progesterone in humans has not been established. Also, several lines of evidence indicate that schizophrenia may be associated with abnormal neurobiological responses to stress, but the effects of stress on progesterone in schizophrenia has not been investigated. The purpose of the present study was to examine the effects of stress on plasma progesterone and cortisol in healthy subjects and to determine if schizophrenic patients have altered stress-induced plasma progesterone levels compared to normal controls. Stress was induced through administration of 2-deoxyglucose (2DG), a glucose analog that impairs glucose metabolism resulting in a clinical state comparable to hypoglycemia. There were significant increases in plasma progesterone and cortisol levels following 2DG-induced glucoprivic stress in healthy controls. There was no relationship between stress related progesterone and cortisol elevations. Schizophrenic patients, in comparison to controls, had significantly greater 2DG-induced elevations in progesterone levels but no differences in stress-related cortisol levels. There was evidence that basal progesterone and cortisol levels were elevated in the schizophrenic patients. The implications of these data are discussed.     

Effects of cryopreservation on Ca2+ signals induced by membrane depolarization, caffeine, thapsigargin and progesterone in boar spermatozoa

Although the fertilizing ability of spermatozoa is greatly reduced after freezing, complete understanding of alterations induced by cryopreservation has not been elucidated. The present study evaluates the effects of cryopreservation on the Ca2+ handling of boar spermatozoa using several sperm activators. Intracellular Ca2+ signals from single spermatozoa were measured using confocal Ca2+ imaging of unfrozen samples and of other spermatozoa after having been frozen. Elevation of the external K+ concentration elicited a three times larger Ca2+ increase in fresh spermatozoa than in cryopreserved spermatozoa. Caffeine elicited Ca2+ transients with some oscillations in the fresh spermatozoa, but not in the thawed spermatozoa. Depletion of the Ca2+ store with thapsigargin induced a rapid rise in Ca2+ in the control but generated a smaller increase of Ca2+ after thawing. Exposure to progesterone induced a biphasic rise of the Ca2+ level in the fresh spermatozoa only. Sperm viability was reduced by cryopreservation. Resting Ca2+ levels in fresh and cryopreserved spermatozoa were similar. Longer incubation (2.5 h) of thawed spermatozoa partly recovered the Ca2+ response to the interventions. These results suggest that cryopreservation reduces the responsiveness of spermatozoa to depolarization, modulators of the internal Ca2+ store and progesterone in terms of the Ca2+ signal, thus providing a possible mechanism for reduced fertility observed in cryopreserved boar spermatozoa.     

Termination of unwanted pregnancy in a llama with cloprostenol and subsequent pregnancy: a case report

A 5-yr-old female llama was presented by its owner for an elective abortion. The llama was accidentally bred to an unknown, and possibly related, male about 2.5 mo prior to presentation. The pregnancy was first confirmed by ultrasonography then cloprostenol (150 microg im) was administered once. Cloprostenol, an analogue of prostaglandin F2alpha, was chosen in preference to natural PGF2alpha due to reported adverse reactions in llamas to this abortifacient. Blood serum progesterone levels decreased rapidly from 5.7 to < 0.2 ng/ml at 0 to 60 h post injection, respectively. The aborted fetus was expelled at approximately 108 h after the injection. Twenty days post abortion the llama was rebred. At 27 and 87 d post breeding, pregnancy was indicated first by male refusal and then by elevated serum progesterone concentrations and was confirmed by ultrasonography. Following a 355 d gestation period, a male cria was born. This case provides evidence that an abortion can be induced with cloprostenol without an adverse effect on future fertility in the llama.     

Progesterone protects normative anxiety-like responding among ovariectomized female mice that conditionally express the HIV-1 regulatory protein,Tat, in the CNS

Increased anxiety is co-morbid with human immunodeficiency virus (HIV) infection. Actions of the neurotoxic HIV-1 regulatory protein, Tat, may contribute to affective dysfunction. We hypothesized that Tat expression would increase anxiety-like behavior of female GT-tg bigenic mice that express HIV-1 Tat protein in the brain in a doxycycline-dependent manner. Furthermore, given reports that HIV-induced anxiety may occur at lower rates among women, and that the neurotoxic effects of Tat are ameliorated by sex steroids in vitro, we hypothesized that 17β-estradiol and/or progesterone would ameliorate Tat-induced anxiety-like effects. Among naturally-cycling proestrous and diestrous mice, Tat-induction via 7 days of doxycycline treatment significantly increased anxiety-like responding in an open field, elevated plus maze and a marble-burying task, compared to treatment with saline. Proestrous mice demonstrated less anxiety-like behavior than diestrous mice in the open field and elevated plus maze, but these effects did not significantly interact with Tat-induction. Among ovariectomized mice, doxycycline-induced Tat protein significantly increased anxiety-like behavior in an elevated plus maze and a marble burying task compared to saline-treated mice, but not an open field (where anxiety-like responding was already maximal). Co-administration of progesterone (4 mg/kg), but not 17β-estradiol (0.09 mg/kg), with doxycycline significantly ameliorated anxiety-like responding in the elevated plus maze and marble burying tasks. When administered together, 17β-estradiol partially antagonized the protective effects of progesterone on Tat-induced anxiety-like behavior. These findings support evidence of steroid-protection over HIV-1 proteins, and extend them by demonstrating the protective capacity of progesterone on Tat-induced anxiety-like behavior of ovariectomized female mice.     

Clinical utility of freeze-all approach in ART treatment: A mini-review

A significant proportion of couples at reproductive age rely on assisted reproductive technology to overcome infertility. In vitro fertilisation (IVF) involves typically the use of exogenous gonadotropins to stimulate the ovary to produce oocytes, which are collected surgically. After fertilization by conventional IVF or intracytoplasmic sperm injection (ICSI), embryos are cultured in the embryology laboratory for a few days before being replaced into the uterus (fresh embryo transfer). Spare embryos can be vitrified and stored in liquid nitrogen to be transferred in a subsequent cycle. Over the years, concerns have arisen about possible adverse outcomes of transferring embryos back to the uterus immediately after controlled ovarian stimulation (COS) as regards to obstetrical and perinatal outcomes. It has been suggested that high hormonal levels during COS could create a relatively hostile environment for embryo implantation whilst increasing the risk of ovarian hyperstimulation syndrome (OHSS). With the remarkable improvement of vitrification as an alternative to the slow-freezing technique for human embryos, a new strategy the so-called “freeze-all” (FA) or “elective frozen embryo transfer” (eFET) was introduced. This approach involves COS, followed by the elective cryopreservation of the entire cohort of viable embryos to be transferred to the uterus in subsequent cycles in a possibly more physiological environment, thus avoiding the supra-physiologic hormonal levels observed during COS. The initial reports suggested that this policy could lead to improved pregnancy rates and reduced perinatal complications, which resulted in a steady increase and widespread use of FA globally. However, as data accumulated, it became clear that the use of FA to unselected couples undergoing ART offered no additional benefits over the conventional approach. Nonetheless, current evidence based on randomized controlled trials and observational studies indicates that FA might be justified in selected clinical scenarios, such as those involving the risk of OHSS. By contrast, there is a lack of evidence to support the FA policy for other indications, such as implantation failure or high progesterone levels on the trigger day. This review summarizes the clinical effectiveness of FA with the main focus on the health of offspring.     

Elective frozen-thawed embryo transfer (FET) in women at risk for ovarian hyperstimulation syndrome

Elective cryopreservation of cultured embryos has become a treatment option for women at risk for ovarian hyperstimulation syndrome (OHSS). The aim of our study was to investigate the outcome of elective cryopreservation and consecutive frozen-thawed embryo transfer (FET) in a large IVF clinic in Austria. A total of 6104 controlled ovarian hyperstimulation cycles (COH) were performed on 2998 patients including 200 patients (6.7%) who were undergoing elective cryopreservation and FET due to high risk of OHSS. We estimated the cumulative live birth rate using the Kaplan-Meier method and evaluated independent predictors for successful live births with a Cox model. A total of 270 frozen-thawed embryo transfers were performed on 200 patients with up to 4 transfers per patient. The first embryo transfer showed a live birth rate of 42.0%, the second transfer showed a cumulative rate of 58.5%. After a total of 4 FETs from the same COH cycle, a cumulative live birth rate of 61.0% per COH cycle could be achieved. Four cases of OHSS occurred amongst these patients (2.0%), all of them of moderate severity. Multivariate analysis identified maternal age, the use of assisted hatching and the number of embryos transferred at the blastocyst stage as independent predictors for cumulative live birth. Our study clearly suggests that elective FET is safe and shows excellent cumulative live birth rates. This concept can, therefore, be used to avoid the severe adverse events caused by COH and the inefficient use of cultured embryos.     

Elevated Progesterone Levels on the Day of Oocyte Maturation May Affect Top Quality Embryo IVF Cycles

In contrast to the impact of elevated progesterone on endometrial receptivity, the data on whether increased progesterone levels affects the quality of embryos is still limited. This study retrospectively enrolled 4,236 fresh in vitro fertilization (IVF) cycles and sought to determine whether increased progesterone is associated with adverse outcomes with regard to top quality embryos (TQE). The results showed that the TQE rate significantly correlated with progesterone levels on the day of human chorionic gonadotropin (hCG) trigger (P = 0.009). Multivariate linear regression analysis of factors related to the TQE rate, in conventional IVF cycles, showed that the TQE rate was negatively associated with progesterone concentration on the day of hCG (OR was -1.658, 95% CI: -2.806 to -0.510, P = 0.005). When the serum progesterone level was within the interval 2.0–2.5 ng/ml, the TQE rate was significantly lower (P <0.05) than when the progesterone level was < 1.0 ng/ml; similar results were obtained for serum progesterone levels >2.5 ng/ml. Then, we choose a progesterone level at 1.5ng/ml, 2.0 ng/ml and 2.5 ng/ml as cut-off points to verify this result. We found that the TQE rate was significantly different (P <0.05) between serum progesterone levels < 2.0 ng/ml and >2.0 ng/ml. In conclusion, the results of this study clearly demonstrated a negative effect of elevated progesterone levels on the day of hCG trigger, on TQE rate, regardless of the basal FSH, the total gonadotropin, the age of the woman, or the time of ovarian stimulation. These data demonstrate that elevated progesterone levels (>2.0 ng/ml) before oocyte maturation were consistently detrimental to the oocyte.     

Inhibition of progesterone secretion by a 3 beta-hydroxysteroid dehydrogenase inhibitor in late pregnant sheep

The role of progesterone in the initiation of parturition in the sheep is unclear. Whether a decrease in plasma progesterone is the essential prerequisite for the initiation of parturition or whether other factors also maintain uterine quiescence until delivery is not known. The effect of withdrawal of progesterone on the initiation of parturition has been investigated by intravenous administration of trilostane, a 3 beta-hydroxysteroid dehydrogenase delta 5-4 isomerase inhibitor, to late pregnant sheep. Twenty-five or 100 mg trilostane caused a precipitous decrease in plasma progesterone to about 30% of preinjection levels. Progesterone remained depressed for up to 7 days after treatment. 13,14-Dihydro-15-keto-prostaglandin F2 alpha (PGFM) became elevated between 7 and 36 h after trilostane injection but gradually returned to preinjection levels during the subsequent 36 h, at a time when plasma progesterone was still depressed. Four of 11 animals treated with 100 or 200 mg trilostane aborted prematurely at a time when plasma PGFM was maximal and plasma progesterone minimal. There were no consistent changes in plasma estradiol-17 beta or ovine placental lactogen concentrations after treatment with trilostane. It is suggested that a decrease in plasma progesterone will cause a transient increase in plasma PGFM concentrations which can lead to the premature initiation of parturition. In some instances the myometrium does not appear to respond to the elevated PGFM concentrations even when the estrogen:progesterone ratio is elevated by a decrease in plasma progesterone.     

Growth hormone-releasing hormone neurons in the anestrus cat do not express progesterone receptors

Ovarian steroids have been implicated in the regulation of growth hormone (GH) secretion in several species and increased progesterone secretion has been associated with elevated circulating GH levels in the cat. These high GH concentrations may be due, at least in part, to a direct action of progesterone on growth hormone-releasing hormone (GHRH) neurons. Using standard immunocytochemical methods coupled to high-temperature antigen retrieval, the objective of this study was to determine whether progesterone receptors were colocalized in GHRH neurons of the anestrus cat. GHRH perikarya were restricted to the infundibular nucleus and the ventral ventromedial nucleus and although frequently surrounded by numerous progesterone receptor-immunoreactive cells, none was colocalized. This study, therefore, provides evidence that, in the adult anestrus female cat, GHRH neurons do not express nuclear progesterone receptors.     

Implicit motives and gonadal steroid hormones: effects of menstrual cycle phase,oral contraceptive use,and relationship status

Implicit motives for power and affiliation, salivary levels of testosterone, estradiol, and progesterone, and relationship status were measured in 18 normally cycling (NC) women, 18 women using oral contraceptives (OC), and 18 men at three assessments, corresponding to the menstrual, midcycle, and premenstrual phases of women's menstrual cycle. NC and OC women had elevated levels of affiliation motivation and decreased levels of power motivation at midcycle. Power motive changes were particularly pronounced in NC women across cycle phases. OC women and participants not engaged in an intimate relationship had significantly heightened levels of affiliation motivation, averaged across all cycle phases. Testosterone and power motivation, both averaged across all cycle phases, were positively correlated in men and in single women, but not in women engaged in an intimate relationship. Averaged levels of estradiol and power motivation were positively correlated in engaged women, but not in single women or men. Averaged levels of progesterone and affiliation motivation were negatively correlated in men, and there was evidence for a positive association between luteal affiliation motivation and periovulatory and luteal progesterone in NC women. This study therefore provides evidence that implicit motivational states fluctuate across the menstrual cycle, that the power motive is associated with testosterone and, in women, with estradiol, and that the affiliation motive and progesterone are associated in different ways in men and NC women.     

Effect of metformin on the fertilizing ability of mouse spermatozoa

Numerous antioxidants have been added to cryopreservation media with varied success. The biguanide, metformin, commonly used for the treatment of type II diabetes, possesses properties impacting metabolism control that have not been yet assessed in cryopreservation protocols. The aim of this experiment was to; (i) determine the effect of metformin on fresh spermatozoa properties; and (ii) to assess positive or negative effects of metformin in post-thaw function and fertilizing capacity of mouse spermatozoa when used in cryopreservation media. The experiments have shown that the presence of metformin in fresh semen did not induce negative effects on spermatozoa quality, except a slight reduction in sperm motility at 5000 μM metformin. However, when metformin was included in a cryopreservation protocol, an improvement in the fertilization rate and a reduction in the percentage of abnormal zygotes after in vitro fertilization was observed. In conclusion, metformin did not affect sperm quality at low concentrations (50 μM), but its presence in the cryopreservation media could represent a benefit to improve the quality of frozen semen.     

Luteinizing hormone secretion in hypophysial stalk-transected pigs given progesterone and pulsatile gonadotropin-releasing hormone

This study was conducted to determine whether progesterone inhibits luteinizing hormone (LH) secretion in female pigs by a direct action on the pituitary gland. Eight ovariectomized, hypophysial stalk-transected gilts were given 1-microgram pulses of gonadotropin-releasing hormone iv every 45 min from Day 0 to 12. On Days 5-12, each of four gilts received either progesterone or oil vehicle im at 12-hr intervals. Serum progesterone concentrations in steroid-treated gilts reached 70 +/- 6.8 ng/ml (mean +/- SE) by Day 8 and remained elevated thereafter, whereas serum progesterone concentrations in oil-treated controls were less than 1 ng/ml for the entire study. Daily serum LH concentrations were not different between gilts treated with progesterone or oil. The 1-microgram pulses of gonadotropin-releasing hormone reliably evoked pulses of LH in both treatment groups. The LH pulse frequency and amplitude, assessed from samples collected every 15 min for 6 hr on Day 12, were similar for progesterone- and oil-treated gilts. These results provide evidence that progesterone does not act at the pituitary gland to alter LH secretion in pigs.     

Progesterone is not responsible for the blood pressure fall in late-pregnant New Zealand genetically hypertensive rats

In various models of experimental and genetic hypertension in rats, blood pressure is markedly reduced during late pregnancy. The period during which the blood pressure reduction occurs is also the period when plasma progesterone is maximally elevated, and administration of progesterone to renal hypertensive rats has been reported to reduce blood pressure (J. Armstrong, 1959, Proc. Soc. Exp. Biol. Med. 102:452-455). To test the possibility that elevated plasma progesterone is responsible for the blood pressure reduction in late pregnancy, on Day 14 of pregnancy a group of New Zealand genetically hypertensive (NZGH) rats was ovariectomized and implanted with progesterone-filled capsules, to maintain plasma progesterone at low levels just sufficient to maintain pregnancy, and compared with intact, pregnant NZGH. Ovariectomy did not alter the characteristic course of blood pressure reduction seen in late-pregnant intact NZGH rats. In addition, daily administration of progesterone (15 mg/kg, sc) for 14 days did not alter blood pressure of either nonpregnant NZGH rats or New Zealand normotensive rats with chronic 1-kidney, 1-clip hypertension. It is concluded that blood pressure of NZGH rats is reduced to near normotensive levels in late pregnancy, as reported for other models of rat hypertension, but that elevated plasma progesterone levels are not requisite for that reduction and do not reduce blood pressure of renal hypertensive rats.     

Plasma estradiol, testosterone, and progesterone levels during the ovulatory cycle of the skate (Raja erinacea)

Amounts of estradiol, testosterone, and progesterone in plasma were measured during the reproductive cycle of female Raja erinacea. Estradiol titers correlated directly with follicle size in females undergoing ovarian recrudescence, while highest concentrations were found in females with preovulatory follicles. These data indicate that as follicles grow, their steroidogenic capacity increases. In mature, nonspawning females, titers of estradiol and testosterone varied markedly. Progesterone was not detected in peripheral plasma of skates that did not produce eggs during the observation period. In females producing eggs, estradiol and testosterone predominated during the follicular phase of each spawning cycle. While estradiol and testosterone were elevated, progesterone was not detectable in the peripheral circulation. As ovulation and formation of capsules approached, plasma estradiol and testosterone declined to near baseline levels. Circulating progesterone rose sharply two days before encapsulation of ovulated eggs and remained elevated for only two days. On the day of encapsulation, concentrations of plasma progesterone had fallen to nearly baseline levels. Progesterone titers remained low throughout egg retention and oviposition. These measurements demonstrate that progesterone titers are elevated at specific times during the reproductive cycle of the skate and clearly suggest that progesterone is critically involved in events occurring at ovulation, encapsulation, and possibly oviposition.     

Porcine uterus cryopreservation: an analysis of contractile function using different uterotonics

Cryopreservation of whole organs has become increasingly successful in recent years, and establishing reliable methods for confirming the success of specific cryopreservation procedures has therefore become extremely important. On the assumption that methods such as histological evaluation do not provide definitive evidence of long-term cryopreservation and that clear signs of conserved function in an organ are good evidence of its viability, contractile function was analysed in porcine uteri (n=60), either after long-term (group A) or short-term (group B) cryopreservation and post-thaw treatment with three different uterotonics. A slow freezing protocol was used to preserve the organs. Fifteen fresh uteri were analysed similarly for contractile function, which was evaluated by measuring intrauterine pressure after administration of oxytocin, prostaglandin E(1) (PGE(1)), and carbachol. After cryopreservation, all but three uteri (95%) showed rhythmic contractions similar to those in fresh uteri except for differences in the heights of contraction peaks, with lower contractions in PGE(1) subgroup B (P<0.05). With the exception of three nonresponsive uteri in group A, there were no differences in contractility between uteri after long-term cryopreservation and fresh uteri. The results of this study thus contribute to the debate on whether slow freezing or vitrification techniques are best for whole-organ cryopreservation. In summary, (1) preservation of muscular function in porcine uteri is feasible with a slow freezing protocol; (2) measurement of contractile function following administration of uterotonics is a useful method of confirming functionality; and (3) long-term cryopreservation does not significantly impair post-thaw contractibility in comparison with fresh uteri.     

The significance of cooling rates and animal variability for boar sperm cryopreservation: insights from the cryomicroscope

Once the first methods for freezing mammalian semen had been established, research aimed at improving cryopreservation procedures became highly focused on the interactions between cooling rates and the permeability of the plasma membrane to water and cryoprotectants. This was based on the premise that cooling rates could be optimized from a theoretical basis for different species of interest. While this approach has stimulated considerable research, it has not achieved its original aim at the species level, largely because it overlooks inter-individual variation in sperm biochemical composition and physiology. If the underlying hypothesis is valid, however, optimal cooling rates should be identifiable for spermatozoa from individual animals. Experiments with the cryomicroscope revealed that while sperm survival after cryopreservation varied considerably between boars, there was little evidence that optimal freezing rates could be identified for individuals. Based on these findings, we tested the hypothesis that sperm susceptibility to cryoinjury is a consistent feature of each individual, but those individuals differ in susceptibility. This hypothesis was supported by evidence from an experiment with >100 boars; moreover, using genetic analyses, we demonstrated genomic differences between individual boars that correlated with post-thaw sperm quality.     

Maintenance of functional corpora lutea in androgenized female rats treated with OMSG

Rats were androgenized by injection of 50 micrograms testosterone propionate on the 5th day after birth and when adult were treated with 5 i.u. PMSG; some of the animals were mated. Serum was obtained daily and the concentrations of progesterone, 20 alpha-dihydroprogesterone and prolactin, estimated by radioimmunoassays, were compared to values found for mated, but not ovulating, androgenized females and those for normal pregnant females. Ovulation and luteinization of follicles occurred. The concentration of progesterone increased after the injection of PMSG and remained elevated for at least 10 days; mating did not alter the progesterone levels. The concentration of 20 alpha-dihydroprogesterone was also elevated but the ratio of the level of progesterone to this steroid was generally greater than unity. Prolatin levels were elevated in the rats which ovulated. It is concluded that the corpora lutea induced in androgenized females by PMSG are functional and maintained.     

Progestin receptor-mediated reduction of anxiety-like behavior in male rats

Background

It is well known progesterone can have anxiolytic-like effects in animals in a number of different behavioral testing paradigms. Although progesterone is known to influence physiology and behavior by binding to classical intracellular progestin receptors, progesterone''s anxiety reducing effects have solely been attributed to its rapid non-genomic effects at the GABA_A receptor. This modulation occurs following the bioconversion of progesterone to allopregnanolone. Seemingly paradoxical results from some studies suggested that the function of progesterone to reduce anxiety-like behavior may not be entirely clear; therefore, we hypothesized that progesterone might also act upon progestin receptors to regulate anxiety.

Methodology/Principal Findings

To test this, we examined the anxiolytic-like effects of progesterone in male rats using the elevated plus maze, a validated test of anxiety, and the light/dark chamber in the presence or absence of a progestin receptor antagonist, RU 486. Here we present evidence suggesting that the anxiolytic-like effects of progesterone in male rats can be mediated, in part, by progestin receptors, as these effects are blocked by prior treatment with a progestin receptor antagonist.

Conclusion/Significance

This indicates that progesterone can act upon progestin receptors to regulate anxiety-like behavior in the male rat brain.     

Effects of cryopreservation on the intracellular calcium concentration of human spermatozoa and its response to progesterone

The intracellular free calcium concentration [Ca²⁺]_i of sperm from 23 ejaculates was measured before and after cryopreservation using the fluorescent probe Fura-2. Spermatozoa were treated with 3.18 μM progesterone so that the regulation of [Ca²⁺]_i in a dynamic situation could be studied. [Ca²⁺]_i (nM) was 290 ± 13 in fresh spermatozoa vs. 550 ± 26 in cryopreserved samples (mean ± S.E.M. P < 0.0001 paired t-test). Progesterone at a dose of 3.18 μM stimulated a large and rapid increase in [Ca²⁺]_i to a peak value > 1 μM after 10–20 seconds. [Ca²⁺]_i then declined to a slightly raised basal level over the next 30–40 seconds. This phenomenon occurred in all the fresh samples, but about half the frozen thawed samples failed to respond. The peak [Ca²⁺] attained by frozen samples which did respond after the addition of progesterone was similar to that observed with fresh sperm. The calcium channel blocker verapamil (200 μM) completely inhibited the transient rise in [Ca²⁺]_i produced by progesterone, but 100 μM verapamil had only a partial effect. We conclude that (1) cryopreservation causes a substantial elevation of the [Ca²⁺]_i in human spermatozoa and (2) damage to the plasma membrane during cryopreservation may result in the loss of the progesterone receptor. Both factors may contribute to the loss of fertility after cryopreservation. © 1994 Wiley-Liss, Inc.