DNA topoisomerases I and II in human mature sperm cells: characterization and unique properties.

BACKGROUND: The condensed state of the human sperm's chromatin is essential for the compact structure of the spermatozoon head, which is important for the fertilization process. The enzymes DNA topoisomerases (topo I and topo II) are responsible for the topological structure of the chromatin in somatic cells. The activities and the characterization of topoisomerases in mature human sperm cells have not been previously investigated. METHODS: Sperm cells were purified from the semen of healthy donors by standard procedures and assays measuring the activities, protein size and sensitivity to inhibitors of topoisomerases were performed. RESULTS: Topo I and topo II DNA relaxation activities are present in nuclear extracts derived from human sperm. The sperm topo I activity is inhibited by camptothecin, similarly to the somatic enzyme. An 85 kDa sperm protein, compared with the 100 kDa somatic topo IB enzyme, reacted with anti-human topo I antibody. Sperm topo II lacks the DNA decatenation activity of the somatic enzyme and a 97 kDa protein, compared with the 170 kDa somatic topo IIalpha enzyme, was detected with anti-human topo II antibody. Sperm nuclear extracts contained inhibitors of somatic topo II decatenation activity. CONCLUSIONS: Topoisomerase I and II activities as well as topo I and topo II proteins are present in mature human sperm cells. These enzymes possess unique properties compared with their somatic counterparts.

Relationship between age and semen parameters in men with normal sperm concentration: analysis of 6022 semen samples.

This study evaluates retrospectively the relationship between age and semen parameters among men with normal sperm concentration. It was based on computerized data and performed in an Academic Fertility and IVF Unit. Six thousand and twenty-two semen samples with sperm concentrations of >or=20 x 10(6) ml(-1) were examined according to WHO criteria and analysed in relation to patients' age. For each age group, mean values +/- SD of semen volume, sperm concentration, percentage of motile spermatozoa, normal morphology, acrosome index, total sperm count/ejaculate, total motile sperm count/ejaculate and sexual abstinence duration were examined. A peak semen volume of 3.51 +/- 1.76 ml(-1) was observed at age >or=30 to <35 years and a lowest volume of 2.21 +/- 1.23 ml(-1) was observed at age >or=55 years (P<0.05). Sperm motility was found to be inversely related to age with peak motility of 44.39 +/- 20.69% at age <25 years and lowest motility of 24.76 +/- 18.27% at age >or=55 years (P<0.05). A reduction of 54% was observed for total motile sperm, between values of 103.34 +/- 107 x 10(6) at age >or=30 to <35 years and 46.68 +/- 53.73 x 10(6) (P<0.05) at age >55 years. A statistically significant and inverse relationship was observed between semen volume, sperm quality and patient age, in spite of prolonged sexual abstinence duration. Top sperm parameters were observed at age >or=30 to <35 years, while the most significant reduction in sperm parameters occurred after the age of 55 years.

Infertility, ovulation induction treatments and the incidence of breast cancer--a historical prospective cohort of Israeli women.

CONTEXT: Ovulation induction drugs may be associated with increased breast cancer risk. Results so far have been inconclusive. OBJECTIVE: To evaluate the association between infertility, exposure to ovulation induction drugs and the incidence of breast cancer. DESIGN: Historical prospective cohort and nested case-control study. SETTING: Institutional practice PATIENTS: About 5,788 women attending five infertility centers in Israel between 1964 and 1984. INTENTION: Abstracting of medical records and telephone interviews. MAIN OUTCOME MEASURE: Breast cancer incidence was determined through linkage with the National Cancer Registry database. Standardized incidence ratios (SIRs) and 95% confidence intervals were computed by comparing the observed to the expected cancer rates in the general population. In addition, a nested case-control study within the cohort was performed with interviews of breast cancer cases and two matched controls. RESULTS: The study cohort included 120,895 women years of follow-up. Compared to 115.2 expected breast cancer cases, 131 cases were observed (SIR = 1.1; 95% CI 0.9-1.4). Risk for breast cancer was significantly higher for women treated with clomiphene citrate (SIR = 1.4; 95% CI 1.0-1.8). Similar results were noted when comparisons were carried out between treated and untreated women, and when multivariate models were applied. In the nested case-control study, higher cycle index (OR = 2.2; 95% CI 1.0-4.8) and treatment with clomiphene citrate (OR=2.7; 95% CI 1.3-5.7) were associated with higher risk for breast cancer. CONCLUSION: Infertility and usage of infertility drugs in general are not associated with increased risk for breast cancer. However, for infertile women treated with clomiphene citrate, breast cancer risk is elevated.

Relationship between sexual abstinence duration and the acrosome index in teratozoospermic semen: analysis of 1800 semen samples.

A high acrosome index (percentage of sperm with normal acrosome morphology--cutoff value > or =10%) is known to be associated with an improved fertilization rate in conventional IVF. A retrospective evaluation of the relationship between duration of sexual abstinence and acrosome index among oligozoospermic and normozoospermic semen samples with teratozoospermia was undertaken. A significant (P = 0.001) decrease in the acrosome index was observed among the normozoospermic samples (n = 1264) between the peak value of 10.2 +/- 3.6% on day 2 and 8.5 +/- 4.0% on day 5 of abstinence, while for the oligozoospermic samples (n = 536) the peak value of 8.7 +/- 3.5% was observed on day 1 and the lowest values of 6.8 +/- 3.7% (P = 0.04) on day 5 of abstinence. The results suggest that an optimal acrosome index will be obtained following a short sexual abstinence.

Effect of cryopreservation on quality and fertilization capacity of human sperm.

OBJECTIVE: To analyze retrospectively the effect of cryopreservation on donor's sperm. STUDY DESIGN: Data were collected on 178 cryopreserved-thawed sperm specimens from 44 donors and 624 oocytes from 62 women, which underwent in vitro fertilization-embryo transfer (IVF-ET) treatments with donor's sperm. Data on fresh sperm, 175 sperm specimens from 76 couples which underwent IVF-ET treatments, were used as a control group. Semen analysis was done by cell concentration, percent of motility, and quality of motility according to the World Health Organization (WHO) recommendation. Sperm quality parameters which had the strongest impact on fertilization capacity were determined using the statistical response surface model and conjoint analysis. RESULTS: Passing sperm through Percoll column decreased sperm concentration, with no improvement in sperm motility but with a slight increase in quality of motility. Quality of motility of donor's sperm had the strongest impact on fertilization capacity. CONCLUSION: Current freezing-thawing protocols of sperm cause a decrease in sperm parameters without affecting fertilization capacity. Furthermore, quality of motility of frozen-thawed sperm seems to be a significant measure of sperm fertilization capacity.

The relationship between occupational psychological stress and female fertility.

OBJECTIVE: To investigate the association between occupational psychological stress and female fertility. METHODS: This was a case-control study including consecutive working female patients attending fertility and in vitro fertilization clinics in the Soroka University Medical Center. We compared occupational stress between 64 working patients who had attended the clinics due to female infertility (case group) and 106 working patients who had attended the clinics due to their partner's reproductive impairment (control group). RESULTS: Patients from the female infertility group were older (31.9 +/- 6.2 versus 30.2 +/- 4.6, P = 0.047) and tended to participate more in sporting activity [23.4 versus 10.4%, odds ratio (OR) = 2.6, 95% confidence interval (CI) = 1.05-6.73, P = 0.022] as compared with patients from the male infertility group. Patients from the case group tended to work more weekly hours as compared with the controls (33.6 +/- 16.8 versus 26.9 +/- 17.4, P = 0.028). High reliability was found, as demonstrated by Cronbach's alpha of 0.81-0.90 for the four burnout parameters. Patients from the female infertility group had significantly lower listlessness scores as compared with the control group, using the Mann-Whitney test (2.6 +/- 1.1 versus 3.1 +/- 1.2, P = 0.013). CONCLUSIONS: Patients admitted due to female infertility tended to have lower listlessness scores as compared with patients admitted due to their partner's infertility problem. No significant association was found between other burnout, job strain and job satisfaction scores and women's fertility status.

Immunohistochemical staining of IL-1 alpha and IL-1 receptor antagonist but not IL-1 beta in cultures of sertoli cells.

The interleukin-1 (IL-1) system has been suggested to be involved in the cell cell cross talk within the testis. To identify a testicular cell source of IL-1 alpha, IL-1 beta and IL-1 receptor antagonist (IL-1ra), immature mouse Sertoli cells were isolated, purified, cultured and examined for the cellular compartment localization of these cytokines by immunohistochemical staining. Our results show that both Germ cells and Sertoli cells in unpurified Sertoli cell cultures (before hypotonic shock) and purified culture of Sertoli cells (after hypotonic shock) were stained for IL-1 alpha. The levels of this cytokine were increased in Sertoli cells when the purified cultures were stimulated with lipopolysaccharide (LPS) (5 microg/mL). However, we could not identify a positive staining for IL-1 beta when Sertoli cell cultures were stained for this cytokine, even after stimulation with various concentrations of LPS (0.1-10 microg/mL). On the other hand, immunohistochemical staining of isolated Sertoli cells without treatment with hypotonic shock (cultures containing Sertoli cells and Germ cells) for IL-1ra showed constitutive positive staining of both cell types (Sertoli cells and Germ cells). Our results, using immunohistochemical staining, may indicate the different expression of IL-1 alpha, IL-1 beta and IL-1ra in Sertoli cells. These results may suggest the involvement of IL-1 system in the autocrine and paracrine regulation of testicular cell functions.

Production of interleukin-1-like molecules by human sperm cells.

OBJECTIVE: To characterize and localize interleukin (IL)-1alpha and IL-1beta in human sperm cells. DESIGN: Prospective and comparative study. SETTING: Andrology clinic of a university hospital. PATIENT(S): Two groups of normogonadotropic men: 17 fertile men (donors with proved fertility) and 8 oligoteratoasthenospermic infertile men. INTERVENTION(S): None. MAIN OTUCOME MEASURE(s): Evaluation of IL-1alpha and IL-1beta levels and expression in sperm cells by immunohistochemical staining, immunoassay, and Western blot analysis. RESULT(S): Both types of IL-1-like molecules (IL-1alpha and IL-1beta) were expressed in the tail, neck, and head of sperm cells of fertile men and patients with oligoteratoasthenospermia. Swim-up sperm cells from fertile men and patients with oligoteratoasthenospermia secreted similar levels of IL-1-like molecules. The levels of IL-1beta-like molecules were higher than those of IL-1alpha-like molecules in both groups. The expressed IL-1-like molecules were characterized by the presence a 60-kd protein for both IL-1alpha-like and IL-1beta-like molecules. In some samples of both fertile men and infertile men with oligoteratoasthenospermia, 17-kd, 33-kd, and 45-kd IL-1beta-like molecules were detected. Impairment of sperm function, such as decreased sperm count and motility and/or impaired morphology, was not related to the capacity of sperm cells to produce IL-1-like molecules. CONCLUSION(S): IL-1 molecules originating in sperm cells may play a role in the physiologic functions of sperm cells (autocrine effect) and/or in cell-cell interactions within the testis (paracrine effect).

Involvement of serum and lipopolysaccharide in the production of interleukin-1- and interleukin-6-like molecules by human sperm cells.

PROBLEM: To examine the capacity of sperm cells from fertile and infertile men to secrete interleukin (IL)-6, and the involvement of serum factors and lipopolysaccharide (LPS) in the regulation of IL-6 and IL-1 production by sperm cells. METHODS: Swim-up sperm cells from fertile (donors) and oligoteratoasthenospermic (OTA)-infertile men were incubated with or without 5% fetal calf serum (FCS) and LPS (10 microg/mL) for 2-24 hr. After incubation, IL-6 and IL-1 bioactivities were measured in supernatants and lysates by specific bioassays (B9 cell proliferation assay and 1A-5 system, respectively). RESULTS: IL-6- and IL-1-like activities were observed to be produced by swim-up sperm cells from both study groups. Stimulation of swim-up sperm cells with either LPS or FCS or both together did not affect their capacity to produce IL-1. However, LPS, but not serum increased the secretion levels of IL-6 by swim-up sperm cells. CONCLUSIONS: Swim-up sperm cells from both study groups constitutively produce IL-6 and IL-1, and serum components did not affect this capacity. However, LPS was shown to increase the capacity of swim-up sperm cells of both study groups to secrete IL-6, but not IL-1. Cytokines may be involved in the physiology and pathophysiology of sperm functions and, thus, may affect male fertility.

Treatment outcome in women with a single ovary versus patients with two ovaries undergoing in vitro fertilization and embryo transfer (IVF/ET).

OBJECTIVE: To compare ovarian response and pregnancy rate between women with one and two ovaries undergoing in vitro fertilization and embryo transfer (IVF/ET). STUDY DESIGN: 20 IVF/ET treatment cycles in ten women with a single ovary were compared with 60 IVF/ET cycles in 47 women with two ovaries. Both groups were matched for age and treated for mechanical infertility. In both groups treatment protocol included gonadotropin releasing hormone/human menopausal gonadotropin/human chorionic gonadotropin (GnRH/hMG/hCG). RESULTS: Effective daily dose of gonadotropins (3.7+/-0.7 vs. 3.6+/-1.0), mean 17beta-estradiol levels on day of hCG administration (1136+/-467 vs. 1343+/-776), number of retrieved oocytes (6.4+/-3.7 vs. 8.3+/-4.2) and number of embryos per transfer (3.0+/-0.7 vs 2.9+/-1.2) were not statistically different between the groups. A significantly higher pregnancy rate was observed among women with one ovary (52.9%) as compared with those with two ovaries (20.8%), (P=0.015). Multivariate logistic regression analysis demonstrated an odds ratio of 5.73 for patients with a single ovary. CONCLUSION: Treatment outcome in patients with a single ovary undergoing IVF/ET is comparable to those with two ovaries. The unexpected significantly higher pregnancy rate observed among these patients need to be further evaluated.

[Assisted reproductive technologies reported in the National Registry of Israel 1995-1996].

National registration of the results of assisted reproductive technology (ART) is maintained by many countries. The Israel Committee for Registry of ART asked 19 in-vitro fertilization (IVF) units in 1995 and 20 in 1996 to report on their activities and results. Data were collected by questionnaires and analyzed by computer. The most common ovarian stimulation was the combination of GnRH agonist and gonadotropins. There were 10.89 treatment cycles in 1995, of which 45% were with intracytoplasmic sperm injection (ICSI). In 1996, of 12.72 cycles, 54% were with the ICSI procedure. Embryos were transferred into the uterine cavity in 90% of the conventional IVF cycles and in 95% of the ICSI cycles. The overall pregnancy rate was 22% per embryo transfer in the conventional IVF and ICSI cycles. The delivery rate was 13.7% and 15.4% per embryo transfer in the conventional IVF and ICSI cycles, respectively. The rates for abortion and tubal pregnancy were 24% and 1.3%, respectively. These results are better than in previous years and are comparable with results in some western European countries. Efforts are being made to convert the registry into a real-time computerized system.

Expression of IL-12, IL-10, PGE2, sIL-2R and sIL-6R in seminal plasma of fertile and infertile men.

The involvement of cytokines and other immunoregulatory factors in male infertility is still unclear. In the present study we compared the levels of IL-12, IL-10, PGE2, sIL-2R and sIL-6R in the seminal plasma (SP) of fertile and infertile men. Four groups were included: fertile donors (FERT), infertile men with azoospermia (AZOO), and infertile men with either oligoterato-asthenoazoospermia (OTA), or OTA with genital infection (OTA-INF). Cytokines and cytokine-soluble receptors in semen were evaluated by specific ELISA commercial kits. The levels of IL-12, sIL-2R and sIL-6R were similar in SP of fertile and infertile men. The mean levels of IL-10 in the SP of FERT, OTA and AZOO were 5.6 +/- 0.9, 4 +/- 2.8 and 8 +/- 3.5 pg ml-1, respectively, and did not differ significantly. The mean level of IL-10 in SP from OTA-INF (0.9 +/- 0.5 pg ml-1) was significantly lower than that for FERT (5.6 +/- 1.9 pg ml-1; P = 0.02) and AZOO (8.2 +/- 3.4 pg ml-1; P = 0.05), but not significantly different from that for OTA (3.7 +/- 2.1 pg ml-1). The mean SP level of PGE2 was significantly lower in SP of OTA-INF than FERT (7.67 +/- 2.26 and 19.67 +/- 3.69 micrograms ml-1, respectively; P < 0.02). In conclusion, the seminal plasma from fertile and infertile men contained similar levels of IL-12, sIL-2R and sIL-6R. However, the levels of IL-10 were significantly lower in SP from OTA-INF compared to FERT and AZOO. Our results indicate that specific cytokines and their soluble receptors are involved in the male reproductive system.

Does transcervical intra-fallopian insemination improve pregnancy rates in cases of oligoteratoasthenozoospermia? A prospective, randomized study.

The relatively low pregnancy rates (PR) after treatment of patients with oligoteratoasthenozoospermia (OTA) result in a search for different treatment modalities. The objective of this study was to assess the efficacy of transcervical intrafallopian insemination (IFI) with husband's semen in comparison to intrauterine insemination (IUI) in couples with OTA. A prospective, randomized study included 30 couples with OTA-related infertility (according to WHO criteria). The female patients underwent individually adjusted controlled ovarian stimulation by gonadotropins. Spermatozoa was prepared using the Percoll 70% technique and insemination was performed 36-40 h after human chorionic gonadotropin (HCG) administration. The Tomcat Catheter was used for IUI and the Jansen-Anderson Catheter for IFI to the fallopian tube leading to the ovary that contained more dominant follicles. The couples were divided according to sperm count, into group A (9 couples): < 10 mill ml-1 and group B (21 couples): > 10 mill ml-1. Within the groups the patients were randomly assigned for IUI or IFI treatment. Among group B couples, two pregnancies out of 15 IUI cycles (13.3% PR) and two pregnancies out of 18 IFI cycles (11.1% PR) were achieved. Group A patients completed 7 IUI and 9 IFI treatment cycles with no pregnancies observed. These data did not demonstrate a statistically significant advantage for either technique.

Fertility drugs and the risk of breast and ovarian cancers: results of a long-term follow-up study.

OBJECTIVE: To investigate a possible linkage between the use of fertility drugs for infertility and the risk of breast and ovarian cancers. DESIGN: Long-term, historic-prospective study. SETTING: Fertility clinic in a university hospital. PATIENT(S): Files of 1,197 infertile women with a mean (+/- SD) follow-up of 17.9+/-5 years (21,407 person-years) were reviewed. Diagnoses, number of courses, and dosage of fertility drugs were extracted from the files. INTERVENTION(S): Cancers were identified by record linkage to the National Cancer Registry. Histopathologic reports and data on estrogen and progesterone receptors in breast cancer tissue were also reviewed. MAIN OUTCOME MEASURE(S): Standardized incidence ratio with 95% confidence interval (CI) were used for risk assessment. RESULT(S): Of 20 breast cancers (standardized incidence ratio, 1.40 [95% CI, 0.83-2.10]), 16 were detected among 780 women who had been exposed to 3,978 cycles of clomiphene citrate (CC) and/or hMG (standardized incidence ratio, 1.65 [95% CI, 0.94-2.68]). The standardized incidence ratio for this cancer was significantly increased only in patients with one or two CC treatments and a dose of < or =1,000 mg (2.6 [1.19-5.0] and 2.52 [1.21-4.64], respectively). Two cases of ovarian cancer (1 patient unexposed) were observed with no evidence of excessive risk. Six of the eight patients with data on estrogen and progesterone receptors were exposed to CC, and all tested positive for these receptors. CONCLUSION(S): An association between the use of fertility drugs and an increased risk of breast and ovarian cancers has not been confirmed.

Obstetric outcome of singleton pregnancies conceived by in vitro fertilization and ovulation induction compared with those conceived spontaneously.

OBJECTIVE: To compare the obstetric characteristics of singleton pregnancies conceived by IVF and ovulation induction with those conceived spontaneously. DESIGN: Case-control study. SETTING: Tertiary care medical center. PATIENT(S): All singleton pregnancies that were achieved by IVF (n = 169) and ovulation induction (n = 646) and were delivered from January 1989 through December 1994 were evaluated. Each group was compared with a separate control group that conceived spontaneously (n = 469 and n = 1,902 for the IVF and ovulation induction groups, respectively) and delivered during the same period and was matched in terms of maternal age, gestational age, and parity. INTERVENTION(S): Ovulation induction, IVF-ET. MAIN OUTCOME MEASURE(S): Obstetric complications. RESULT(S): Multivariate analysis showed that patients who conceived by IVF and ovulation induction had a significantly higher risk for gestational diabetes mellitus (odds ratio [OR] = 2.0; 95% confidence interval [CI] = 1.23-3.30 and OR = 1.9, 95% CI = 1.09-1.79, respectively), pregnancy-induced hypertension (OR = 2.1, 95% CI = 1.04-4.10 and OR = 1.5, 95% CI = 1.04-2.02, respectively), and cesarean section (OR = 3.6, 95% CI = 2.44-5.29 and OR = 1.4, 95% CI = 1.09-1.79, respectively) compared with their matched controls. CONCLUSION(S): After controlling for maternal age, gestational age, and parity, we demonstrated that singleton pregnancies conceived by IVF and ovulation induction are at increased risk for maternal gestational diabetes mellitus and pregnancy-induced hypertension, and at greater risk for delivery by cesarean section.

Do changes in growth hormone levels correlate with IGF-I levels in patients undergoing IVF-ET?

It has been suggested that adjunctive growth hormone (GH) therapy improves ovarian response and in vitro fertilization (IVF) outcome in specific groups of patients. The correlation between insulin-like growth factor (IGF) and GH is well established. The aim of this study was to determine whether changes in plasma GH correlate with IGF blood levels in patients during IVF treatment. Thirty-six women undergoing IVF and embryo transfer (ET) were examined. Ovarian stimulation was carried out by gonadotropin-releasing hormone agonists (GnRHa) and gonadotropins. Blood was drawn at the early and late follicular phase, on the day of human chorionic gonadotropin (hCG) injection and at the mid- and the late luteal phases. The samples were assayed for IGF-I, IGF-II, IGF-binding protein-3 (IGF BP-3), GH and estradiol. According to the IGF-I and GH plasma levels, patients were divided into three major groups: Group I consisted of patients in whom peak levels of GH reached more than 4 ng/ml and IGF-I decreased significantly. In this group, estradiol levels were 1863 +/- 149 pg/ml. Group II consisted of patients in whom peak blood GH levels did not exceed 2.5 ng/ml and the IGF-I level remained unchanged. In this group estradiol levels were 630 +/- 57 pg/ml. Group III consisted of patients in whom blood GH levels were low and remained unchanged while estradiol levels were 1600 +/- 420 pg/ml. In this group no significant increase in IGF-levels were observed. There was no significant change in the levels of either IGF-II or IGF BP-3 in any of the groups. We can conclude that (1) there is a negative correlation between GH and IGF-I plasma levels in patients undergoing controlled ovarian hyperstimulation (COH)-IVF, when levels of estradiol and GH are elevated; (2) plasma levels of IGF-I under ovarian hyperstimulation are probably regulated by a multifactorial system; and (3) no correlation was found between the plasma levels of IGF-I and those of IGF-II and IGF BP-3 in all patient groups.

Distinct expression of cytokines and mitogenic inhibitory factors in semen of fertile and infertile men.

PROBLEM: To assess the effect of seminal plasma (SP) of fertile and infertile men on leukocyte mitogenic response, and the capability of sperm cells to produce IL-1. METHODS: This study included four groups: fertile men (donors, normal), infertile men with azoospermia (azoo), oligo-terato-asthenozoospermia (OTA), and OTA with genital infection (OTA-inf). Mouse spleen cell proliferation in response to lipopolysaccharide (LPS) or Concanavalin-A (Con-A) was examined in the presence of SP from the above four groups. Supernatants (sup) and lysates (lys) of sperm cells from fertile and oligoteratoasthenospermic (OTA) men were evaluated for IL-1 bioactivity by specific bioassay. RESULTS: Seminal plasma (SP) of the four groups were shown to inhibit the mitogenic response of mouse spleen cells to LPS and Con-A. SP of fertile men was significantly more inhibitory than SP from infertile men. Sperm cells from fertile and OTA infertile men constitutively produced IL-1. Sperm cells of both groups produced similar levels of IL-1 as examined in the supernatants and lysates. CONCLUSIONS: Seminal plasma of fertile men had more inhibitory mitogenic activity than that of OTA. Sperm cells constitutively produce IL-1. It is possible that the factors involved in this inhibition are not only anti-proliferative immune factors. Cytokines and inhibitory factors of mitogenesis in the seminal plasma may be involved in the physiology and pathophysiology of sperm functions and thus affect male fertility.

Intraovarian blood flow during spontaneous and stimulated cycles.

This study was undertaken to investigate intraovarian blood flow during the early follicular, periovulatory and mid-luteal phases, during spontaneous and stimulated ovarian cycles. Transvaginal ultrasonography with colour flow imaging was used to measure the pulsatility index in eight patients with spontaneous cycles, 20 patients undergoing induction of ovulation with clomiphene citrate and 11 patients undergoing controlled ovarian hyperstimulation for in-vitro fertilization (IVF)-embryo transfer with gonadotrophin-releasing hormone analogue, human menopausal gonadotrophin, human chorionic gonadotrophin. All patients were studied during the early follicular, periovulatory and mid-luteal phase. Intraovarian blood flow velocity waveforms were found in 20% of cases at the early follicular phase, in 56% during the periovulatory phase and in 85% during the mid-luteal phase (P < 0.001). Pulsatility index at the early follicular phase was found to be 1.05 +/- 0.22, during the periovulatory phase 0.99 +/- 0.22 and during the mid-luteal phase 0.85 +/- 0.22. The appearance of intraovarian flow velocity waveforms may suggest either ovarian neovascularization or final follicular maturation or luteinization, which can be detected or measured by Doppler technology.