Follicular Phase Days Research Articles

Changes in Aquaporin 1, 5 and 9 Gene Expression in the Porcine Oviduct According to Estrous Cycle and Early Pregnancy.

Aquaporins (AQPs) are a group of small, integral membrane proteins which play an important role in fluid homeostasis in the reproductive system. In our previous study, we demonstrated AQP1, 5 and 9 protein expression and localization in the porcine oviduct. The presence of these isoforms could suggest their role in the transport of the ovum to the uterus by influencing the epithelial cells’ production of oviductal fluid. The aim of this study was to evaluate the expression of AQP1, AQP5 and AQP9 in the infundibulum, ampulla and isthmus in the porcine oviduct during the estrous cycle (early luteal phase, days 2–4, medium luteal phase, days 10–12, late luteal phase days 14–16, follicular phase days 18–20) and pregnancy (period before implantation, days 14–16 and after the implantation, days 30–32) using the Real-Time PCR technique. As clearly demonstrated for the first time, AQP1, 5, and 9 gene expression is influenced by the estrus cycle and pregnancy. Furthermore, expression of AQPs in the porcine oviduct may provide the physiological medium that sustains and enhances fertilization and early cleavage-stage embryonic development. Overall, our study provides a characterization of oviduct AQPs, increasing our understanding of fluid homeostasis in the porcine oviduct to successfully establish and maintain pregnancy.

The effect of follicular phase length on cycle outcomes and endometrial development in gonadotrophin ovarian stimulation/intrauterine insemination cycles.

Does a shorter follicular phase length (FPL) affect cycle outcomes and endometrial development among women undergoing gonadotrophin ovarian stimulation/intrauterine insemination (OS/IUI)? Retrospective cohort study of 4773 OS/IUI cycles among 2054 patients. FPL was analysed first continuously, then dichotomously using an arbitrary cut-off at the 15th percentile (8 days) to divide cycles into shorter and longer FPL groups. Receiver operating characteristic (ROC) curves were constructed to further analyse the impact of FPL on all outcomes. Primary outcomes included clinical pregnancy, spontaneous abortion, multiple pregnancy and non-viable (ectopic/biochemical) pregnancy rates (CPR, SABR, MPR and NVPR, respectively). Secondary outcomes included endometrial thickness. All analyses controlled for age, day 3 FSH and body mass index. When analysing FPL continuously, CPR increased by 6.0% (adjusted odds ratio [aOR] 1.06, 95% CI 1.03-1.09, P<0.001) with each additional follicular phase day. Similarly, in the dichotomous analysis, cycles with a longer FPL resulted in higher CPR with 45% higher odds of clinical pregnancy (aOR 1.45, 95% CI 1.07-1.97, P = 0.018). No effect of FPL was noted on NVPR, SABR or MPR. Endometrial thickness increased by 0.09mm (95% CI 0.06-0.12, P<0.001) with each additional FPL day and was increased in the longer compared with the shorter FPL group (adjusted mean difference 1.08mm, 95% CI 0.81-1.34, P<0.001). The data suggest that in gonadotrophin OS/IUI cycles, FPL might impact both chance of clinical pregnancy and endometrial thickness, independent of maternal age and ovarian reserve.

Reproductive endocrine functions in women with epilepsy

Women with epilepsy (WWE) have higher incidence of hormonal disturbance in comparison to the general female population. We analyzed the reproductive endocrine functions in patients with idiopathic generalized epilepsy and compared with controls. The study was conducted in the outpatient department of Neurology at a superspeciality hospital from south India. Women of reproductive age with epilepsy were recruited for the study. Patients in post menopausal state, pregnancy, who have undergone oophorectomy, use of oral contraceptive pills, patients with systemic illness, long term medication for other diseases or progressive or degenerative diseases were excluded. Five ml of venous blood was collected from 3rd day of follicular phase in fasting condition from the women study subjects. Free T3(fT3), Free T4(fT4) and Thyroid Stimulating Hormone(TSH), Follicular Stimulating Hormone(FSH). Luteinizing Hormone(LH), Prolactin, Testosterone and Estrodiol were measured. Hormones like serum Androstenedione, dehyroepiandrosterone(DHEA) and serum17-OH Progesterone were measured. Mean age of patients (N = 100) 22.78 + 7.08 and controls (N = 75) was 24.45+ 4.52. BMI was higher in patients (23.87 + 5.18) than in controls (21.87 + 2.11) p = .05. There was no significant difference in thyroid hormone levels and prolactin. There was a lower level of progesterone(0.54 + 0.27) vs. 1.01 + 0.34 ng/ml), p = .00; higher levels of dehydroepiandrostenedione (0.69 + 0.25vs. 0.54 + 0.38μg/mL), p = .05 and luteinizing hormone-LH (6.01 + 3.90mIU/mL vs. 2.85 + 1.36mIU/mL), p = .00, higher LH/FSH ration (0.87 + 0.50 vs 0.32 + 0.16, p = .00). AED'S were Phenytoin (70%), Carbamazepine(26%), Valproate (4%). There was no significant difference between individual AED's. This study demonstrates that WWE have reproductive endocrine abnormalities. The role of epilepsy versus EIAEDS needs further evaluation.

The effect of follicular phase length on endometrial development and the outcomes of ovulation induction/intrauterine insemination (OI/IUI) cycles

Few, limited studies have reported that in IUI cycles a shorter follicular phase is associated with poorer pregnancy outcomes compared to a longer follicular phase [1,2]. However, these studies have been limited by small sample sizes and inadequate control of potential confounders. To evaluate the effect, if any, of follicular phase length on endometrial development and cycle outcomes among patients undergoing OI/IUI. Design: Retrospective cohort study Intervention: Data from 3404 IUI cycles derived from 1502 patients in which gonadotropins were used for OI were analyzed and stratified by follicular phase length (hCG trigger day ≤8 vs. >8). Outcome Measures: Primary outcomes included clinical pregnancy, multiple pregnancy, spontaneous abortion, and non-clinical pregnancy (ectopic/biochemical) rates (CPR, SABR, MPR, and non-CPR, respectively). A secondary outcome was endometrial thickness prior to hCG trigger. Statistics: Demographic factor comparisons between follicular phase length groups were analyzed with Student t-tests, Mann Whitney U-tests, and χ2- tests, as appropriate. Multilevel random and fixed effects regression models were used to calculate odds ratios (ORs) for pregnancy outcomes and regression coefficients for endometrial thickness first by comparing the follicular phase length groups (≤8 vs. >8 days), then by analyzing follicular phase length as a continuous variable. All models adjusted for potential confounders (age, BMI, day-3 FSH) determined a priori. Demographic and cycle characteristics of the study population, stratified by follicular phase length, are outlined in Table 1. After adjusting for potential confounders, cycles with a longer compared to those with a shorter follicular phase resulted in significantly higher CPR (14.0 vs. 9.0%, p = 0.02; respectively). The odds for clinical pregnancy (CP) were 38% higher in the former compared to the latter group, with the difference nearing significance (OR: 1.38, 95%CI: 0.96-1.99, p = 0.082). When follicular phase length was analyzed as a continuous variable, the odds of CP increased by 6.0% (95%CI: 3.0-10.0, p = 0.001) with each additional follicular phase day. Cycles with a longer compared to those with a shorter follicular phase had comparable SABR (21.0 vs. 17.0%, p = 0.54), MPR (11.0 vs. 9.0%, p = 0.58), and non-CPR (12.0 vs. 16.0%, p = 0.39). The former compared to the latter group had similar odds for SAB (OR 1.56, 95%CI: 0.68-3.58, p = 0.293), MP (OR 1.40, 95%CI: 0.47-4.15, p = 0.544), and non-CP (OR 0.66, 95%CI: 0.28-1.58, p = 0.355). Likewise, when analyzed as a continuous variable, follicular phase length did not significantly impact the odds for SAB (OR 1.07, 95%CI: 0.99-1.14, p = 0.07), MP (OR 1.01, 95%CI: 0.94-1.10, p = 0.743), or non-CP (OR 0.98, 95%CI: 0.90-1.07, p = 0.687). Endometrial thickness was increased in cycles with a longer compared to shorter follicular phase with an adjusted mean difference of 1.12 mm (95% CI: 0.93-1.31, p < 0.001). Furthermore, endometrial thickness increased by 0.12 mm (95% CI: 0.09-0.15, p <0.001) with each additional follicular phase day. In gonadotropin-induced IUI cycles, a longer follicular phase is associated with increased endometrial thickness and possibility of clinical pregnancy.

Human fallopian tube epithelium co-culture with murine ovarian follicles reveals crosstalk in the reproductive cycle.

Do interactions between human fallopian tube epithelium and murine follicles occur during an artificial reproductive cycle in a co-culture system in vitro? In a co-culture system, human fallopian tissues responded to the menstrual cycle mimetic by changes in morphology and levels of secreted factors, and increasing murine corpus luteum progesterone secretion. The entire fallopian tube epithelium, including ciliated and secretory cells, can be regulated in the reproductive cycle. Currently, there are no in vitro culture models that can monitor fallopian tissues in real time in response to factors produced by the ovary. In addition, there are no reports on the impact of fallopian tissue on ovarian function during the menstrual cycle. Human fallopian tissue (n=24) was obtained by routine hysterectomies from women (aged 26-50 years, mean age=43.6) who had not undergone exogenous hormonal treatment for at least 3 months prior to surgery. CD1 female mice were used for ovarian follicle isolation. The human fallopian epithelium layers were either co-cultured with five murine multilayer secondary follicles (150-180 μm follicles, encapsulated in one alginate gel bead) for 15 days or received stepwise steroid hormone additions for 13 days. The fallopian tissue morphology and cilia beating rate, as measured by an Andor Spinning Disk Confocal, were investigated. Oviduct-specific glycoprotein 1 (OVGP1), human insulin-like growth factor 1 (hIGF1), vascular endothelial growth factor A (VEGF-A) and interleukin 8 (IL8) as biological functional markers were measured either by ELISA or western blot to indicate dynamic changes in the fallopian epithelium during the reproductive cycle generated by mouse follicles or by stepwise steroid hormone induction. Three or four patients in each experiment were recruited for replicates. Data were presented as mean±SD and further analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. The cultured fallopian tube epithelium responded to exogenous steroid hormone stimulation, as demonstrated by enhanced cilia beating rate (~25% increase, P=0.04) and an increase in OVGP1 secretion (P=0.02) in response to 1 nM estradiol (E2) treatment when compared with 0.1 nM E2. Conversely, 10 nM progesterone plus 1 nM E2 suppressed cilia beating rate by ~30% (P=0.008), while OVGP1 secretion was suppressed by 0.1 nM E2 plus 50 nM progesterone (P=0.002 versus 1 nM E2 alone). Human fallopian tube epithelium was co-cultured with murine secondary follicles to mimic the human menstrual cycle. OVGP1 and VEGF-A secretion from fallopian tissue was similar with stepwise hormone treatment and when cultured with murine follicles. However, the secretion patterns of hIGF1 and IL8 differed in the luteal phase when comparing steroid treatment with follicle co-culture. In co-culture, hIGF1 secretion was suppressed in the luteal versus follicular phase (P=0.005) but stepwise hormone treatment had no effect on hIGF1. In co-culture, IL8 secretion was also suppressed on luteal phase day 15 (P=0.013) versus follicular phase day 7, but IL8 secretion increased continuously under high E2/progesterone treatment (P=0.003 for D13 versus D3). In the co-culture system, the corpus luteum continuously produced progesterone in the presence of fallopian tube tissue until Day 18 while, without fallopian tissue, progesterone started to drop from Day 13. One limitation of this study is that murine follicles were used to mimic the human menstrual cycle. However, although secretion patterns of peptide hormones such as inhibins and activins differ in mice and humans, the co-culture system used here did reveal interactions between the tissues that govern reproductive function. In vitro co-culture models of fallopian reproductive tissues with ovarian follicles can provide an important tool for understanding fertility and for uncovering the mechanisms responsible for reduced fertility. In addition, the role of oviductal secretions and how they influence ovarian function, such as the production of progesterone during the menstrual cycle, can be uncovered using this model. None. This work was funded by grants from the NIH (UH3TR001207), the American Cancer Society (RSG-12-230-01-TBG) and NIH (R01EB014806). The authors declare no competing financial interest.

Change of anti-Mullerian-hormone levels during follicular phase in PCOS patients

Anti-Mullerian-hormone (AMH) does not seem to fluctuate significantly during the menstrual cycle in healthy women. However, little is known about cycle fluctuations of AMH levels in patients with polycystic ovarian syndrome (PCOS). The purpose of this study was to examine AMH fluctuations during the follicular phase in PCOS patients receiving antiestrogens or recombinant follicle-stimulating hormone (FSH). About 40 PCOS patients diagnosed according to Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group 2003 and 19 controls were prospectively enrolled. PCOS patients received either antiestrogens or recombinant FSH for monoovulation induction and controls received antiestrogens. AMH levels were determined (1) between the 2nd and the 5th day of follicular phase and (2) when a single large dominant follicle ≥18 mm had appeared. Our study shows that AMH levels do not change during follicular development in controls as well as in PCOS patients with AMH levels < 5 ng/ml, irrespective of antiestrogen or FSH therapy. However, in PCOS patients with AMH levels ≥5 ng/ml, AMH declines significantly during follicular development (p < 0.01). We conclude that AMH levels should be determined in the early follicular phase in PCOS patients without the influence of antiestrogens or exogenous FSH, because these interventions may lower AMH values in patients with high levels.

Conditioned Pain Modulation in Women With Irritable Bowel Syndrome

Evidence suggests that patients with irritable bowel syndrome (IBS) are more vigilant to pain-associated stimuli. The aims of this study were to compare women with IBS (n = 20) to healthy control (HC, n = 20) women on pain sensitivity, conditioned pain modulation (CPM) efficiency, and salivary cortisol levels before and after the CPM test and to examine the relationship of CPM efficiency with gastrointestinal pain, somatic pain, psychological distress symptoms, and salivary cortisol levels in each group. Women, aged 20-42 years, gave consent, completed questionnaires, and kept a symptom diary for 2 weeks. CPM efficiency was tested with a heat test stimulus and cold water condition stimulus in a laboratory between 8 and 10 a.m. on a follicular phase day. Salivary cortisol samples were collected just before and after the experimental testing. Compared to the HC group, women with IBS reported more days with gastrointestinal and somatic pain/discomfort, psychological distress, fatigue, and feeling stressed. During the CPM baseline testing, women with IBS reported greater pain sensitivity compared to the HC group. There was no significant group difference in salivary cortisol levels nor in CPM efficiency, though a post-hoc analysis showed a higher prevalence of impaired CPM efficiency among IBS subjects with more severe lower-GI symptoms. In the IBS group, reduced CPM efficiency was associated with daily abdominal pain/discomfort and psychological distress. Overall, women with IBS exhibited an increased sensitivity to thermal stimuli. Impaired CPM was present in a subset of women with IBS.

Changes in circulatory FSH of Barbari goats following treatment with high molecular weight inhibin isolated from buffalo follicular fluid

The objective of the present study was to isolate and purify high MW inhibin (∼129kDa) from buffalo ovarian follicular fluid (buFF) and to investigate its biological activity. Throughout the process of purification, the inhibin fractions were evaluated for bioactivity by a specific, sensitive and uniformly reproducible bioassay in mice. The final biological activity of this preparation was tested in normal cycling adult female Barbari goats. Eight animals, randomly divided into two groups, were synchronized for estrus by administering PGF2α twice at an interval of ten days. Following synchronization, the treatment group (n=4) received (i.m.) 0.4ml (240μg protein total dose) of purified inhibin (MW∼129kDa) of buFF in the morning at 08.00h for the four consecutive days of follicular phase, while the control group (n=4) received only saline (0.4ml). Blood samples were collected from jugular vein immediately before the first injection and subsequent collections were made daily in the afternoon until day 8 of the experiment including four days (0, 1, 2, & 3days) of the next cycle. FSH was assayed in all the samples by ELISA. The peripheral FSH concentration sharply declined from 1.854±0.137 to 0.979±0.02 u/l, 8h after the administration of inhibin on the first day. The value in controls was 2.004±0.132 u/l. For the duration of treatment of four consecutive days, the FSH level in experimental group remained significantly low (p<0.05) compared to control group. After cessation of treatment, the FSH level remained low on day 0 and 1 of the next cycle in the experimental and control animals. However, a significant rebound increase in plasma FSH levels occurred on day 2 & 3 (2.73±0.179 & 1.849±0.128u/l) only in the experimental group compared to control animals (P<0.05). This increment might be caused by the rebound surge of FSH from anterior pituitary which further corroborates the effect of inhibin in treated animals. In conclusion, the present study demonstrates that the high MW form of inhibin (∼129kDa) isolated from buFF has comparable biological activity as revealed by 31–32kDa inhibin from other species.

Use of FTA card for dry collection, transportation and storage of cervical cell specimen to detect high-risk HPV

Background The FTA elute micro card™, which enable the collection, transport, and archiving of DNA could be an attractive alternative to a liquid based collection system for detection of human papillomavirus (HPV). Objectives To develop a method based on the FTA elute micro card™ for dry collection of cervical epithelial cell samples, suitable for subsequent PCR-based HPV testing. Study design The method was evaluated by a comparison of the DNA collected by cytobrush and the regular FTA elute micro card™ from 50 cervical cell samples. The method was then used to estimate the DNA amount in 1040 samples applied to the indicating FTA elute micro card™. Result The agreement in HPV positivity between the cytobrush and FTA samples (94%) was excellent (kappa = 0.88, 95% CI 0.748–1). All the 1040 samples on the indicating FTA card™ had sufficient amounts of genomic DNA (>10 copies of a single copy gene) to be suitable for HPV typing. In 53 of the 1040 women the day in the menstrual cycle was noted, and the copy number during follicular phase day 9–13 was found to be statistically significantly lower than for the other three stages in the menstrual cycle (day 4–8, 14, >14) and during menopause. Conclusion The indicating FTA elute micro card™ represents a suitable medium for collection of cervical cell samples, although follow-up studies are needed to verify the detection of low frequency HPV types.

High frequency of luteal phase deficiency and anovulation in recreational women runners: blunted elevation in follicle-stimulating hormone observed during luteal-follicular transition.

The purposes of this investigation were to evaluate the characteristics of three consecutive menstrual cycles and to determine the frequency ofluteal phase deficiency (LPD) and anovulation in a sample of sedentary and moderately exercising, regularly menstruating women. For three consecutive menstrual cycles, subjects collected daily urine samples for analysis of FSH, estrone conjugates (E1C), pregnanediol-3-glucuronide (PdG), and creatinine (Cr). Sedentary (n=11) and exercising (n=24) groups were similar in age (27.0+/-1.3 yr), weight (60.3+/-3.1 kg), gynecological age (13.8+/-1.2 yr), and menstrual cycle length (28.3+/-0.8 days). Menstrual cycles were classified by endocrine data as ovulatory, LPD, or anovulatory. No sedentary women (0%) had inconsistent menstrual cycle classifications from cycle to cycle, but 46% of the exercising women were inconsistent. The sample prevalence of LPD in the exercising women was 48%, and the 3-month sample incidence was 79%. In the sedentary women, 90% of all menstrual cycles were ovulatory (SedOvul; n=28), whereas in the exercising women only 45% were ovulatory (ExOvul; n=30); 43% were LPD (ExLPD; n=28), and 12% were anovulatory (ExAnov; n=8). In ExLPD cycles, the follicular phase was significantly longer (17.9+/-0.7 days), and the luteal phase was significantly shorter (8.2+/-0.5 days) compared to ExOvul (14.8+/-0.9 and 12.9+/-0.3 days) and SedOvul (15.9+/-0.6 and 12.9+/-0.4 days) cycles. Luteal phase PdG excretion was lower (P < 0.001) in ExLPD (2.9+/-0.3 microg/mg Cr) and ExAnov (0.8+/-0.1 microg/mg Cr) cycles compared to SedOvul cycles (5.0+/-0.4 microg/mg Cr). ExOvul cycles also had less (P < 0.01) PdG excretion during the luteal phase (3.7+/-0.3 microg/mg Cr) than the SedOvul cycles. E1C excretion during follicular phase days 2-5 was lower (P=0.05) in ExOvul, ExLPD, and ExAnov cycles compared to SedOvul cycles and remained lower (P < 0.02) in the ExLPD and ExAnov cycles during days 6-12. The elevation in FSH during the luteal-follicular transition was lower (P < 0.007) in ExLPD (0.7+/-0.1 ng/mg Cr) cycles compared to SedOvul and ExOvul cycles (1.0+/-0.1 and 1.1+/-0.1 ng/mg Cr, respectively). Energy balance and energy availability were lower (P < 0.05) in ExAnov cycles than in other menstrual cycle categories. The blunted elevation in FSH during the luteal-follicular transition in exercising women with LPD may explain their lower follicular estradiol levels. These alterations in FSH may act in concert with disrupted LH pulsatility as a primary and proximate factor in the high frequency of luteal phase and ovulatory disturbances in regularly menstruating, exercising women.

Proliferative index of human luteinized granulosa cells varies as a function of ovarian reserve

We examined whether the proliferative index of luteinized granulosa cells, as determined by flow cytometry, varied as a function of a woman's ovarian reserve, as reserve, as reflected by follicular-phase day 3 serum follicle-stimulating hormone. This prospective cohort study consisted of 19 women of similar chronologic age preparing for in vitro fertilization-embryo who met specific day 3 serum follicle-stimulating hormone criteria. The "low follicle-stimulating hormone" group consisted of 11 women with day 3 serum follicle-stimulating hormone levels < or = 6 IU/L. The "high follicle-stimulating hormone" group consisted of eight women with day 3 serum follicle-stimulating hormone levels > or = 18 IU/L. A total of 56 preovulatory follicles containing > or = 10(4) luteinized granulosa cells were examined by flow cytometry. The low follicle-stimulating hormone group was compared with the high follicle-stimulating hormone group to examine proliferative index as a function of serum day 3 follicle-stimulating hormone levels. The low follicle-stimulating hormone group had a greater proliferative index (11.1% +/- 0.4%) than did the high follicle-stimulating hormone group (8.3% +/- 0.6%), p < 0.001). This study demonstrates that in spite of the same chronologic age, luteinized granulosa cells from preovulatory follicles of women with day 3 serum follicle-stimulating hormone levels > or = 18 IU/L have a 25% decreased proliferative index compared with luteinized granulosa cells from women with day 3 serum follicle-stimulating hormone levels < or = 6. This suggests that granulosa cell proliferation is influenced by ovarian reserve and may explain in part the more favorable response to ovulation induction protocols that younger women demonstrate compared with women of more advanced reproductive age.

Effect of depo-medroxyprogesterone acetate on serum progesterone levels when administered on various cycle days

Depo-medroxyprogesterone acetate (DMPA) in the conventional dose of 1.50 mg, was administered intramuscularly to 49 healthy, already sterilized, Thai women on cycle day 5 (13 subjects), day 7 (12 subjects), day 9 (13 subjects) and day 11 (11 subjects) of normal menstrual cycles. Serum progesterone levels were then monitored in order to ascertain the latest follicular phase day in the cycle (up to day 11) when ovulation would be inhibited in the first month after injection. Among the 25 subjects who received DMPA on day 5 and 7, no longitudinal serum progesterone level rises indicative of ovulation were detected. When DMPA was given on day 9 and 11, 2 out of 13 subjects (15.39%) and 3 out of 11 subjects (27.28%), respectively, had serum progesterone levels characteristic of ovulation. It is concluded that the initial cycle's ovulation can also be inhibited when DMPA is administered on day 7 of that cycle. However, DMPA administered on day 9 and 11 failed to inhibit ovulation of that cycle in some of the subjects.

Follicular function in the domestic cat as determined by estradiol-17 beta concentrations in plasma: relation to estrous behavior and cornification of exfoliated vaginal epithelium.

Follicular function was estimated by radioimmunoassay of estradiol-17β in jugular vein plasma of female cats. The mean base level of estradiol-17β in plasma was 11.7 pg/ml (SD = 4.8; n = 106). The follicular phase, defined as the time interval when estradiol-17β levels were >20 pg/ml plasma, was 7.4 days (SD = 2.3; n = 108), range 3–16 days. The duration of follicular function was not affected by the occurrence of coitus or ovulation. Estradiol-17β fell from peak levels to <20 pg/ml plasma within a mean of 2.6 days (SD = 1.1; n = 105). The interval between follicular phases was 8.1 days (SD = 3.1; n = 41), ranging from 3–15 days. Proestrus was not observed with regularity and was seen in only 27/168 cycles, averaging 1.2 days (SD = 0.8). Duration of estrous behavior without regard for coitus was 7.4 days (SD = 3.7; n = 168; range 2–19 days). Duration of estrus in cats that experienced coitus and ovulation was 8.6 days (SD = 4.1; n = 32), in those that had coital contact but failed to ovulate, it was 8.3 days (SD = 4.3; n = 43), but in those without coital contact, it was 6.2 days (SD = 2.9; n = 93) (coitus vs no coitus, P<0.01). Estrous behavior was seen in 8, 36, 52, 80 and 85% of cats on follicular phase Days 1–5, respectively. All cats were in estrus by follicular phase Day 6. The interval between estrous periods was 9.0 days (SD 7.6; n = 162; range 4–22 days) if ovulation did not occur. Ovulation and the subsequent pseudopregnancy prolonged return to estrus for 45.0 days (SD = 10.3; n = 32). No conspicuous changes in size and appearance of external genitalia were observed. Clearing of the vaginal smear (absence of noncellular debris) was the earliest sign of follicular activity. Vaginal epithelial changes were characterized as follows: Anuclear cells (fully cornified) increased from∼ 5% to ∼40% of total cell population and intermediate cells (partially cornified with intact nucleus) decreased from ∼45% to 6% progressively from the first to the fourth day of the follicular phase; parabasal cells (noncornified), generally low throughout a cycle (1–6%), were absent on follicular phase Days 4–7 and the following day; superficial cells (partially cornified with signs of degeneration of the nucleus) did not change much during the cycle (range, 40–50%) except for a slight increase during the follicular phase (range, 50–60%).