Low P4 Research Articles

Corpus luteum proximity alters molecular signature of the small extracellular vesicles and cumulus cells in the bovine ovarian follicle environment

Progesterone (P4) is predicted to act as a negative regulatory hormone for oocyte maturation events; however, its local effects during follicular development remain poorly understood in bovine. The complex process of oocyte meiosis progression is dependent on cellular communication among follicular cells. Besides, the breakdown of this communication, mainly between cumulus cells (CC) and oocyte, through the retraction of cumulus projections connecting these cells can impact oocyte maturation. In our study, we observed that follicles from the ovary ipsilateral to the corpus luteum (CL) containing high intrafollicular P4 concentrations enhance the abundance of proteins detected in follicular-derived small extracellular vesicles (sEVs) predicted to be involved in the retraction of membrane projections based on actin filaments, such as transzonal projections (TZPs). Conversely, we found that follicles from the ovary contralateral to the CL, which contained low intrafollicular P4 concentrations, had a high detection of proteins predicted to regulate the maintenance of TZPs. We also performed RNAseq analysis which demonstrated that 177 genes were differentially expressed in CC under the different P4 environments. Bioinformatic analysis points to changes associated to cell metabolism in cells from follicles ipsilateral to the CL in comparison to genes involved in cell communication in CC from follicles contralateral to the CL. Our functional analysis experiment confirmed that supplementation of cumulus-oocyte complexes during in vitro maturation with P4 at concentration similar to ipsilateral follicles reduces the number of TZPs. In summary, our study underscores a direct association between P4 concentration and cumulus-oocyte interaction, with potential consequences for the acquisition of oocyte competence.

P-653 Impact of dydrogesterone use in cycles with low progesterone levels on the day of frozen embryo transfer

Abstract Study question Assessing if Frozen Embryo Transfer (FET) outcomes in hormonal replacement treatment with dydrogesterone (DYD) for low progesterone levels match those with normal progesterone (P4) values Summary answer The live birth rate showed no significant difference between patients with low and normal serum P4 levels in FET, utilizing DYD as rescue therapy What is known already The number of IVF/ICSI cycles and frozen embryo transfers (FET) is increasing. The preparation for FET can occur in natural or artificial cycles, showing no significant differences in clinical pregnancy and live birth rates. Artificial cycles involve exogenous estradiol supplementation, starting after menses, and progesterone (P4) addition post-endometrial proliferation. Numerous studies consistently indicate that diminished P4 levels are correlated with inferior outcomes in hormonal replacement treatment, showing a potential decrease of up to 30% in ongoing pregnancy rates. Despite a suggested rescue strategy, evidence supporting its efficacy using an oral route is insufficient. Study design, size, duration This was a retrospective cohort study, of single embryo transfers (SET) from FET cycles conducted between July 2019 and March 2022, after artificial endometrial preparation with valerate-estradiol and micronized vaginal P4. In cases of low serum P4 (up to 12,5ng/dl) on the planned transfer day, 10 mg of dydrogesterone (DYD) three times daily was added. Solely blastocyst single embryo transfers (SET) were included. The primary endpoint was the live birth rate> 24 weeks. Participants/materials, setting, methods 535 FET-SET cycles were analyzed. Patients with an endometrial layer thinner than 7 mm, endocavitary pathology or serum P4 values< 4ng/dl were excluded. The explanatory variables evaluated were: relevant female characteristics, previous IVF/ICSI cycle and relevant FET cycle parameters (endometrial thickness, serum P4 value on the FET day, days in culture before blastocyst freezing) and use of DYD as rescue therapy. Statistical analysis was performed using IBM®SPSS® statistics v22.0 and R4.2.2 for Windows. Main results and the role of chance Out of the 535 FET cycles analyzed, 136(25.4%) underwent treatment with DYD. There were 337 pregnancies (63%), 207 live births (38.6%) and 130 miscarriages (38.5%). Serum P4 varied from 4.2-32.3 ng/dl, with an average of 14.93 ng/dl (SD 4.52). The best model to explain serum P4 distribution considered a gamma distribution for P4. Female age on the FET day, female ethnicity and female weight seemed to explain P4 variation. A sensitivity analysis on the P4 values was performed. The data were divided into 5 groups according to P4 levels and the rate of live birth per quintile was evaluated. There were no statistically significant differences between the groups. Patients with low P4 which had DYD added had no statistical differences regarding deliveries, 40% vs 36% (p = 0,354). The multivariable model revealed that older females at pick up, a thinner endometrial measure and transferring a day 6 blastocyst were negativey associated with live birth (p < 0,05), but not P4 levels or medication with DYD. A sub-group analyses was performed according to the median value of P4 (9.6 ng/ml) of those to whom DYD was added. No statistically significant differences were identified for biochemical pregnancy 59% vs 60% (p = 1) and live birth rate 34%vs38% (p = 0,653). Limitations, reasons for caution This retrospective study lacks an untreated control group, causing inherent heterogeneity. It’s unclear if there’s an upper limit for P4 levels to restrict DYD supplementation or a minimum level rendering this strategy unsuitable. Monitoring DYD’s fetal/neonatal safety is imperative. Wider implications of the findings The supplementation with oral DYD in hormonal replacement FET cycles is an easy and cheap strategy in patients with for low P4 levels. The duration of DYD administration should be further evaluated. Trial registration number not applicable

P-626 Progesterone rescue is effective for clinical pregnancy outcome in women presenting low circulating progesterone levels at frozen embryo transfer day

Abstract Study question Is subcutaneous progesterone (prolutex® 25mg daily) addition effective for clinical pregnancy outcome in women presenting low progesterone(P4) levels at frozen embryo transfer(FET) day? Summary answer Subcutaneous progesterone rescue improved ongoing pregnancy rate and clinical pregnancy rate in women with low P4 results at FET day. What is known already FET has been increased, because preimplantation genetic testing for aneuploidies is performed widely, and FET presents a significantly low risk of ovarian hyperstimulation syndrome.In FET, there is no established optimal protocol for preparing endometrium, but hormonal replacement therapy (HRT) is widely used due to its convenient schedule adjustment. HRT protocol induce anovulation, functioning corpus luteum is lacking and exogenous progesterone administration is very important. Therefore, women showing low P4 levels around FET day have lower pregnancy rate. However, there are currently no data on the effectiveness of progesterone rescue. Study design, size, duration Primary outcomes from a randomized cohort retrospective study investigating subcutaneous progesterone (prolutex® 25mg) daily addition for whom had low P4 levels (<12ng/mL) at FET day. The study was conducted at one infertility center in Korea from January 2022 to December 2023 and pregnancy outcomes of FET transfers registered until Oct. 2023,. 194 patients fulfilled the inclusion criteria. Participants/materials, setting, methods One hundred ninety four women divided into two groups based on progesterone levels, with 12ng/ml as the threshold. Under 12ng/mL group were randomized to subgroup with and without addition of subcutaneous progesterone. In total 278 FET cycles, the effect of progesterone rescue on cumulative clinical pregnancy rate (CPR), Ongoing pregnancy rate (OPR) was reported as primary outcomes. Main results and the role of chance In total 278 cycles, there were 137 cycles with adequate serum P4 levels, while 141 cycles presented with low P4 levels in which randomly rescue subcutaneous progesterone administration added from next day of embryo transfer. When comparing the groups, there was no statistical difference in maternal age, BMI, smokers, duration of infertility, type of infertility, indication of in vitro fertilization(IVF), endometrial preparation method. In low P4 group, with and without subcutaneous progesterone addition subgroups included 51 and 90 cycles, retrospectively. CPR were 52.9% versus 35.6% (P = 0.044; chi-squared test) with a statistically significant higher rate of OPR in progesterone rescue group (45.1% versus 28.9%, p = 0.052). When comparing the group with adequate P4 levels and the group with low P4 levels without progesterone rescue, OPR were 18.2% versus 28.9% (p = 0.06, chi-squared test), CPR was significantly higher in low P4 levels group (23.4% versus 35.6%,p=0.046, chi-squared test). Adequate P4 level group had more IVF attempts (4.6 versus 2.2, p < 0.001, chi-squared test). There is tendency to use more luteal phase support medication with repeated attempt (serum P4 concentration on ET day 39.3 ng/mL versus 8.7 ng/mL, p < 0.001, t-test) Limitations, reasons for caution The main limitation of the study was retrospective, and had small sample size. A control group without progesterone rescue in low P4 levels did not receive any intervention. Wider implications of the findings The progesterone rescue for women presenting low P4 on FET day improved clinical outcome and excessively raising progesterone medication may have adverse effects. Therefore rather than using excessive progesterone administration from luteal support start day, progesterone rescue might be effective when low P4 levels presenting at FET day. Trial registration number not applicable

P-418 What is the optimal midluteal phase circulating progesterone level during a fresh IVF cycle? The answer differs from what one expects in a natural cycle

Abstract Study question Do midluteal phase circulating progesterone (P4) levels relate to the fresh embryo transfer clinical outcome and is rescue P4 useful in such cases? Summary answer Live birth rate (LBR) was significantly lower when P4 levels were below 20ng/ml and did not seem to benefit from rescue intramuscular P4. What is known already Preceding literature on the predictive value of midluteal P4 for LBR has shown conflicting results and frequently included small sample sets and heterogeneous ovarian stimulation protocols. The findings reported ranged from the lack of an association between P4 serum levels and clinical outcome to worse success rates seen either in the groups with the highest or the lowest P4 in midluteal phase. Furthermore, while rescue P4 has been empirically proposed, it has lacked formal assessment thus far. Study design, size, duration In total, 652 patients who underwent a fresh single blastocyst transfer in an IVF cycle in our center between 2014 and 2021 were included in this retrospective cohort study. All were GnRH-antagonist cycles using hCG trigger and luteal support with micronized vaginal progesterone 200mg twice daily. Rescue intramuscular progesterone was systematically administered whenever low midluteal P4 (below 1 standard deviation of the center’s mean) levels were detected. Participants/materials, setting, methods The cycles were subdivided into three groups according to the midluteal P4 levels: <10ng/ml, 10-20ng/ml and >20ng/ml. The primary outcome assessed was LBR, with secondary outcomes including clinical pregnancy (CPR) and pregnancy loss rates. Maternal age and body mass index (BMI), year of treatment, ovarian response, endometrial thickness, cause of infertility and embryo quality were controlled for using multivariable logistic regression. Main results and the role of chance The mean age ± standard deviation of patients was 36.8±3.8 years, while mean weight and BMI were 63.2±11.5kg and 23.3±3.9kg/m2, respectively. LBR in the group with P4>20ng/ml (46.5%) was significantly higher than in the groups with low and intermediate P4 serum levels (23.1% and 28.1%, respectively, p < 0.001). The same was true for CPR (54.5%, 30.8% and 37.9%, respectively, p < 0.001). Conversely, the rate of pregnancy loss in the group with P4>20ng/ml (23.8%) was significantly lower than in the groups with low and intermediate P4 levels (42.6% and 37.5%, respectively, p = 0.018). The multivariable logistic regression confirmed that, after controlling for the confounding variables described above, P4>20ng/ml remained significantly associated with a higher LBR (OR: 2.285; 95% CI: 1.506-3.469) and a lower pregnancy loss rate (OR: 0.488; 95% CI: 0.273-0.873), irrespective of the use of rescue IM P4. Finally, a multivariable linear regression analysis of the patients baseline characteristics suggested that the only parameter predictive of midluteal P4 levels was the female patients’ weight, with an inverse relationship (adjusted regression coefficient: -0,012; 95% CI: -0,016/-0,009). Limitations, reasons for caution As the pituitary suppression, ovulation trigger and luteal support protocols used in the cycles included in the study were homogeneous (in order to maximize the internal validity of the results), this limits the extrapolibility of the results to other stimulation protocols. Furthermore, we caution that this is a retrospective study. Wider implications of the findings The usefulness of both midluteal P4 serum measurements and rescue P4 should continue to be assessed. The reasons behind the need for higher levels of P4 in a fresh cycle, when compared to natural cycles, must also be investigated. Luteal support may require personalization according to female body weight. Trial registration number Not Applicable

P-437 Are serum progesterone levels predictive for reproductive outcomes in artificial cycles if individualized luteal phase support is only administered when progesterone is below 5 ng/ml?

Abstract Study question Do serum progesterone levels predict reproductive outcomes in artificial cycles when individualized luteal phase support is administered only if progesterone falls below 5 ng/ml? Summary answer The low 5 ng/ml progesterone threshold appears to be sufficient for the patient not to require additional progesterone injections when progesterone is above 5 ng/ml. What is known already Serum progesterone (P4) level is negatively associated with ongoing pregnancy rate (OPR) and miscarriage rate in artificially prepared frozen embryo transfer (FET) cycles using standard micronized vaginal progesterone (MVP) for luteal phase support. Individualized luteal support is considered a successful rescue strategy in cycles with low serum progesterone levels eliminating the effect of P4 levels on outcome. Cut-off levels for this rescue strategy range from 8.8 to 10 ng/ml. Study design, size, duration A retrospective single-centre cohort study including a total of 1,016 vitrified-warmed single blastocyst transfers performed at a tertiary fertility centre between October 2021 and December 2022. Blastocysts of ≥ 3CC Gardner score were eligible for vitrification on day 5 or day 6 (Vit-kit or Vit-kit NX, FuijiFilm). Embryos were warmed and subsequently transferred on the same day which was day 5 in the cycle, regardless if the embryo was vitrified on day 5 or day 6. Participants/materials, setting, methods We included 595 patients undergoing an artificially prepared embryo transfer (ET) cycle using MVP 800mg BID for luteal phase support. Serum P4 levels were determined on the day of ET with the initiation of additional injections of subcutaneous P4 from the day after ET in patients with P4 < 5ng/ml. Primary endpoint was OPR. Multivariable logistic regression was performed to correct for confounders. Generalized estimating equations (GEE) models were fitted for primary and secondary outcomes. Main results and the role of chance OPR was comparable for the 3 groups: 9/40 (22.5%) for patients with P4 <5ng/ml, 64/275 (23.3%) for patients with P4 5-10 ng/ml and 181/701 (25.8%) for patients with P4 >10ng/ml (p = 0.377). No significant association between P4 category and likelihood for OPR was found after adjusting for confounders. Early clinical miscarriage rate was significantly different between the 3 groups: 5/40 (12.5%) in patients with P4 <5ng/ml, 26/275 (9.5%) with P4 5-10ng/ml and 37/701 (5.3%) with P4 >10ng/ml (P = 0.005). Limitations, reasons for caution Higher clinical miscarriage rate in cycles with a P4 level under 10 ng/ml questions the efficacy of rescue progesterone in cycles with a low P4. The patient population also exists of a subgroup of PGT-a tested cycles which was not included as a potential confounder in this analysis. Wider implications of the findings The 5 ng/ml P4 threshold appears to be sufficient for the patient not to require additional P4 injections when P4 is above 5 ng/ml. Consequently, a significantly lower proportion of cycles would need to be supplemented with rescue progesterone; 3.8% versus 31.3% for the 10 ng/mL cut off. Trial registration number ONZ-2023-0223

Impact of dydrogesterone use in cycles with low progesterone levels on the day of frozen embryo transfer.

This study aims to evaluate whether the clinical outcomes of cycles with frozen embryo transfer (FET) in hormonal replacement treatment supplemented with dydrogesterone (DYD) following detection of low circulating levels of progesterone (P4) were comparable to the results of cycles with otherwise normal serum P4 values. Extended analyses of a retrospective cohort that included FET cycles performed between July 2019 and March 2022 after a cycle of artificial endometrial preparation using valerate-estradiol and micronized vaginal P4 (400mg twice daily). Whenever the serum P4 value was considered low on the morning of the planned transfer, 10mg of DYD three times a day was added as a supplement. Only single-embryo transfers of a blastocyst were considered. The primary endpoint was live birth rate. Five-hundred thirty-five FET cycles were analyzed, of which 136 (25.4%) underwent treatment with DYD. There were 337 pregnancies (63%), 207 live births (38.6%), and 130 miscarriages (38.5%). The P4 values could be modeled by a gamma distribution, with a mean of 14.5ng/ml and a standard deviation of 1.95ng/ml. The variables female age on the day of FET, ethnicity, and weight were associated with a variation in the serum P4 values. There were no differences in the results between cycles with or without the indication for DYD supplementation. Live birth rate did not vary significantly in females with low and normal serum P4 levels on the day of FET when DYD was used as rescue therapy.

Effect of 200 μg of gonadorelin hydrochloride at the first GnRH of a CO-Synch program on ovulation rate and pregnancies per artificial insemination in Holstein heifers

The initial ovulatory response during synchronization programs is often low in dairy heifers, largely due to follicular dynamics and hormonal dynamics. Specifically, the progesterone (P4) concentration at the time of the first GnRH treatment in a breeding program can influence the LH response, often resulting in a suboptimal ovulatory response. The objective of this study was to determine the effect of the highest label dose 200 μg (100 μg vs. 200 μg) of GnRH (50 μg of gonadorelin hydrochloride per mL; Factrel, Zoetis Inc. Madison, NJ) at the first GnRH of a 6-d CO-Synch plus P4 device program on ovulatory response and pregnancy per AI (P/AI) in first service in Holstein heifers. A total of 1,308 Holstein heifers were randomly allocated at the beginning of a 6-d CO-Synch program at day 0 to receive either i.m. treatment of 100 μg (2CC, n = 655) or 200 μg (4CC, n = 653) of GnRH. Also, at d 0, heifers received an intravaginal insert with 1.38 g of P4 (Eazi-Breed CIDR Cattle Insert, Zoetis Inc.). On day 6, the insert was removed, and i.m. treatment of 25 mg of PGF2α (12.5 mg of dinoprost tromethamine/mL; Lutalyse HighCon Injection, Zoetis Inc.) was administered. On d 7, a second i.m. treatment of 25 mg of PGF2α was given, followed on d 9 by concurrent i.m. treatment of 100 μg of GnRH, and timed AI. A subset of 396 heifers had their ovaries scanned to evaluate ovulatory response, and blood samples were collected to measure the serum concentration of P4 at d 0 and d 6 of the study. The P4 concentrations at d 0 were categorized as low (≤3 ng/mL) or high (>3 ng/mL). The ovulatory response was greater for heifers receiving 4CC than 2CC at d 0 (54.7% vs. 42.8%). The ovulatory response was greater for low P4 than high P4 at d 0 (54.3% vs. 37.8%). However, we did not observe an interaction between treatment and P4 concentrations (low P4 2CC = 48.6% vs. high P4 2CC = 30.0%; low P4 4CC = 60.0% vs. high P4 4CC = 45.5%). The receiver operating characteristic curve analysis indicated that P4 concentrations at d 0 treatment could predict the ovulatory response, although the area under the curve was only 0.6. As expected, heifers that ovulated had increased P/AI (no = 55.6% vs. yes = 67.7%); however, we found no effect of treatment on P/AI (2CC = 63.3% vs. 4CC = 59.6%), and no interactions between treatment and ovulation and treatment and P4 (high vs. low) for pregnancy outcomes. In summary, P4 concentration and increasing the dose of GnRH at d 0 positively affected ovulatory response in Holstein heifers. However, there was no interaction between treatment and P4 on ovulation and no subsequent impact of GnRH dose on P/AI.

The combination of dydrogesterone and micronized vaginal progesterone can render serum progesterone level measurements on the day of embryo transfer and rescue attempts unnecessary in an HRT FET cycle.

To evaluate the role of serum progesterone (P4) on the day of embryo transfer (ET) when dydrogesterone (DYD) and micronized vaginal progesterone (MVP) are combined as luteal phase support (LPS) in a hormone replacement therapy (HRT) frozen ET (FET) cycles. Retrospective study, including single euploid HRT FET cycles with DYD and MVP as LPS and P4 measurement on ET day. Initially, patients with P4 levels < 10 ng/ml increased MVP to 400 mg/day; this "rescue" was abandoned later. 560 cycles of 507 couples were included. In 275 women, serum P4 level was < 10 ng/ml on the ET day. Among those with low P4 levels, MVP dose remained unchanged in 65 women (11.6%) and was increased in 210 women (37.5%). Women with P4 levels ≥ 10 ng/ml continued LPS without modification. Overall pregnancy rates in these groups were 61.5% (40/65), 54.8% (115/210), and 48.4% (138/285), respectively (p = n.s.). Association of serum P4 levels with ongoing pregnancy rates was analyzed in women without any additional MVP regardless of serum P4 levels (n = 350); multivariable analysis (adjusted for age, BMI, embryo quality (EQ)) did not show a significant association of serum P4 levels with OPR (OR 0.96, 95% CI 0.90-1.02; p = 0.185). Using inverse probability treatment weights, regression analysis in the weighted sample showed no significant association between P4 treatment groups and OP. Compared to fair EQ, the transfer of good EQ increased (OR 1.61, 95% CI 1.22-2.15; p = 0.001) and the transfer of a poor EQ decreased the odds of OP (OR 0.73, 95% CI 0.55-0.97; p = 0.029). In HRT FET cycle, using LPS with 300 mg/day MVP and 30 mg/day DYD, it appears that serum P4 measurement and increase of MVP in patients with P4 < 10 ng/ml are not necessary.

Reproductive Seasonality, Estrous Cycle, Pregnancy, and the Recurrence of Postpartum Estrus Based on Long-Term Profiles of Fecal Sex Steroid Hormone Metabolites regarding Zoo-Housed Female Golden Takins (Budorcas taxicolor bedfordi).

This study investigates the non-invasive monitoring of the endocrine ovarian activities of captive female golden takins (Budorcas taxicolor bedfordi) based on long-term fecal sex steroid hormone metabolite dynamics. Fecal progesterone (P4) metabolite dynamics were monitored in nine females for 0.5-15 years between 2004 and 2022. Fecal estradiol-17β (E2) and estrone (E1) metabolites were measured during certain estrous cycles, and fecal E1 metabolite concentrations were measured during all gestation periods. The breeding season of the captive animals was mainly between May and December, and they were polyestrous animals whose breeding season begins during the long-day period. The onset of the breeding season occurred slightly earlier as age increased. The mean age (±SD) at puberty based on fecal P4 metabolite dynamics was 4.1 ± 2.9 years. The first conception ages ranged from 2.3-10.2 years. The mean estrous cycle period (±SEM) was 25.4 ± 1.1 days, and mounting and mating occurred in periods of low fecal P4 metabolite levels during the breeding season. The mean gestation period (±SD) from the estimated mating date to the calving date was 253.9 ± 5.7 days, and the fecal P4 metabolite distribution during pregnancy was bimodal. Fecal estrone metabolite levels gradually increased 21 weeks before delivery, peaked during the week of delivery, and then markedly decreased in the first week after delivery. Estrus resumed in the first April-August period after delivery (mean ± SD; 103.5 ± 40.9 days) or in May of the year after delivery (421.0 ± 16.5 days). This study revealed that the estrous cycle and pregnancy of female golden takins can be determined by fecal progesterone metabolite dynamics and that fecal estrone metabolite dynamics increases toward parturition and are useful for predicting the date of delivery. This endocrinological information is important for planned breeding efforts for the golden takins.

Possibility of early pregnancy detection in alpacas (Vicugna pacos) based on fecal steroid hormone concentrations.

Early pregnancy detection in alpacas, whose breeding season is limited to the rainy season and has a long gestation period, is important for reproductive management. Conventional detection methods such as ultrasonography cannot be used to detect pregnancy before 30 days after mating. In this study, we examined the feasibility of using fecal steroid hormones as an early detection method in pregnant and non-pregnant alpacas. Fecal and blood samples were collected from pregnant and non-pregnant alpacas after mating. Progesterone (P4) and estradiol 17-β were extracted and quantified from blood and fecal samples. A positive correlation exists between the steroid hormones in serum and feces, indicating that serum steroid hormone concentrations can be estimated from fecal steroid hormones. Within 10days after mating, both pregnant and non-pregnant alpacas had fecal P4 concentrations greater than 1.0ng/mg dry matter (DM), but by 15 days after mating, fecal P4 concentrations decreased to the pre-mating concentration in non-pregnant alpacas. From 15 days after mating, non-pregnant alpacas had a low fecal P4 concentration (< 1ng/mg DM), whereas a high fecal P4 concentration indicated the possibility of pregnancy, suggesting that this test is clinically beneficial as a supportive test for pregnancy detection.

#362: Importance of Monitoring Progesterone(P4) Values a Day Before D5 FET and Effect of Intervention with Progesterone Supplements in Cases with Low P4 Values

Background and Aims: Our study was carried out at a tertiary fertility Centre- Hyderabad, to study the importance of monitoring P4 values, day before D5FET and intervention with progesterone supplements in case of low levels and its outcome. Methods: This is a retrospective cohort study done by analyzing all D5FET cases and cases with low P4 values were given progesterone supplements and its effect on outcome were studied. Done between 1/01/22-31/03/23, a period of 15 months. Excluded donor, D3nD6 cycles. Results: In a 15 months period, there were 466 D5FET cycles which were included in our study. The age group of women were between 21-45 years and their P4 values, day before D5FET were studied. Around 290(62.2%) cases had levels between 11-20 ng/ml. Among them, 217 conceived being 74.8%. There were 79(16.9%) people who had levels between 21-30 ng/ml among whom 60 conceived with success rate being 75.9%. There were 97 cases who had levels less than 10 ng/ml. If their levels were less than 8ng/ml, they received aqueous progesterone 25 mg subcutaneously twice, 12 hours apart. If levels were between 8-10 ng/ml, they received micronized progesterone 200 mg per rectum twice, 12 hrs apart. The positive pregnancy rate came up to 76.2% among people who received supplementation. In fact, there were 4 people who had levels &lt;5 ng/ml, all four received aqueous progesterone and conceived. Conclusion: Our study concludes that evaluating P4 values day before D5FET is of vital importance. Timely supplementation helps in positive pregnancy rate at par with people with optimum levels of P4.

Rectal progesterone administration secures a high ongoing pregnancy rate in a personalized Hormone Replacement Therapy Frozen Embryo Transfer (HRT-FET) protocol: a prospective interventional study.

Can supplementation with rectal administration of progesterone secure high ongoing pregnancy rates (OPRs) in patients with low serum progesterone (P4) on the day of blastocyst transfer (ET)? Rectally administered progesterone commencing on the ET day secures high OPRs in patients with serum P4 levels below 35 nmol/l (11 ng/ml). Low serum P4 levels at peri-implantation in Hormone Replacement Therapy Frozen Embryo Transfer (HRT-FET) cycles impact reproductive outcomes negatively. However, studies have shown that patients with low P4 after a standard vaginal progesterone treatment can obtain live birth rates (LBRs) comparable to patients with optimal P4 levels if they receive additionalsubcutaneous progesterone, starting around the day of blastocyst transfer. In contrast, increasing vaginal progesterone supplementation in low serum P4 patients does not increase LBR. Another route of administration rarely used in ART is the rectal route, despite the fact that progesterone is well absorbed and serum P4 levels reach a maximum level after ∼2 h. This prospective interventional study included a cohort of 488 HRT-FET cycles, in which a total of 374 patients had serum P4 levels ≥35 nmol/l (11 ng/ml) at ET, and 114 patients had serum P4 levels <35 nmol/l (11 ng/ml). The study was conducted from January 2020 to November 2022. Patients underwent HRT-FET in a public Fertility Clinic, and endometrial preparation included oral oestradiol (6 mg/24 h), followed by vaginal micronized progesterone, 400 mg/12 h. Blastocyst transfer and P4 measurements were performed on the sixth day of progesterone administration. In patients with serum P4 <35 nmol/l (11 ng/ml), 'rescue' was performed by rectal administration of progesterone (400 mg/12 h) starting that same day. In pregnant patients, rectal administration continued until Week 8 of gestation, and oestradiol and vaginal progesterone treatment continued until Week 10 of gestation. Among 488 HRT-FET single blastocyst transfers, the mean age of the patients at oocyte retrieval (OR) was 30.9 ± 4.6 years and the mean BMI at ET 25.1 ± 3.5 kg/m2. The mean serum P4 level after vaginal progesterone administration on the day of ET was 48.9 ± 21.0 nmol/l (15.4 ± 6.6 ng/ml), and a total of 23% (114/488) of the patients had a serum P4 level lower than 35 nmol/l (11 ng/ml). The overall, positive hCG rate, clinical pregnancy rate, OPR week 12, and total pregnancy loss rate were 66% (320/488), 54% (265/488), 45% (221/488), and 31% (99/320), respectively. There was no significant difference in either OPR week 12 or total pregnancy loss rate between patients with P4 ≥35 nmol/l (11 ng/ml) and patients with P4 <35 nmol/l, who received rescue in terms of rectally administered progesterone, 45% versus 46%, P = 0.77 and 30% versus 34%, P = 0.53, respectively. OPR did not differ whether patients had initially low P4 and rectal rescue or were above the P4 cut-off. Logistic regression analysis showed that only age at OR and blastocyst scoring correlated with OPR week 12, independently of other factors like BMI and vitrification day of blastocysts (Day 5 or 6). In this study, vaginal micronized progesterone pessaries, a solid pessary with progesterone suspended in vegetable hard fat, were used vaginally as well as rectally. It is unknown whether other vaginal progesterone products, such as capsules, gel, or tablet, could be used rectally with the same rescue effect. A substantial part of HRT-FET patients receiving vaginal progesterone treatment has lowserum P4. Adding rectally administered progesterone in these patients increases the reproductive outcome. Importantly, rectal progesterone administration is considered convenient, and progesterone pessaries are easy to administer rectally and of low cost. Gedeon Richter Nordic supported the study with an unrestricted grant as well as study medication. B.A. has received unrestricted grant from Gedeon Richter Nordic and Merck and honoraria for lectures from Gedeon Richter, Merck, IBSA and Marckyrl Pharma. P.H. has received honoraria for lectures from Gedeon Richter, Merck, IBSA and U.S.K. has received grant from Gedeon Richter Nordic, IBSA and Merck for studies outside this work and honoraria for teaching from Merck and Thillotts Pharma AB and conference expenses covered by Merck. The other co-authors have no conflict of interest to declare. EudraCT no.: 2019-001539-29.

Effects of prostaglandin F2α treatment at follicular wave emergence on endometrial epidermal growth factor concentration on day 3 of the next estrous cycle and on fertility in dairy cows

Inadequate exposure to estradiol (E2) and progesterone (P4) may be the main causes of altered endometrial epidermal growth factor (EGF) profile, leading to reduced fertility in dairy cows. We hypothesized that PGF2α administration at different timings of the estrous cycle and stages of follicular development could change the profile of steroid hormones between luteolysis and estrus. This results in reduction in the peak concentration of endometrial EGF on day 3 (day 0 = estrus) in the next estrous cycle. In study 1, lactating Holstein cows were treated with PGF2α either on days 12–14 (selection phase group, n = 20) or on days 16–17 (control group, n = 24) of the estrous cycle. Blood samples were obtained before PGF2α treatment, 24 and 48 h after treatment, and on the day of estrus and ovulation for E2 and P4 assays. Endometrial tissues were collected by biopsy on day 3 for EGF assays. The duration from PGF2α treatment to both estrus and ovulation was longer in the selection phase group than in the control group (P < 0.05). The time between estrus and ovulation was longer in the selection phase group (P < 0.05). E2 concentrations were higher in the control group on the day of estrus (P < 0.05). P4 concentrations were not different between the groups. Endometrial EGF concentrations were lower in the selection phase group than in the control group (P < 0.05). In study 2, lactating Holstein cows were treated with PGF2α either on day 12 (selection phase group, n = 76) or day 16 (control group, n = 80). They were subjected to artificial insemination (AI) at estrus, and those that failed to ovulate by 24h after the first AI were subjected to second AI. On day 3, endometrial EGF concentration was determined. At estrus and on days 3 and 7, blood was collected for E2 and P4 assay from 20 randomly selected cows in each group before PGF2α treatment. The cows in the selection phase group exhibited lower EGF concentration, proportion of cows with normal EGF profile, and conception rate than cows in the control group (P < 0.05). On the day of estrus, E2 concentrations tended to be lower in the selection phase group than in the control group (P = 0.08). P4 concentrations were not different between the groups. These findings suggest that low E2 concentrations at estrus and low P4 concentrations for a prolonged period due to premature termination of CL in the absence of a dominant follicle are potential causes of altered endometrial EGF profile in dairy cows.

The effect of progesterone concentrations during superovulation of Holstein heifers in a randomized trial

The aim of this study was to evaluate the effect of different progesterone (P4) concentrations during the follicular growth on the intensity of estrous expression, ovarian response to the superovulatory treatment, and embryo production and quality in superovulated heifers. A total of 63 Holstein heifers were randomly assigned into 2 experimental groups: Low P4 (n = 31) and High P4 (n = 32). Animals received a pre-synchronization protocol followed by a protocol of superovulation that included the allocated P4 treatment. Activity was monitored continuously by an automated activity monitor, and estrus characteristics (maximum intensity and duration) were recorded. Embryo collection was performed 7 d post artificial insemination (AI). Embryos were counted and graded from good or excellent (1) to degenerated (4). The outcomes of interest were: number and diameter of follicles at the time of AI, ovulation success (confirmed 7 d post-AI), time to estrus event, maximum intensity and duration of estrus, number and quality of embryos. Data were analyzed according to the type of outcome variable using logistic, linear, or Poisson regression models. A total of 105 embryos (High P4: n = 42; Low P4: n = 63) were graded for quality. Different P4 levels did not affect the maximum intensity (High P4 = 497.8 ± 23.9%; Low P4 = 542.2 ± 23.5%) or the duration (High P4 = 13.5 ± 1.5 h; Low P4 = 14.3 ± 1.4 h) of estrus. Heifers in the High P4 treatment had greater number of follicles at time of AI (High P4 = 16.6 ± 1.6 follicles; Low P4 = 13.9 ± 1.2 follicles), but with smaller diameter (High P4 = 11.3 ± 0.1 mm; Low P4 = 12.0 ± 0.1 mm) compared with Low P4. High P4 heifers tended to have better embryo quality compared with Low P4 heifers (odds ratio = 1.98; 95% CI = 0.90-4.35). High P4 heifers had less embryos than Low P4 heifers, but this was modified by the CIDR (intravaginal implant of P4) removal to estrus interval (interval 0-21 h: mean ratio = 1.15, 95% CI = 0.42-1.87; interval 22-46 h: mean ratio = 0.58, 95% CI = 0.27-0.96). Although estrous expression was not associated with embryo quality, as the duration and the maximum intensity of estrous expression increased, the number of embryos recovered 7 d post-AI increased (duration: mean ratio = 1.04; 95% CI = 1.03-1.05; maximum intensity: mean ratio = 1.50; 95% CI = 1.42-1.58). In conclusion, P4 during the follicular growth, and intensity of estrus, are playing a role in regulating the quality and the number of embryos produced by superovulated heifers. This study was supported by contributions from Resilient Dairy Genome Project and the Natural Sciences and Engineering Research Council.

P-596 Rectal progesterone administration secures high ongoing pregnancy rate in a personalized Hormone Replacement Therapy Frozen Embryo Transfer (HRT-FET) protocol - a prospective interventional study

Abstract Study question Can supplementation with additional rectal administration of progesterone secure high ongoing pregnancy rates (OPR) in patients with low serum progesterone (P4) at blastocyst transfer (ET)? Summary answer Rectally administered progesterone starting on the blastocyst transfer day secures high OPR in patients with serum P4 levels lower than 35 nmol/l (11ng/ml). What is known already Low serum P4 levels at peri-implantation in HRT-FET cycles impact reproductive outcomes negatively. However, studies have shown that patients with low P4 after a standard vaginal progesterone treatment can obtain live birth rates (LBR) comparable to patients with optimal P4 levels if they receive additional subcutaneous progesterone, starting close to the day of blastocyst transfer. In contrast, increasing vaginal progesterone supplementation in low serum P4 patients does not increase LBR. Another route of administration rarely used in ART is the rectal route, although progesterone is well absorbed and serum P4 levels reach a maximum level after approximately two hours. Study design, size, duration This prospective interventional study included a cohort of 488 patients treated with single blastocyst HRT-FET, in which a total of 374 patients had a serum P4 level ≥35nmol/l (11ng/ml) at ET, and 114 patients had a serum P4 level &amp;lt;35nmol/l (11ng/ml). The study was conducted from January 2020 to November 2022. Participants/materials, setting, methods Patients undergoing HRT-FET in a public Fertility Clinic. Endometrial preparation included oral oestradiol (6mg/24hours), followed by vaginal micronized progesterone, 400mg/12hours. Single blastocyst transfer and P4 measurements were performed on the 6th day of progesterone administration and patients with serum P4 &amp;lt;35nmol/l (11ng/ml) additional rectal administration of progesterone (400mg/12hour) was started the same day. In pregnant patients, vaginal and rectal administration continued until week 8, and oestradiol and vaginal progesterone treatment continued until week 10. Main results and the role of chance Among 488 HRT-FET single blastocyst transfers, the mean age of the patients at oocyte retrieval (OR) was 30.9 ±4.6 years and the mean BMI at ET 25.1 ±3.5 kg/m2. The mean serum P4 level after vaginal progesterone administration on the day of blastocyst transfer was 48.9 ±21.0 nmol/l (15.4 ±6.6ng/ml), and a total of 23% (114/488) of the patients had a serum P4 level lower than 35nmol/l (11ng/ml). The overall, positive hCG rate, clinical pregnancy rate, OPR week 12, and total pregnancy loss rate were 66% (320/488), 54% (265/488), 45% (221/488), and 31% (99/320), respectively. There was no significant difference in neither OPR week 12 nor total pregnancy loss rate between patients with P4 ≥35nmol/l (11ng/ml) and patients with P4 &amp;lt;35nmol/l, who received rescue in terms of rectally administered progesterone, 45% vs. 46%, p = 0.77 and 30% vs. 34%, p = 0.53, respectively. OPR did not depend on whether patients had initially low P4 and rectal rescue or were above the P4 cut-off. Logistic regression analysis showed that only age at OR and blastocyst scoring correlated with OPR week 12, independently of other factors like BMI, and vitrification day of blastocysts (day 5 or 6). Limitations, reasons for caution In this study vaginal progesterone (Cyclogest®), a solid pessary with progesterone suspended in vegetable hard fat, was used vaginally as well as rectally. It is unknown whether other vaginal progesterone products could be used rectally as easily with the same rescue effect. Wider implications of the findings A substantial part of HRT-FET patients receiving vaginal progesterone treatment has low serum P4. Adding rectally administered progesterone in these patients increases the reproductive outcome. Importantly, rectal progesterone administration is considered convenient by the majority of patients; moreover, progesterone pessaries are easy to administer rectally and of low cost. Trial registration number EudraCT no.: 2019-001539-29

P-502 Higher progesterone levels on the day of frozen embryo transfer is associated to better reproductive outcomes and higher obstetric complications: a retrospective cohort study

Abstract Study question Can serum progesterone (P) levels on the day of frozen embryo transfer (ET) compromise reproductive outcomes and/or obstetric complications? Summary answer Serum P levels was related to higher clinical pregnancy rate on frozen ET cycles. However, women with P level≥8.8 ng/ml presented higher obstetric complications. What is known already P levels &amp;lt;9.2 ng/ml on the day of ET significantly decreased ongoing pregnancy rate (OPR) in 211 donated oocyte cycles. In another study of the same group, serum P levels &amp;lt;8.8 ng/ml on the day of ET presented lower OPR in both own or donated oocyte cycles. Another study shown that low serum P4 the day prior to euploid FET can benefit from association of injectable P and achieve similar reproductive outcomes compared to those with initial higher P levels. Study design, size, duration This is a retrospective cohort study that included 509 women who have undergone frozen embryo transfer. In this study was included only first cycle of each patient. The women were divided into two groups according P levels: 1) P level &amp;lt;8.8 ng/ml (n = 388) and 2) P level≥8.8 ng/ml (n = 121). The analysis included the period between 01 January 2020 to 31 December 2021. Participants/materials, setting, methods All participants of study who underwent frozen ET after an artificial endometrial preparation cycle with estradiol valerate or 17βestradiol and vaginal micronized progesterone(600 mg/day). Age, body mass index(BMI), cause of infertility, and reproductive outcomes(clinical pregnancy, miscarriage, twin pregnancy, live birth rates) and obstetric complications (prematurity, ectopic pregnancy, acretism, hypertensive syndrome in pregnancy, gestational diabetes, intrauterine growth restriction rates) were analyzed. Only women without uterine abnormalities and with endometrium≥6.5 mm were included in this study. Main results and the role of chance Clinical pregnancy rate did not significant statistical between the 1 and 2 groups. Live birth rate (p = 0.01) was significantly higher in group 2. Other reproductive outcomes were similar between the groups. In relation to obstetric complications, miscarriage rate was significantly higher in group 1 (p &amp;lt; 0.01) and prematurity (&amp;gt;0.01), acretism (&amp;gt; 0.01), and gestational diabetes (p = 0.01) rates were higher in group 2. The P level did not present relation to cause of infertility. The logistic regression analysis shown that only good embryo quality was predictor to clinical pregnancy rates. Limitations, reasons for caution These outcomes were shown only in women without uterus abnormality and that used vaginal progesterone. It is necessary caution to considerer these results to general population. Furthermore, serum P level was analyzed in single moment without definition of interval between last vaginal P dose administration and blood sample collected. Wider implications of the findings Monitoring P levels on FET day should be performed routinely on artificial cycles because it can be related to higher live birth rate. However, new studies can confirm if higher P levels on FET day or adjust of P level according P value will be related to better reproductive outcomes. Trial registration number not applicable

Doppler ultrasonography: an improvement in real-time recipient selection in equine ET programs

In this study, we test the hypothesis that a CL with greater blood perfusion has a more significant impact than luteal size and P4 concentrations on pregnancy per embryo transfer (P/ET). The study was carried out from November/2021 to May/2022 on a stud farm of the Mangalarga Marchador breed in Itaperuna-RJ, Brazil. Recipient mares (n=110), aged between 3-16 years, weighing 370-460 kg, with adequate BCS and on days 3 to 9 after spontaneous ovulation, received a single fresh grade I or II blastocyst, retrieved by uterine flushing of donor mares. Immediately before the ET, the CL characteristics were evaluated by B-mode and Color-Doppler ultrasonography (US) and a blood sample was collected for plasma P4 assay. Recipients were retrospectively classified according to CL area [small (≤6 cm2) or large (>6 cm2)], CL blood perfusion [low ≤55 % or high >55%] and plasma P4 concentration [low ≤9.98 ng/mL or high >9.98 ng/mL]. Pregnancy diagnosis was performed 30 days after ET and P/ET analysis was performed using the GLIMMIX of SAS procedure. The overall P/ET was 73% (83/110). The P/ET was not influenced by the luteal area, but effects of CL blood perfusion class (P=0.007) and the P4 plasma concentration class (P=0.02) on P/ET were observed. The P/ET was greater in the high luteal perfusion group [89.1%, 41/46 vs.65.6%, 42/64] and in the high P4 group [85.4%, 41/48 vs. 67.7%, 42/62]. When area, luteal blood perfusion and P4 concentration were compared with the probability of pregnancy through ROC curve analysis, luteal blood perfusion (AUC=0.66) was the most significant (P=0.01) predictor of pregnancy compared to area (AUC=0.53) and plasma P4 (AUC=0.60). Finally, recipients were grouped into four different groups by analyzing CL blood perfusion classes and P4 (high CLBP and high P4 vs. High CLBP and low P4 vs. low CLBP and high P4 vs. low CLBP and low P4) and compared with the P/ET using the Fisher test. P/ET differed between groups (P=0002), as indicated by the higher P/ET in the high CLBP and high P4 95% (19/20) and high CLBP and low P4 84.6% (22/26) groups, compared to animals with low CLBP and low P4 55.5% (20/36). In conclusion, although the P4 concentration is a potential indicator of pregnancy success, it cannot be used in real-time when selecting the most appropriate recipient in a commercial ET program. Also, luteal blood perfusion determined by Doppler-US is an innovative and easily accessible tool that can be used in real-time to select the recipients with the greatest chances of maintaining pregnancy in ET programs in horses.

Progesterone initiates tendril formation in the oviducal gland during egg encapsulation in cloudy catshark (Scyliorhinus torazame)

The diverse reproductive strategies of elasmobranchs (sharks, rays, and skates) have attracted research attention, but the endocrine control of reproduction is still incompletely known in elasmobranchs. By long-term monitoring of the egg-laying cycle in cloudy catsharks (Scyliorhinus torazame), we recently demonstrated a transient increase in plasma progesterone (P4) levels just prior to the appearance of the capsulated eggs in the oviducts. In the present study, we examined the in vivo effects of P4 administration in mature female cloudy catsharks. Although no capsulated eggs were observed following the implantation of P4-containing silicone tubing, we did find dark swollen oviducts in the abdominal cavity, in which clumps of long and coiled tendrils were observed. The tendril is an extension of the egg capsule, and the formation of the egg capsule begins with the tendril before main capsule formation. During the period of P4 implantation, the tendrils elongated, and their diameters were significantly increased on day 2 of treatment. Tendril formation was also confirmed on the day of endogenous P4 surge. Tendrils were not formed in catsharks implanted with estradiol-17β or testosterone. Histological analysis of the oviducal gland revealed that P4 implantation induced the secretion of tendril materials from the secretory tubules in the baffle zone, while the tendril materials were stored in the cytoplasm of the secretory cells under low P4 condition. Morphometrically, the area of secreted luminal materials in the secretory tubules was highly correlated to the diameter of tendrils. Our results suggest that the P4 surge during the egg-laying cycle serves as a trigger for egg capsule formation in the oviducal gland of cloudy catshark, but the hormonal signals were incomplete as the main capsule was not formed. Further studies are required to identify the hormones required for ovulation and formation of the main egg capsule.

Influence of Ovarian Status and Steroid Hormone Concentration on Day of Timed Artificial Insemination (TAI) on the Reproductive Performance of Dairy Cows Inseminated with Sexed Semen.

This study aimed to evaluate the effect of the ovarian status and steroid hormone concentration on the day of TAI on the reproductive performance of dairy cows subjected to estrus synchronization treatment and timed artificial insemination with sexed semen. Seventy-eight cyclic Holstein cows pre-treated with PGF2α-GnRH were divided in two groups-I (Preselect-OvSynch, n = 38) and II (OvSynch+PRID-7-day+eCG, n = 40)-and inseminated with sexed semen. The presence of preovulatory follicle (PF) with or without corpus luteum (CL), the PF diameter, the estradiol (E2) and progesterone (P4) concentrations on the day of TAI, the pregnancy rate (PR) and embryo loss were determined. On the day of TAI, 78.4% of all the pregnant cows presented a PF (mean size 1.80 ± 0.12 cm) without CL, low P4 (0.59 ± 0.28 ng/mL) and high E2 (12.35 ± 2.62 pg/mg) concentrations. The positive correlation between the size of the PF and the level of E2 in the pregnant cows from group II was stronger than that of group I (R = 0.82 vs. R = 0.52, p < 0.05). The pregnancy rate on day 30 (57.5% vs. 36.8%) and day 60 (50% vs. 26.3%; p < 0.05) and the embryo losses (13% vs. 28.5%) showed better effects of treatment in group II. In conclusion, the ovarian status and the steroid hormone concentration on the day of TAI influence the pregnancy rates of dairy cows subjected to estrus synchronization and timed artificial insemination with sexed semen.

Effects of additional gonadotropin-releasing hormone and prostaglandin F2α treatment to an estradiol/progesterone-based embryo transfer protocol for recipient lactating dairy cows

This study was designed to evaluate whether the utilization of a second PGF2α treatment at the end of an estradiol/progesterone (E2/P4)-based protocol with or without GnRH at the beginning of the protocol would improve pregnancy rates of lactating Holstein cows assigned to timed embryo transfer. A total of 501 lactating Holstein cows in 5 farms were enrolled in the experiment. Within farm, cows were blocked by parity and, within block, were assigned randomly to (1) insertion of an intravaginal P4 device (controlled internal drug-releasing device; CIDR) and estradiol benzoate on d -11, PGF2α on d -4, CIDR withdrawal and an injection of estradiol cypionate on d -2, and timed embryo transfer on d 7 (1-PGF; n = 164); (2) the same treatments as 1-PGF, but with PGF2α administered on d -4 and -2 (2-PGF; n = 171); and (3) 2-PGF with the addition of a GnRH treatment on d -11 (GnRH+2-PGF; n = 166). Ovaries were scanned by transrectal ultrasonography on d -11, -4, and 7, and blood samples were collected on d -11, -4, 0, and 7 for P4 determination. Treatment comparisons were performed using contrasts. The proportion of cows with a new corpus luteum on d -4 was greater in GnRH+2-PGF cows. Cows in 1-PGF had a greater P4 concentration on d 0 but lesser P4 on d 7 compared with cows in the other groups. Cows assigned to receive 2-PGF (2-PGF and GnRH+2-PGF) had greater estrus expression, and a greater proportion of cows ovulated to estradiol cypionate. No further contrast effects were observed for follicle diameter, double ovulation rate, pregnancy per embryo transfer (P/ET) on d 32 and 60, or pregnancy loss. As P4 concentration on d -4 increased, P/ET on d 60 tended to increase. Cows with P4 ≥3.66 ng/mL on d -4 had greater P/ET on d 32 and 60 than those with P4 below that threshold. Regardless of treatment, cows with P4 concentration ≥3.66 ng/mL also had a greater pregnancy per synchronized protocol (P/SP) on d 60. Also, a P4 concentration on d -4 (low or high) × follicle diameter (continuous) interaction tendency was observed when evaluating P/ET. Although P/ET did not differ among cows with different follicles sizes with reduced P4 concentration on d -4 (<3.66 ng/mL), it increased in cows with larger follicles exposed to increased P4 concentration (≥3.66 ng/mL). When P4 on d 0 was evaluated, P/ET on d 32 and 60 was greater for cows with low (≤0.09 ng/mL) versus high (>0.21 ng/mL) P4; as P4 concentration on d 0 increased, P/ET linearly decreased. In summary, cows with increased P4 concentrations during growth of the ovulatory follicular wave had improved P/ET. Administering a second PGF2α dose reduced P4 concentration on d 0 and increased ovulatory response to the protocol, but no benefits were observed on P/ET or P/SP.