Higher Pregnancy Research Articles

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 <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 <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 <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-479 Prognosis for ongoing pregnancy after recurrent implantation failure (RIF) following IVF/ICSI treatment

Abstract Study question What is the cumulative ongoing pregnancy rate within one year after visiting the RIF outpatient clinic in patients with RIF after IVF/ICSI treatment? Summary answer The cumulative ongoing pregnancy rate was 42.9% with a mean time to pregnancy of 8.8 months (95% CI 7.5-10.1 months). What is known already An estimated portion of 10-15% of IVF patients experience RIF. Multiple add-on treatments have been proposed, however, evidence for effective clinical therapeutic options still remain scarce. Although it is described that RIF patients might eventually benefit most from a ‘keep calm and carry on’ approach, in practice patients often seek clearer answers on their pregnancy chance and request further investigations/interventions. It would be helpful to give patients insight in their chances of achieving pregnancy after RIF, but at this moment little is known about their prognosis. This is further complicated by the lack of consistency in the definition of RIF. Study design, size, duration A prospective cohort study on 42 RIF patients after one-year follow-up was performed, as part of the MURIM (Multidisciplinary Research on Repeated Implantation Failure and Recurrent Miscarriages) study. Baseline characteristics including an endometrial assessment and ReceptIVFity (vaginal microbiome) test were collected at the RIF outpatient clinic. After a follow-up duration of minimal one year participants were asked to complete a questionnaire regarding fertility treatment and pregnancy outcome. Missing information was received via medical files. Participants/materials, setting, methods RIF patients aged 18 to 38 years old visiting the RIF outpatient clinic at Maastricht University Medical Centre+ between April 2019 until September 2021 were included. RIF was defined as consecutive implantation failure of three high quality embryos or ten embryos without a quality criterion. Clinical characteristics, pregnancy outcome and time to pregnancy was analyzed by survival analysis and cox hazard regression analysis via SPSS version 20.0. A p-value of < 0.05 was considered statistically significant. Main results and the role of chance Forty-two out of 44 contacted patients responded to the questionnaire (response rate = 95.5%). The ongoing pregnancy rate was 42.9% (Standard Deviation (SD): 0.50%) during the first year after visiting the RIF outpatient clinic. Mean time to ongoing pregnancy was 8.8 months (95%-CI: 7.5-10.1 months). The mean amount of embryo transfers (ETs) during this year was 2 (SD: 1.78). The ongoing pregnancy rate per ET (by survival analysis) for the first, third and sixth ET was 33.1%, 49.3% and 63.8%, respectively. To correct for a possible overestimation of the cumulative ongoing pregnancy rate per ET by normal survival analysis, pregnancy rates were calculated again using the number at risk of the total group during the first ET (pessimistic cumulative pregnancy rate). The pessimistic cumulative ongoing pregnancy rates were 33.1%, 42.9% and 48.6%, respectively. When comparing baseline characteristics by univariable cox hazard regression between women with and without ongoing pregnancy, no significant differences were found. Nineteen pregnancies were reported during the one-year follow-up, one conceived spontaneously (5.3%); four via fresh ET (IVF=15.8% and ICSI=5.3%); and 14 by frozen ET (IVF=26.4% and ICSI=47.4%). The pregnancies resulted in 14 livebirths (73.7%); one miscarriage (5.3%); one stillbirth (5.3%); and three ongoing pregnancies (15.8%). Limitations, reasons for caution Although this is the first study that prospectively describes pregnancy prognosis after RIF, it is limited by the short follow-up period and relatively small sample size. The RIF outpatient clinic consultation might have contributed to the obtained pregnancy results but the proportionality is unclear as there is no control group. Wider implications of the findings The obtained ongoing pregnancy prognosis after RIF is encouraging and justifies a conservative approach after three failed ETs. Furthermore, the high pregnancy rate indicates the need of well-defined, individualized diagnostic criteria to define RIF and to be able to deviate between couples with 'bad luck' and an underlying (treatable) cause. Trial registration number NL66835.068.18/METC18-040

P-599 The use of serum test to detect the LH surge in ultrasound-monitored intrauterine insemination (IUI) significantly increases pregnancy rates.

Abstract Study question Could a serum test to detect LH surge in ultrasound-monitored IUI cycle improve pregnancy rates? Summary answer Serum test to detect LH surges with ultrasound monitoring during IUI cycle significantly increased pregnancy rates. What is known already IUI is known as the first line of treatment for infertile couples and is usually administered with human chorionic gonadotropin (hCG) before ovulation with clomiphene citrate (CC) or/and human menopausal gonadotropin (hMG) to ovarian known to stimulate. One of the key factor for IUI is that insemination should be performed close to ovulation time, as there are many reports that sperm injection at the ovulation time has a high pregnancy rates. However, there are still few reports on the optimal timing of ovulation for performing IUI. Study design, size, duration The IUI performed from January 2016 through November 2022 (n = 3187) was included in this study. A total of 875 patients underwent the IUI by inferring LH surge with procedure with serum test, while 2312 patients underwent the procedure IUI without the serum test (control). Participants/materials, setting, methods In this study included 3187 IUI cycle were analyzed. Controlled ovarian hyperstimulation was conducted with CC (100 mg/day) and hMG. Patients were divided into two groups with serum test or not test (control). The serum test group was monitored LH surge by inferring LH peak during IUI cycle. When the sufficient follicular size and endometrial thickness had been reached, hCG was administrated and insemination was performed. Pregnancy rates were compared by Chi-squared test. Main results and the role of chance In each group, there were no significantly difference in age (34.4±3.7 vs. 34.7±3.7 years old) and endometrial thickness (8.8±2.2 vs. 8.9±2.2 mm) for IUI cycle condition. The serum test group was significantly higher clinical pregnancy rates compared to control group (19.0 vs. 15.7 % respectively) during IUI cycle (P < 0.05). In addition, patients were classified according to the history of at least one or more failed IUI cycle. The clinical pregnancy rates was significantly higher in the serum test group than control group (21.6 vs. 10.8 % respectively) who history of at least one or more failed IUI cycle (P < 0.001). Limitations, reasons for caution A limitation of this study has a too small size of the intervention group compared with the control group. Wider implications of the findings In our results, the serum test group had a significantly higher clinical pregnancy rates than the control group. Therefore, detecting a surge in LH with a serum test during the IUI cycle could predict the optimal timing of ovulation and thus increase the pregnancy rates. Trial registration number not applicable

P-140 Relationship between re-expansion rate of vitrified blastocysts during recovery culture post-warming and pregnancy rate after single blastocyst transfers

Abstract Study question What is the relationship between re-expansion rate of vitrified-warmed blastocysts and pregnancy rate after single blastocyst transfers (SVBT)? Summary answer Re-expansion rate of vitrified-warmed blastocysts was higher with increasing the number of TEs and yielded higher pregnancy rate after SVBT. What is known already The increased use of blastocyst transfer has emphasized the importance of selecting high-quality embryos. Pre-vitrification morphological grading: Gardner’s criteria or time-lapse imaging of embryo morpho kinetics and developmental speed, is established a tool for selection of high-quality blastocysts. The recovery culture of vitrified blastocysts post-warming is performed before embryo transfer. Embryos that have dehydrated and shrunk during vitrification are restored to their pre-vitrification state during the recovery culture. Developmental competence in vivo of re-expanded blastocysts after thawing was higher than that of non-expanded blastocysts, but it is not clear the relationship between the re-expansion rate and the pregnancy rate, quantitatively. Study design, size, duration We analyzed 1034 vitrified-warmed blastocysts in 758 patients from July 2017 to June 2021. All embryos were monitored by a time-lapse system (EmbryoScope+, Vitrolife). Expanded blastocysts which reached the blastocoel cavity of 160 µm or more with obvious ICM were vitrified and cryopreserved. The blastocysts were transferred in the following cycle or later. Fetal heartbeat was detected as the pregnancy confirmation. Participants/materials, setting, methods The re-expansion rate was defined as the percentage of re-expanded blastocyst diameter after the recovery culture to the blastocyst diameter at the time of vitrification. The time of recovery culture was about 3 hours. The re-expansion rate and pregnancy rate were analyzed by logistic regression analysis. We also examined the association between re-expansion rate and the number of trophectoderm cells at equatorial region (eTE) or iDAScore: the parameters for selecting high-quality embryos. Main results and the role of chance Logistic regression analysis showed a significant relationship between re-expansion rate and pregnancy rate (P < 0.0001), with an AUC of 0.58 and a cutoff value of 88.5%. Pregnancy rates in re-expansion rates ≥ 88.5% were significantly higher than those in < 88.5% (51.7 vs. 37.4%). Therefore, we classified re-expansion rates ≥ 88.5% as positive and < 88.5% as negative, and performed logistic regression analysis of women age, vitrification time of blastocysts, eTE, and iDASCore. The results showed that women age, vitrification time, and iDAScore all did not differ significantly from the re-expansion rate. The number of eTE was significantly higher with increasing re-expansion rates (P < 0.0001), with an AUC of 0.58 and a cutoff value of 7.5. Limitations, reasons for caution We used only the expanded blastocysts with 160 µm or more in diameter and obvious ICM for vitrification. In addition, this study was retrospective, and further research is needed to confirm the reproducibility of the study in other clinics with different vitrification protocols and patient populations. Wider implications of the findings Quantitative analysis of re-expansion rate of vitrified blastocysts indicated that higher re-expansion rate (≥ 88.5%) had high pregnancy rate. It is useful to exploring the influence factors of re-expansion for producing high-quality embryos. Here, we found that the number of eTE is as an important factor for re-expansion. Trial registration number not applicable

P-369 An innovative approach for FET that does not rely on the luteinizing hormone surge or human chorionic gonadotropin: a natural cycle with luteal support

Abstract Study question Can progestin supplementation in natural cycle, without reliance on LH surge or hCG ovulation triggering, effectively prepare the endometrium for a frozen embryo transfer (FET)? Summary answer High pregnancy rates can be achieved in natural cycle using luteal support as the main method for endometrial synchronization, without relying on the LH surge What is known already In recent years, frozen-thawed embryo transfer has become widely adopted and plays a significant role in assisted reproductive technology. To date, the evidence suggests that natural cycles result in higher pregnancy rates and fewer obstetric complications compared to hormonal replacement therapy, however, the most effective method for preparing the endometrium has not yet been determined. For transfer in the natural cycle specifically, conflicting results have been reported in terms of the outcome following spontaneous or triggered ovulation. No research is available that supports a natural cycle FET without monitoring the endogenous LH surge or using hCG trigger. Study design, size, duration This is a prospective analysis of 508 natural FET cycles between March 2019 and December 2022, including patients receiving embryos with own oocytes (339) and donated oocytes (169). Participants/materials, setting, methods All patients underwent ultrasound monitoring and serum hormone levels determination of estrogen and progesterone in 3 moments of the cycle (the day of the start of luteal support, 1 week before B-HCG and the day of B-HCG). Upon ultrasound confirmation of the endometrium's optimal thickness and appearance and the presence of the pre-ovulatory follicle, patients began progesterone supplementation and the day for FET was scheduled. Main results and the role of chance A clinical pregnancy rate of 65.48% was reported with a miscarriage rate of 9.09%. A higher rate of miscarriage (22.22%) was found to be associated with an endometrial thickness of less than 7mm at the time of progesterone initiation. The size of the dominant follicle did not affect pregnancy outcome, but a higher pregnancy rate was observed when the follicle measured 15 mm or larger (CPR of 66.83% in cycles with a follicle ≥15mm versus CPR of 60.22% in cycles with a follicle <15mm). Estrogen levels decline in the second measurement but increase again on the day of a positive beta test. This protocol requires less monitoring compared to true and modified natural cycles and is convenient to coordinate with oocyte recipients. A thin endometrial lining, measured below 7mm, increases the risk of a clinical miscarriage. A lack of increase in estrogen levels on the day of B-hCG is an unfavorable sign for a positive outcome. Limitations, reasons for caution Our analysis of data has a prospective design, although it may be limited by unmeasured factors such as the number, stage, and chromosomal testing of transferred embryos. Wider implications of the findings Supplementary luteal support does not affect the natural progression of the endometrium towards receptiveness and results in a key intervention for controlling the start, length and functionality of the window of implantation. A natural cycle, with progesterone supplementation but without monitoring the LH surge, results in high pregnancy rates. Trial registration number Not applicable

P-189 Systematic management and confirmation of safety (Safety management manual; 7 CHA IVF laboratory instructions; Field training; Lecture; Inspection) can prevent embryo mix-up in IVF laboratory

Abstract Study question Is systematic patient safety management training effective in improving awareness in safety confirmation and preventing errors in Embryo mix-up for embryologists? Summary answer From survey for 116 embryologists, they responded that they followed safety management instructions and regulations, and that they improved their safety work capability and awareness What is known already The cases of embryo mix-up known through the media include errors of sperm change during IUI, fertilization, Freeze-thawing, PGT, and Embryo transfer process, but there will be more due to statistical difficulties. Errors mostly occur because clinics do not have the right protocol. Currently, there aren’t any regulations or policies to track eggs, sperm, embryos or frozen embryos during ART. For this reason, infertile couples have to choose clinics with high pregnancy rates and safe clinics on their own to perform assisted reproductive techniques. Study design, size, duration This study was conducted through a survey of 116 embryologists who worked at five clinics of CHA fertility centers. The patient safety management system was created from the CHA fertility Center itself. From August 2021 to December 2022, safety confirmation training was conducted for 16 months. All embryologists were anonymously surveyed twice with 32 questions. The first survey was conducted in April 2022, and the second survey was in December 2022. Participants/materials, setting, methods The survey was conducted in 32 items with 10 categories. Interviewees’ made response for each item in five steps. Each item asked 1) Learning in the standard safety manual 2) Changes in safety awareness before and after training, 3) Compliance to the seven safety instruction (double checks) for each work part, 4) Level of difficulty in education, 5) Effects of education and 6) Suggestions for safety confirmation. Main results and the role of chance In the first survey, 61 out of 112 responded (54.5%) 60 out of 116 (51.7%) in the second. The answers were scored using a 5-point Likert scale (1 = strong disagree, 5 = Strong agree). 1. The mean score for work satisfaction was 3.1 ± 0.8 out of 5 in the first survey and 3.1 ± 0.7 out of 5 in the second. Work satisfaction remained unchanged. 2. The perception of patient safety confirmation was 3.7/5 before training and 4.0/5 after training (p < 0.03) in the second which improved the perception of safety confirmation. 3. The safety confirmation ability through training was improved to 4.1/5(p < 0.01) after training compared to 3.8/5 in both the first and second surveys. 4. The necessity and importance of safety training were considered important in the first survey as it scored 3.6/5 and 4.5/5 (p < 0.001) in the second survey. 5. In the course of work, they responded that their safety management skills were improved (1st: 3.4/5, 2nd: 4.5/5(p < 0.05)) by the effects of training. As for CHA IVF lab, the embryologists were well aware of the guidelines (scoring 4.5/5 in the first and second survey) to know seven safety instructions (which are double check, outloud, RI witness, monitoring, document, information) should be followed well. Limitations, reasons for caution Embryo mix-up during ART procedures can be a major mistake to be avoided. The confirmed documentations can make errors any time. Establishing the safety protocol of the same process as ART procedure, the system, and education are important, but the most important is the safety management capability of the manager. Wider implications of the findings The safety management manual, the 7 CHA IVF laboratory patient safety instructions, online lecture training, field training, and inspection have raised awareness of the safety errors and the importance of double checking. Errors in safety can be prevented through systematic system development and repeated safety training. Trial registration number non-clinical trials

From menarche to menopause: psychoneuroendocrinology across the female reproductive lifespan

As reproductive hormones rise and begin to fluctuate with puberty and the beginning of the menstrual cycle (i.e., menarche), there is substantially increased risk of affective illness for females compared to males. Additional risk is associated with reproductive transition events characterized by dramatic hormone changes, including the decrease in progesterone and estradiol during the premenstrual phase, the abrupt withdrawal from high pregnancy hormone levels during the postpartum period, and the turbulent hormone flux that occurs during pubertal and menopausal transitions. As such, a differential sensitivity to normal changes in reproductive hormones during specific reproductive transition events may precipitate or exacerbate affective symptoms in susceptible women. Using a model of mood disorder susceptibility in which rapid and abrupt changes in reproductive hormones may trigger the onset of affective illness in vulnerable females, this symposium will present novel findings on psychosocial and biological mechanisms underlying hormone-related, transdiagnostic affective state change spanning the female reproductive lifespan – from menarche to menopause. Following a brief introduction by the Chair, Elizabeth Andersen, the first speaker, Hannah Klusmann, will present methods and recommendations for determining ovulation status in peripubertal females within one year of menarche and how mood sensitivity to hormone flux across the menstrual cycle differs by ovulation status. Katja Schmalenberger will present recent findings on the coupling of heart rate variability suppression and the cyclical increase in mood symptoms during the midluteal menstrual cycle phase. The third speaker, Crystal Schiller, will discuss findings from three studies that used a sex steroid manipulation in women with a history of postpartum depression to determine functional neuroimaging correlates of postpartum irritability symptoms. Danielle Swales’ presentation on the interaction of life stress and estradiol on reward processing during the menopause transition will conclude the symposium.

Proteomic profile of stallion seminal plasma

Proteins mediate all physiological changes. Seminal plasma (SP), the product of testes, epididymis, and accessory sex glands, is a fluid released during ejaculation and represents up to 98% of the voluminous stallion ejaculate. The SP plays an essential role in reproduction as a transit medium and a source of energy, antioxidants, enzymes, and minerals. This study aimed to investigate proteomic profile of the seminal plasma of stallions. Twenty-four Criollo stallions with a known reproductive history and per-cycle concept rate data calculated from at least 30 inseminations were used. One ejaculate was collected from each stallion during the breeding season. Pregnancy rates ranged at day 16 after artificial insemination from 20.2% to 95.6%. The animals were divided into two groups: High Pregnancy (HP), with a pregnancy rate per cycle ≥51%, and Low Pregnancy (LP), with a pregnancy rate of <50%. Sperm concentration, kinetics, morphology, and plasma membrane integrity and functionality were evaluated. The sample was centrifuged at 400xg for 10min. SP was then transferred to a 2 mL microcentrifuge tube and centrifuged again (10.000xg, 60min, 4°C). Proteins were separated using 2D SDS PAGE gel electrophoresis in duplicate. Spots in at least 80% of the gels in one of the groups, with significant abundance (P<0.05) and at least a 1.5-fold magnitude difference between groups, were selected. Thespectra were acquired in a nano LC-MS/MS spectrometer, and proteins were identified by the MASCOT application and validated by SCAFFOLD. From a total of 716 analyzed spots, four were selected. For non-parametric data the Kruskal Wallis and for parametric data Anova and t-test were used. The cysteine-rich secretory protein 3 (CRISP3) and SP protein HSP-1-like (HSP1) showed higher abundance in the SP in group LP than HP (P< 0.05). However, kallikrein-1E2 (KLK) and phospholipase A2 (PLA2) showed higher abundance in HP than LP (P<0.05). In the horse, CRISP-3 is synthesized in great amounts in the accessory sexual glands, ampulla and seminal vesicle. HSP1 has an essential role as a molecular chaperone for sperm motility and viability, in the formation of the oviductal sperm reservoir, in the regulation of cell volume, and possibly in the interaction between gametes. KLK is one of the major proteins found in seminal equine plasma. The kallikreins are serine proteases, and the glandular kallikrein is regulated by androgens and secreted by the prostate. The PLA2 plays an essential role in the late maturational events of spermatozoa, the acrosomal reaction, and sperm egg fusion. In conclusion, the proteins are essential to the biological processes related to stallion fertility, providing insight into SP proteins that could serve as biomarkers for fertility in stallions.

Metabolomics profile of equine seminal plasma

Seminal plasma (SP) is the product of testes, epididymis, and accessory sex glands released during ejaculation, making up 98% of the volume of the stallion ejaculate. Analyzing the entire metabolome allows to assess the final products of the biological system metabolism. The present study aimed to investigate the metabolomics of stallion SP. Twenty-four Criollo stallions with a known reproductive history and ≥30 inseminated mares were once collected during the breeding season. Pregnancy rates (day 16 after artificial insemination) ranged from 20.2% to 95.6%. Two groups were formed: High Pregnancy (HP; pregnancy rate per cycle ≥50%), and Low Pregnancy (LP; pregnancy rate of ≤49%). Microscopic semen analysis was performed using a computer-assisted sperm analysis system. Membrane physical integrity was assessed by fluorescent probes, and the hypoosmotic test analyzed membrane functional integrity. After centrifugation at 400xg for 10min centrifugation, SP plasma was transferred to a 2mL microcentrifuge tube and centrifuged again (10.000xg, 60min, 4°C). The supernatant was mixed 1:1 with deionized water after filtering with 0.45μm filters to remove cell sediment. Each sample was again filtered through a 0.22μm syringe filter before profile acquisition. The metabolic profile was acquired using liquid chromatography coupled with time-of-flight mass spectrometry. A total of 18 target metabolites were identified after analysis. The metabolites were tryptophan, lactic acid, ornithine, stearic acid, oleic acid, palmitic acid, melatonin, acetylcarnitine, carnitine,phenylalanine, isoleucine, isovaleryl, citric acid, hippuricacid, 1,3-dioxan-5-ol, taurine, phosphoric acid, and fructose. Groups were considered independent factors while the metabolite profile was considered a dependent factor. The Shapiro-Wilk Test was used to evaluate data distribution of SP metabolomics. The Kruskal Wallis test for nonparametric data and ANOVA and Tukey test for parametric data were used. Pearson's correlation was performed with a significance level of P<0.05. Four metabolites: oleic acid, isoleucine, taurine, and phosphoric acid, presented a difference (P<0.05) between the HP and LP groups. The oleic acid and isoleucine were higher in the LP group, while taurine and phosphoric acid were higher in stallions from the HP group. Phosphoric acid presented a relevant negative correlation with immobile spermatozoa (r=-0.7206) and a positive correlation with functional integrity (r=0.6162), physical integrity (r=0.6306), fertility (r=0.6471), progressive motility (r=0.5145), total motility (r=0.7157), VCL (r=0.6034), VSL (r=0.5491), VAP (r=0.5629), BCF (r=0.7055), ALH (r=0.5945), the total volume of the ejaculate (r=0.5941) and total protein/ejaculated (r=0.5182). Metabolites are products of cell signaling pathways and can be used as biological markers. Phosphoric acid contributes to the energy metabolism of sperm by breakdown of an inorganic pyrophosphate molecule. In conclusion, phosphoric acid in stallion SP should be considered as a marker of fertility.

A Brief History and Advancement of Contraceptive Multipurpose Prevention Technology (cMPT) Products.

The high incidence of HIV and other sexually transmitted infections (STIs), and an unmet need for modern contraception resulting in a high unintended pregnancy rate, are major problems in reproductive health. The concept of multipurpose prevention technology (MPT) was introduced following the failure of several leading microbicide candidates to prevent human immunodeficiency virus type 1 (HIV-1) transmission in large clinical trials in the early 2000s. MPTs are defined as products designed to simultaneously prevent at least two of the following conditions: unintended pregnancy, HIV-1, or other major STIs. The goal of contraceptive MPT products (cMPTs) is to provide contraception and protection against one or more major STI pathogen (eg, HIV-1, herpes simplex virus (HSV) type 2, Neisseria gonorrhoeae (gonorrhea), Treponema pallidum (syphilis), Trichomonas vaginalis, Chlamydia trachomatis (Chlamydia). This new field has great potential and will benefit from lessons learned from the early microbicide trials. The cMPT field includes candidates representing various categories with different mechanisms of action including pH modifiers, polyions, microbicidal peptides, monoclonal antibodies, and other peptides that target specific reproductive and infectious processes. More preclinical research is being conducted to ensure minimal side effects and maximum efficacy in vivo. Effective proven and novel candidates are being combined to maximize efficacy, minimize side effects, and avoid drug resistance. More attention is being paid to acceptability and new delivery systems. cMPTs have a very promising future if adequate resources can be mobilized to sustain the effort from preclinical research to clinical trials to bring effective, acceptable, and affordable products to market.

Embryo transfer technique affects pregnancy rates in recipient mares

Embryo transfer in horses started in the 1970′s and has evolved since then in several technical and biological areas that may influence the overall outcome. The transfer method evolved from surgical to non-surgical (NSET), with pregnancy rates for NSET related closely to operator skills. Recently, high pregnancy rates have been reported by non-specialized operators using the cervical forceps method (Cuervo Arango et al., Veterinary Record. 2018,183;10,1-5). The aim of this retrospective study was to compare pregnancy and early embryonic loss rates between conventional and cervical forceps transfer techniques (Wilsher and Allen, Equine Vet. Educ. 2004. 16; 1, 39-44), operated by two veterinarians with different degrees of experience. During the last 12 breeding seasons in a commercial equine embryo transfer program in Chile, 345 embryos were transferred by two operators with low / moderate (n=86) or high experience (n=259), using a conventional manual technique and 228 embryos were transferred by the same two operators (n=81) and (n=147) respectively, using a bovine cervix forceps/Polansky speculum technique. Embryo flushes were performed at 8 or 9 days after ovulation on 102 donor mares aged 4 to 24 years old and transferred to recipient mares (n=387) aged 3 to 12 years old. Ovulation synchrony ranged from -1 to +4 days when comparing the recipient to the donor. No medications were used during embryo transfer and procedures were done at the samefacilities. Data were analyzed by a binary logistic regression model in which pregnancy and embryo loss were treated as dependent variables, and the experience of the operator and the ET technique as independent variables. Pregnancy rates at first diagnosis (12 to 14 days post ovulation) and embryo loss rate until 60 days of gestation were compared between and within operator groups. Overall pregnancy rates were higher (P<0.001) by the cervical forceps / speculum embryo transfer technique (195/228; 85%) than with the conventional technique (236/345; 65%) in both groups combined. Early embryonic loss rate did not differ (P>0.1) between the groups (6.0 vs. 4.6%, respectively). For the conventional technique, there was a significant operator effect (P=0.015) on the pregnancy rates at the first diagnosis (57 vs. 72%); no operator effect (P>0.1) was observed when using cervical forceps (84 vs. 86 %). These results agree with the report of Cuervo-Arango et al. (2018). We also confirmed that simple bovine cervical forceps work as well as the equine specific forceps reported by Wilsher and Allen (2004). In conclusion, the cervical forceps technique yielded higher pregnancy rates, with no effect of the operator's experience compared to the conventional technique in which the skill and experience of the operator is crucial and needs time and losses to achieve.

Vitrification and direct warming in iso-osmotic holding medium of equine in vitro embryos supports competitive pregnancy rates

Improved efficiency of ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) resulted to a steep increase in the number of in vitro produced (IVP) equine embryos worldwide. These IVP blastocysts are routinely cryopreserved and both slow freezing and vitrification result in high pregnancy rates. The use of minimal volume cryodevices and fast, multistep warming protocols can be challenging for veterinary practitioners receiving vitrified embryos, but direct warming in embryo holding medium supports embryo viability (Canesin et al. Theriogenology. 2020;151:151-158). The aim of this study was to compare pregnancy rates in a clinical OPU-ICSI program using a three-step vitrification/warming protocol vs. a commercial vitrification kit and direct warming in embryo holding medium. Embryos were produced in autumn and winter of 2021-2022 by OPU-ICSI (Papas et al. Animals. 2021;11:2004). Day of vitrification (7-12 post ICSI) was similar for both vitrification groups. Blastocysts were vitrified in a minimal volume on a Cryolock (Irvine Scientific) following two methods: (1) a three-step protocol (n=21): holding in basal vitrification medium (BVM; DMEM-F12 with 20% FCS), vitrification in BVM with 1.5M ethylene glycol (5 min), followed by BVM with 7M ethylene glycol and 0.6M galactose (40 s), and warming in warming medium (WM; DPBS with 0.1% glucose,36 mg/L pyruvate, and 0.4% BSA) with 0.3M sucrose (1 min), followed by WM with 0.15M sucrose (5 min) and holding in WM (5 min), all performed at 38.2°C (Choi and Hinrichs. Theriogenology. 2017; 87:48-54); (2) direct warming (n=21): vitrification at 23°C with a commercial VIT-Kit (Minitube, Germany) in VS1 (5 min), VS2 (5 min) and VS3 (40 s), containing increasing concentrations of glycerol and/or ethylene glycol, based on Eldridge-Panuska et al. (Theriogenology. 2005; 63:1308-1319), and direct warming at 38°C for 5 min in 4 ml of Emcare holding medium (Spervital, The Netherlands). For both protocols, warmed embryos were washed four times in Emcare and transferred immediately to a day 4 recipient mare. The effect of vitrification method on pregnancy rate at 14 days and 25 days was determined using binomial logistic regression analyses. Pregnancy rates at 14 and 25 days were respectively 66.7% and 47.6% for the three-step method and 76.2% and 52.4% for the direct warming. No difference was found between the two methods (p=0.496 and p=0.882, respectively). In conclusion, vitrification and warming of IVP blastocysts using commercial media and direct warming supports pregnancy and provides an easy method for embryo warming and transfer by equine veterinary practitioners.

Successful vitrification of embryos ≤500µm without puncture or aspiration of the blastocoele

To achieve good pregnancy rates following vitrification, embryos >300µm require micromanipulator-assisted puncture and blastocoele collapse. Without using a micromanipulator, Wilsher et al (Equine Veterinary Journal.2021;53:1227-1233) reported high pregnancy rates following vitrification of embryos 300-560µm using manual puncture but failed to achieve any pregnancies with non-punctured embryos. These manually punctured embryos were exposed to an equilibration medium (EM; HEPES culture medium + 7.5% DMSO + 7.5% ethylene glycol [EG]) for 6min. Extending the time to 8min in the non-puncture group did not improve results. The aim of this study was to determine if non-punctured embryos, exposed to considerably longer in EM (i.e.,15min) would, upon warming and transfer to recipients, result in acceptable pregnancy rates. Thirty, Grade 1 embryos were recovered on Day 7-8 post ovulation and divided into three groups based on size, G1 (≤300 µm; n = 8), G2 (>300 to ≤500µm; n=16) and G3 (>500µm; n=6). Median (range) embryo diameters for Groups 1 to 3 were 237.5 (180–280), 410 (310–500) and 575 (510–660) µm, respectively. Embryos were vitrified with a commercial human vitrification kit (Kitazato) utilising 2-step concentrations of DMSO and EG (7.5-15% v:v) in a HEPES culture medium containing gentamycin, trelahose and hydroxypropyl cellulose. At room temperature (RT) embryos were placed in EM for 15min prior toa vitrification solution for ≤90 sec, during which time they were loaded onto a Cryolock device, capped and plunged into LN2. Embryos were warmed in 1M sucrose solution of holding media (HM; H199 + 20% FBS + antibiotics) at 42°C for 1min before being transferred to HM + 0.5M sucrose at RT for 4min, then into Vigro Hold for a further 4min. Within 15min, embryos were transferred to recipient mares 6 days post ovulation. Mares were checked thrice for pregnancy and to ensure continuing embryonic development before administration of prostaglandin. Two mares were left pregnant until the heartbeat and expanding allantois stage in groups achieving pregnancies. G1 and G2 embryos achieved a higher pregnancy rate than G3 in which no embryos survived (87.5% [7/8] vs. 75% [12/16] vs. 0% [0/6], respectively; P<0.001). One early embryonic death (EED) occurred in G2 (350µm) between Days 12-14. The four embryos in G2 that did not result in a pregnancy were 330, 380, 425 and 500µm. This study shows that non-punctured embryos <500µm can survive vitrification and result in excellent pregnancy rates (19/24 [79%]) following a 15min exposure to equilibration medium containing 7.5%EG and 7.5%DMSO. Larger numbers of pregnancies are required to ensure that the longer exposure time to cryoprotectants does not increase the risk of EED. Nevertheless, this protocol negates the need to puncture embryos <500µm providing an easy and successful way to vitrify them.

Progress of research on chromosomal mosaicism embryos

Chromosomal mosaicism (CM) is a common phenomenon in preimplantation genetic testing (PGT). In embryos with CM, genetic contents of trophoblastic ectodermal (TE) cells may be different from that of the inner cell mass (ICM) which will develop into the fetus. Embryos with low mosaic proportion could give rise to healthy live births after transplantation, but are accompanied with high pregnancy risks such as high abortion rate. In order to provide a more comprehensive understanding for CM embryos, this article has systematically summarized the recent progress of research on the definition, mechanism, classification, PGT techniques, self-correction mechanism, transplantation outcome and treatment principles for CM embryos.

Patterns of contraceptive use through later reproductive years: A cohort study of Australian women with chronic disease.

Pregnancies among women with chronic disease are associated with poor maternal and fetal outcomes. There is a need to understand how women use or don't use contraception across their reproductive years to better inform the development of preconception care strategies to reduce high risk unintended pregnancies, including among women of older reproductive age. However, there is a lack of high-quality longitudinal evidence to inform such strategies. We examined patterns of contraceptive use among a population-based cohort of reproductive aged women and investigated how chronic disease influenced contraceptive use over time. Contraceptive patterns from 8,030 women of reproductive age from the Australian Longitudinal Study on Women's Health (1973-78 cohort), who were at potential risk of an unintended pregnancy were identified using latent transition analysis. Multinomial mixed-effect logistic regression models were used to evaluate the relationship between contraceptive combinations and chronic disease. Contraception non-use increased between 2006 and 2018 but was similar between women with and without chronic disease (13.6% vs. 12.7% among women aged 40-45 years in 2018). When specific contraceptive use patterns were examined over time, differences were found for women with autoinflammatory diseases only. These women had increased odds of using condom and natural methods (OR = 1.20, 95% CI = 1.00, 1.44), and sterilisation and other methods (OR = 1.61, 95% CI = 1.08, 2.39) or no contraception (OR = 1.32, 95% CI = 1.04, 1.66), compared to women without chronic disease using short-acting methods and condoms. Potential gaps in the provision of appropriate contraceptive access and care exist for women with chronic disease, particularly for women diagnosed with autoinflammatory conditions. Development of national guidelines as well as a clear coordinated contraceptive strategy that begins in adolescence and is regularly reviewed during care management through their main reproductive years and into perimenopause is required to increase support for, and agency among, women with chronic disease.

Effectiveness of a Postpartum Breastfeeding Protocol for Avoiding Pregnancy.

The uses of cervical mucus and basal body temperature as indicators of return to fertility postpartum have resulted in high unintended pregnancy rates. In 2013, a study found that when women used urine hormone signs in a postpartum/breastfeeding protocol this resulted in fewer pregnancies. To improve the original protocol's effectiveness, three revisions were made: (1) women were to increase the number of days tested with the Clearblue Fertility Monitor, (2) an optional second luteinizing hormone test could be done in the evening, and (3) instructions were given to manage the beginning of the fertile window for the first six cycles postpartum. The purpose of this study was to determine the correct and typical use effectiveness rates to avoid pregnancy in women who used a revised postpartum/breastfeeding protocol. A cohort review of an established data set from 207 postpartum breastfeeding women who used the protocol to avoid pregnancy was completed using Kaplan-Meier survival analysis. Total pregnancy rates that included correct and incorrect use pregnancies were eighteen per one hundred women over twelve cycles of use. For the pregnancies that met a priori criteria, the correct use pregnancy rates were two per one hundred over twelve months and twelve cycles of use and typical use rates were four per one hundred women at twelve cycles of use. The protocol had fewer unplanned pregnancies than the original, however, the cost of the method increased.

Pregnancy and delivery characteristics and outcomes in Northwestern Syria: A prospective cohort study.

Detail a picture of the current pregnancy and delivery characteristics and outcomes in Northwestern Syria. This is a prospective observational study, using an electronic health information system, from four healthcare centers in two provinces in Northwestern Syria. We classified the population into two groups: Displaced people are those who live inside refugee camps; and residents are housed outside the camps. We analyzed data from 21 294 pregnant women. Among them 69.6% were displaced. Less than 1% of all care met the standard WHO's antenatal guidelines, and 3.9% met the focused WHO guidelines. Displaced women were more likely to have no antenatal visits (35.8% vs. 25.4%). We found a high rate of anemia (45.8%), prevalence of adolescent pregnancy (20.7%), cesarean-section (25.2%), preterm birth (4.2%), low birth weight (8.3%), and newborn mortality rate within 24 hours of birth (1.2%). Preterm births were more likely to occur in women who had no record of antenatal visits (4.2%) and the prevalence decreased with the increasing number of antenatal visits. We found huge deficiency in perinatal care among all women in this region. We found high cesarean-section rate, anemia, premature birth, newborn mortality within 24 h of birth, and high adolescent pregnancy.

Implementation of reproductive health education in a Filipino city: A case study

The Philippines has adopted reproductive health education (RHE) in schools with the passing of the Responsible Parenthood and Reproductive Health Law in 2012 which promised multi-dimensional support on reproductive health (RH) including RHE. Although there is urgent need for RH policy to enhance family planning and to reduce high teenage pregnancy, this law continues to be extremely controversial, conflicting with Filipino socio-cultural norms, mainly based on Catholic beliefs. Based on this context, this research aims to examine how RHE is implemented in Filipino schools. For the research, a qualitative case study was conducted in a Filipino city. Results show that each local stakeholder has a different concept of RH and delivers their own perceived concept of RH. Teachers lack RH pedagogy and training on RH while they also tend to minimise and modify their teaching in fear of further stimulating student interest in sex. Lastly, conflict between the Department of Education and the Department of Health was observed. This study implies that policy making alone is not a sufficient for effective implementation of RHE. Endorsement of various local stakeholders is necessary to accomplish proper RHE.

Pregnancy Risks of Young Mothers in Medical Discourse and Cultural Settings

East Lombok Regency, despite implementing pregnancy health programs, grapples with a persistently high pregnancy risk. This study investigates the disparity between medical and Sasak cultural perspectives on young mothers’ pregnancy risks. Qualitative case study research reveals a conflict between community sociocultural narratives and medical viewpoints. Cultural norms normalize medical narratives, shaping pre-marital reproductive arrangements, body perceptions, and idealized pregnancy concepts. Young mothers, influenced by cultural values, accept pregnancy as a collective social process within the family. The study emphasizes the impact of cultural determinism on shaping perceptions and preferences for young mothers’ pregnancy health, highlighting the need for an integrated approach that bridges medical and cultural perspectives to address and mitigate pregnancy risks effectively.

An Acute Crisis adds to Unresolved Chronic Crisis

Residents in a community in the South Peninsula of the Western Cape, South Africa live with the reality of unresolved challenges. The women and children of this community face a burden from the prevailing inequality and structural violence that plays out in their daily lives as high levels of unemployment, substance abuse, violence in many forms, food insecurity, high school dropout rates and teenage pregnancy. The complexities of living in this community were exacerbated by the arrival of the Covid-19 pandemic and the resultant lockdown regulations that destabilised all previous notions of normality. The pre-existing social challenges can be viewed as the residents’ experiences of hazards and stressors that have been in place for years in the unequal social structures of South Africa (The World Bank 2022). In this environment, of what can be interpreted as a community in chronic crisis (Dekker et al., 2021), the addition of the acute crisis of the pandemic, posed further cumulative effects on households that impacted more seriously on those who are already the most vulnerable. This paper presents reflections on the stories of women talking about their experiences of the global pandemic to this coastal community. We aim to show the impact on their lives as members of families within their community. The analysis will utilise the phases of a crisis (Emergency Management, 2022) where the pre-crisis or prodromal phase was recognised as life in the chronic crisis of structural violence. The acute or crisis phase of the pandemic became a lens for the effects of an existing long-term crisis compounded by athe. The response or chronic phase of the acute crisis was again found to be exacerbated by the existence of a long drawn out response phase to the chronic crisis. This paper is a window into the innovative attempts by this community to cope with the immense challenges of Covid-19, followed now by the post-crisis (resolution) phase that reflects the ongoing efforts to rebuild the community in the face of the cumulative effects of the pandemic, as but a temporary diversion from the challenges of a continuous chronic crisis. The post-crisis phase remains incomplete, however, a deeper understanding of crisis upon crisis provides knowledge that can assist those implementing social interventions in this community to tackle challenges as a state of chronic crisis for residents.