In vivohuman uterine temperature, pH, and uterine fluid composition analysis

28 Study question: What are the temperature, pH and uterine fluid composition in the human uterus 29 three days following a positive LH test or ovum pick-up? 30 Summary answer: The mean uterine temperature was 36.94±0.26oC, the mean uterine pH was 31 6.76±0.22, and the concentrations of 37 components in aspirated uterine fluid were successfully 32 determined. 33 What is known already: Embryo culture conditions in the laboratory impact key outcomes of IVF/ICSI 34 treatments, such as the quality of the embryos and the live birth rate after treatment, and child 35 outcomes, such as birth weight. Currently used conditions, including temperature, pH, and culture 36 medium composition, are largely derived from clinical experience and experimental studies using 37 animal models. Limited studies have been performed to determine the natural human preimplantation 38 embryo environment in vivo during the physiologically relevant time of the menstrual cycle. This type 39 of fundamental knowledge is required for evidence-based optimization of the in vitro embryo culture 40 environment and improving IVF/ICSI outcomes. 41 Study design, size, duration: In this cross- sectional study, conducted between April 2015 and March 42 2016, temperature and pH were measured in the human uterine cavity on the third day following a 43 positive LH test or ovum pick -up, and uterine fluid was simultaneously aspirated for composition 44 analysis. Uterine temperature was measured in fifty eight women, uterine pH was determined in fifty 45 three women, and twenty two samples of aspirated uterine fluid were analysed for the concentrations 46 of thirty-seven components. 47 Participants/materials, setting, methods: This study involved 61 healthy reproductive -aged women: 48 53 without ovarian stimulation and 8 who underwent ovarian stimulation . We measured uterine 49 temperature using a probe inserted into the uterine cavity directly, and uterine pH after inserting a 50 probe through the outer sheath of an IVF catheter. Uterine fluid was then aspirated using this outer 51 IVF catheter and a 10 ml syringe, and subsequently analysed with a Cobas 8000 chemistry analyser and 52 ultra-performance liquid chromatography-tandem mass spectrometry. 53 Main results and the role of chance: The mean uterine temperature on the third day following a 54 positive LH test or ovum pick -up was 36.9 4 ± 0.26oC and correlated with the women’s core body 55 temperature. The mean pH in the uterine cavity was pH 6.76 ± 0.22, clearly lower than the standard 56 pH used for human preimplantation embryo culture in vitro (pH 7.3 ± 0.1). Concentrations of important 57 energy sources were 0.8 ± 0.02 mM pyruvate, 5.1 ± 1.78 mM glucose and 6.60 ± 1.12 mM lactate . 58 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 3 Glutamic acid (1162 ± 183 μM), glycine (955 ± 156 μM) and alanine (513 ± 82 μM) were the most 59 abundant amino acids in uterine fluid. 60 Limitations, reasons for caution: In absence of a preimplantation embryo, synergistic influences on 61 the uterine environment may be overlooked. Single centre and specific population limitations may 62 hinder broader generalization of the results. Uterine fluid likely contains additional components. 63 Wider implications of the findings: The in vivo uterine characteristics identified in this study are 64 foundational to develop an in vivo evidence-based culture medium for human embryos. Further 65 research is necessary to evaluate whether such a medium can improve human preimplantation 66 embryo development and quality, thereby increasing cumulative live birth rate s and improving child 67 outcomes. 68 Key words: preimplantation embryo environment, in vivo embryo environment, human uterine 69 characteristics, temperature, pH, uterine fluid composition, IVF/ICSI 70 71 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 4

72 Since the first live birth following IVF in 1978, more than ten million babies have been born worldwide 73 through IVF (ESHRE, 2023). Despite the large number of live births, the IVF success rates remain 74 suboptimal, with approximately two-thirds of treatment cycles not leading to a successful pregnancy. 75 In 2019, the cumulative live birth rate was reported to be 30.7% (ESHRE, 2023). A potential limiting 76 factor may be a suboptimal in vitro culture environment that is not fully equipped to support human 77 preimplantation embryo development (Chronopoulou and Harper, 2015) . Primary elements of this 78 environment, including CO2/O2 levels, temperature and the composition, pH and osmolality of the 79 culture medium, influence the quality of preimplantation embryos – a key predictor for the chance of 80 a live birth (Feuer and Rinaudo, 2012, Swain, 2015, Gardner and Kelley, 2017, Cairo Consensus Group, 81 2020). Comparative clinical trials on human embryo culture media have demonstrated that the choice 82 of embryo culture medium affects cumulative live birth rates and child outcomes, such as birth weight 83 (Dumoulin et al., 2010, Nelissen et al., 2012, Youssef et al., 2015, Kleijkers et al., 2016). Optimizing the 84 in vitro embryo culture environment may thus improve preimplantation embryo development and 85 quality, and subsequently increase the chances of a live birth and improve child outcomes. 86 Current human embryo culture conditions primarily rely on laboratory experience and experimental 87 studies with animal models on the effects of altering culture parameters on embryo development 88 and/or IVF outcomes. Most of these studies have been conducted with mouse embryos or other animal 89 models, due to the fact that human preimplantation embryos are generally unavailable . The mouse 90 embryo assay is often used for development and testing of commercial human embryo culture media. 91 However, designing and optimizing a human preimplantation embryo culture environment based on 92 experimental mouse or other animal embryo studies may lead to a culture system tailored to mouse 93 preimplantation embryos that is less applicable to human preimplantation embryos (Menezo and 94 Herubel, 2002, Chronopoulou and Harper, 2015). Given that embryo culture media consist of various 95 components with interactive effects, obtaining empirical evidence would also require examining 96 potentially endless possibilities (Summers and Biggers, 2003) . E ven if human embryos had been 97 available, achieving a truly optimized human preimplantation embryo culture environment following 98 an approach of stu dying individual culture medium components or culture parameters alone, 99 therefore seems implausible (Summers and Biggers, 2003, Pool et al., 2012, Chronopoulou and Harper, 100 2015). A “back to nature approach” – striving to mimic the natural in vivo human preimplantation 101 embryo environment in vitro (Leese, 1998) – has emerged as essential for achieving true optimization. 102 This approach requires a comprehensive understanding of the in vivo human preimplantation embryo 103 environment. However, limited studies have been performed to elucidate the in vivo characteristics of 104 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 5 the preimplantation embryo environment in humans, such as temperature, pH and uterine fluid 105 composition. 106 Temperature 107 The commonly used temperature for human embryo culture in IVF laboratories is 37.0°C and is based 108 on the estimated core body temperature (Swain, 2015). Observations in animal models and a study 109 measuring human follicle fluid temperature 2.3°C lower than the ovarian stroma, suggested 110 optimization of in vitro culture by (partially) culturing human embryos at temperatures below 37.0°C 111 (Grinsted et al., 1985) . But studies investigating the impact of human embryo culture at lower or 112 gradual temperatures failed to yield conclusive results (Baak et al., 2019). Further in vivo evidence on 113 the temperature in the human reproductive tract is limit ed to a single study reporting a uterine 114 temperature of 37.08 ± 0.18°C in the ovulatory phase (n = 10) and 37.47 ± 0.35°C in the secretory phase 115 (n = 11) (Yedwab et al., 1976) . A limitation of this study is its reliance on alterations in core body 116 temperature to estimate menstrual cycle phases, introducing uncertainty regarding the specific timing 117 of temperature measurements during the menstrual cycle . Also, a limited number of measurements 118 were performed and temperature meter accuracy has likely been improved since then. F urther 119 research into the temperature in the human reproductive tract seems required to determine the 120 temperature preimplantation embryos naturally develop in. 121 pH 122 Embryologists generally culture human embryos within a range of pH 7.2-7.4. Commercial embryo 123 culture media manufacturers commonly recommend this pH range in the instructions accompanying 124 the embryo culture media. The current standard pH of 7.2-7.4 appears to originate from the initial use 125 of a culture medium with a stable pH of 7.6 developed for hamster embryos, for the culture of human 126 gametes to enable fertilization, and further refinement lowering the pH through studies on different 127 culture media , pH, and bicarbonate buffering conditions for human embryo culture (Edwards and 128 Steptoe, 1974, Edwards et al., 1981, Bavister, 2002). The embryo culture medium pH is important as it 129 influences the intra cellular pH (pHi), impacting cellular processes with potential consequences for 130 embryo development (Busa and Nuccitelli, 1984, Squirrell et al., 2001, Swain and Pool, 2009, Gu et al., 131 2023). Induced changes in the culture medium pH for a short period during embryo culture have been 132 shown to affect the birthweight of mouse pups (Banrezes et al., 2011). Despite its importance , the 133 optimal pH for the human preimplantation embryo environment remains to be determined (Swain, 134 2010, Swain, 2012, Gatimel et al., 2020) . Only three studies have previously measured the pH in the 135 human reproductive tract, all reporting pH values lower than the pH range of 7.2-7.4 that is commonly 136 used for in vitro human embryo culture worldwide (Feo, 1955, Sedlis et al., 1967, Yedwab, et al., 1976). 137 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 6 pH measurements in these studies were conducted on different days throughout the menstrual cycle 138 and did therefore only part ly represent the natural environment of a preimplantation embryo. 139 Additional in vivo pH measurements are requir ed to identify the pH of the natural preimplantation 140 embryo environment and to assess the suitability of the pH range currently used for in vitro human 141 embryo culture. 142 Oviduct and uterine fluid composition 143 Many different human preimplantation embryo culture media are commercially available and globally 144 used in IVF laboratories. The varying concentrations of components across brands and types of media 145 indicate that the optimal composition for human preimplantation embryo culture medium still remains 146 unknown (Morbeck et al., 2014, Morbeck et al., 2017, Tarahomi et al., 2019, Zagers et al., 2023). While 147 some of these concentrations seem to be based on limited available evidence on the composition of 148 human oviduct and uterine fluid, most concentrations appear to be derived from experimental findings 149 (Morbeck, et al., 2014, Morbeck, et al., 2017, Tarahomi, et al., 2019, Zagers, et al., 2023) . Sixteen 150 studies have previously determined the concentrations of several or more components of human 151 oviduct or uterine fluid (Lippes et al., 1972, David et al., 1973, Lippes, 1975, Shams et al., 1977, Borland 152 et al., 1980, Casslen and Nilsson, 1984, Dickens et al., 1995, Gardner et al., 1996, Srivastava et al., 1996, 153 Tay et al., 1997, Chen et al., 2002, Strandell et al., 2004, Boomsma et al., 2009, Hannan et al., 2011, 154 Kermack et al., 2015, Utsunomiya et al., 2022). Most of these studies aspirated oviduct or uterine fluid 155 throughout the menstrual cycle instead of specifically within a few days after ovulation – the time a 156 preimplantation embryo would reside in the oviduct or uterus. Other limitations were obtaining fluid 157 from the oviduct or uterus ex vivo , analysing hydrosalpinx fluid, collecting oviduct fluid after vascular 158 perfusion, analy sing samples of low volume, analys ing a limited amount of samples , or aspirating 159 uterine or oviduct fluid from women who were either suspected of or diagnosed with uterine diseases 160 and infertility. Eleven other studies focused on identifyin g proteins present in human oviduct fl uid 161 and/or human uterine fluid (Moghissi, 1970, Lippes et al., 1981, Lippes et al., 1983, Lippes and Wagh, 162 1989, Wagh and Lippes, 1989, Lippes and Wagh, 1993, Wagh and Lippes, 1993, Parmar et al., 2008, 163 Salamonsen et al., 2013, Canha- Gouveia et al., 2019, Fujii et al., 2021) . Nevertheless, a thorough 164 understanding of the composition of human oviduct and uteri ne fluid is still lacking. Reliable oviduct 165 and uterine fluid analyses from healthy women of reproductive age are required to ascertain the 166 composition during the menstrual cycle phase when preimplantation embryos naturally reside in the 167 oviducts and uterus. 168 To summarize , the in vivo evidence underlying the current culture conditions for human 169 preimplantation embryos is scarce. A better understanding of the in vivo preimplantation embryo 170 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 7 environment in humans is essenti al to enable mimicking of the natural embryo environment for 171 optimization of in vitro human embryo culture conditions. Therefore, we measured the temperature 172 and pH in the human uterine cavity on the third day following a positive LH test or ovum pick-up, and 173 simultaneously aspirated human uterine fluid to analy se the concentrations of thirty -seven 174 components. 175

176 Ethical approval 177 This study was conducted according to the principles of the Declaration of Helsinki (Oct 2013). Ethical 178 approval was received in 2015 from the Ethics Committee of the Avicenna Research Institute (ARI) , 179 Teheran, Iran (decision number 258-15-95). 180 Study population 181 Women between 18 to 43 years old with a regular ovulatory cycle and a normal uterine function who 182 were visiting the Avicenna Infertility Clinic , Teheran, Iran, for fertility treatment due to severe male 183 factor subfertility (azoospermia or a total motile sperm count lower than eight hundred thousand per 184 ml of semen) or for PGT-M or sex selection treatment, were asked to participate in this study. Women 185 with a history of HIV or any sexually transmitted disease, malformation of the urogenital system, 186 borderline or invasive ovarian cancer or any other cancer, a history of dilatation and curettage, 187 premature ovarian failure, PCOS or severe psychopathology were not eligible for this study. All women 188 were informed about the study during their visit to the clinic and received an informative letter about 189 the study and their personal fertility treatment. In total, sixty-one women provided written informed 190 consent to participate in this study between April 2015 and March 2016 . Fifty three women 191 participated during a natural menstrual cycle before the start of their planned fertility treatment. Eight 192 women participated during an ovarian stimulated cycle during their fertility treatment, three days after 193 the ovum pick-up. Participating in this study during these stimulated cycles was possible because the 194 freeze all embryos strategy was applied in these fertility treatments. Seven women did not receive a 195 fresh embryo transfer due to ovarian hy perstimulation syndrome (OHSS) and one woman opted for 196 elective freeze all. 197 Timing in the menstrual cycle 198 To determine the time of ovulation in an un stimulated menstrual cycle, participating women were 199 trained for daily utilization of the “Ovulation Pred iction Kit” (OPK) (Sensitest ). The OPK is a urine 200 dipstick kit to ascertain the time of the LH surge, which predicts ovulation. Participants started testing 201 on day nine of the menstrual cycle and tested daily until a test was positive . The women were asked 202 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 8 to pay an extra visit to the clinic three days after the positive reading of the ovulation test for the 203 uterine measurements and uterine fluid collection. The eight women participating during a stimulated 204 cycle payed an extra visit to the clinic for the uterine measurements and uterine fluid collection on the 205 third day following the ovum pick up. 206 Uterine temperature and pH measurements 207 All women were positioned in lithotomic position and examined in the ovum pick-up room. Ultrasound 208 imaging was used to evaluate the follicles, the pattern of the ovaries, possible cysts, the length of the 209 uterus (distance between external orifice and fundus), the anterior to posterior uterine diameter, 210 possible uterine malformations, endometrial aspects and thickness. 211 Without any cleansing of the vagina, a medically approved disposable general purpose temperature 212 probe (81-020409EU; DeRoyal), connected to a general vital signs monitor, was then inserted in the 213 posterior vaginal fornix to record the body temperature. We selected this probe because of the 214 following characteristics described by the supplier: 1) its versatile design allows for oral, nasal or rectal 215

for routine monitoring of patient core body temperature, 2) it has a low -friction surface 216 for ease of insertion, 3) it has a completely enclosed sensor wires for improved stability and hygiene, 217 4) the diameter is very thin (ø = 3mm) and its flexible design ensured optimal patient safety, 5) it is 218 fully compatible with most monitors, 6) it was not made with natural rubber latex, 7) it is sterile and 219 8) it has a short response time of only 60 seconds. After this, the temperature probe was inserted 220 through the cervix into the uterus following the standard procedure of a routine embryo transfer. To 221 prevent any contact with the fundus or bleeding of the endometri um, the temperature probe was 222 inserted into the uterine cavity up to four to five centimeters from the external orifice of the cervix. 223 After removing the temperature probe from the uterine cavity, it was checked for the presence of 224 blood on the tip. 225 The standard procedure for embryo transfer (ET) was then applied for insertion of the outer sheath of 226 an ET catheter (IVFETFLEX). This was followed by insertion of a calibrated micro pH probe (pHersaflex 227 S1I catheter; Medical Measurement System Company (MMS)/ Laborie) through this outer sheath of 228 the ET catheter into the uterine cavity to one cm beyond the end of the outer sheath of the ET catheter 229 and up to four to five centimeters from the external orifice of the cervix. This micro pH probe is a 230 flexible, medically approved disposable micro probe with a diameter of 1.67 mm and a response time 231 of 30 seconds at the latest. Therefore, the pH was measured and recorded every second until 60 232 seconds after insertion in the uterine cavity. The pH probe was connected to an Orion II pH recorder 233 and used with MMS Software for c alibration of each pH probe and the pH measurement. Calibration 234 of each pH probe was performed with two known pH solutions in sachets (pH = 4 and 7; Mettler Toledo 235 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 9 BV) prior to each measurement , following the company’s instructions. After removing the pH probe 236 from the uterine cavity, it was checked for the presence of blood on the tip. 237 Uterine fluid aspiration 238 Directly after pH measurement, a five ml syringe was connected to the outer end of the outer sheath 239 of the ET catheter . Aspiration of uterine fluid was performed gently by applying a negative pressure 240 with the syringe and withdrawing the outer sheath of the ET catheter from the uterine cavity 241 simultaneously. Aspirated, often highly viscous, uterine fluid was immediately expelled from the outer 242 sheath of the ET catheter into a 500 µ l Eppendorf micro -tube using positive air pressure supplied 243 through the syringe. The sample was spun down at 4000 rpm for 30 minutes at 37°C in an Eppendorf 244 5810R centrifuge to separate possible aspirated endometrial cells from the uterine fluid. Supernatant 245 and pellet were snap frozen separately and stored in -80°C until composition analysis. 246 Uterine fluid analysis 247 On the day of composition analysis, t wenty-two collected uterine fluid samples (supernatants) were 248 thawed and the volume of every sample was determin ed. To decrease the viscosity, the volume of 249 each sample was increased up to 300 μl by add ing Milli-Q water (Millipore) and mechanically shaken 250 (1800 oscillations per minute for two minutes using a Tissue lyser II (Qiagen)). Concentrations of ions 251 (calcium, chloride, iron, magnesium, sodium, phosphate, potassium), glucose, uric acid, 252 immunoglobulins (IgA, IgG and IgM), albumin and total prot ein were measured using a Cobas 8000 253 chemistry analyser (Roche Diagnostics). Concentrations of lactate (L and D), pyruvate and 21 amino 254 acids (alanine, arginine, asparagine, aspartic acid, citrulline, glutamic acid, glutamine, glycine, 255 histamine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, 256 tryptophan, tyrosine and valine) were determined using Ultra -Performance Liquid Chromatography 257 tandem Mass Spectrometry (UPLC -MS/MS; Acquity -Quattro Premier XE, Waters, Milford, 258 Massachusetts, USA). Iron was measured in micromolar (μM) and all other components were 259 measured in millimolar (mM). After composition analysis, the measured concentrations were 260 recalculated to the original volume of the uterine fluid sample. All measurements were performed at 261 once by two technicians who were blinded to the source and characteristics of each sample. To achieve 262 this, coded labelling was applied for all samples. 263 Statistical analysis 264 The pH and temperature determined in the uterine cavity are presented as the mean ± Standard 265 Deviation (SD). The concentrations of components of uterine fluid are presented as the mean ± 266 Standard Error of the Mean (SEM) . To test whether there was a correlation between core body 267 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 10 temperature and uterine temperature we performed a Pearson’s correlation test in IBM SPSS Statistics 268 28. To determine whether uterine temperature and pH significantly differed between unstimulated 269 and stimulated women, we performed independent t-tests in IBM SPSS Statistics 28. 270

271 One hundred and fifty nine women attending the Avicenna Infertility Clinic (Tehran, Iran) for IVF/ ICSI 272 treatment due to severe male factor subfertility or for PGT-M or sex selection were asked to participate 273 in this study between April 2015 and March 2016. In total, one hundred and eight women signed the 274 informed consent form. Eleven participants withdrew from participation before the appointment for 275 uterine measurements and fluid aspiration. Thirty six participants were excluded due to absence of a 276 positive reading of the LH tests or because they did not show up for t he appointment due to travel 277 difficulties from other cities or regions. Altogether, sixty one participants came to the clinic for uterine 278 temperature and pH measurements and uterine fluid aspiration on the third day following a positive 279 LH test (n = 53) or ovum pick -up (n = 8) (Table 1). Fifty three participants did not receive ovarian 280 stimulation and were examined during their natural menstrual cycle before the start of their personal 281 IVF/ICSI treatment. Eight participants did receive ovarian stimulation for IVF/ICSI treatment, but did 282 not receive an embryo transfer in the same cycle as the ovum pick up due to ovarian hyper-stimulation 283 syndrome (OHSS; participants 14, 18, 20–22, 26 and 34) or elective freeze-all (participant 16), and their 284 uterine environment was examined instead. All participants were between 20 and 40 years old, with a 285 mean age of 29.9 ± 4.4 years old. The mean BMI was 24.8 ± 3.3 kg/m2, within a range of 17.2 -32.8 286 kg/m2. Nine women have been proven to be fertile, as they delivered one or two times before. 287 Prior to uterine temperature measurement, the core body temperature of each woman was 288 determined using a similar disposable temperature catheter ( 81-020409EU; DeRoyal) placed in the 289 vagina. The mean core body temperature was 37.08 ± 0.26oC. 290 Uterine temperature 291 We determined the temperature in the uterine cavity of 58 participants ( 51 unstimulated and 7 292 stimulated women; Table 1 and Figure 1a). The mean temperature in the human uterine cavity on the 293 third day following a positive LH test or ovum pick-up was 36.94 ± 0.26oC (temperature range: 36.0oC 294 to 37.6oC). One stimulated participant, participant 34, felt discomfort during temperature catheter 295 insertion and therefore temperature measurement and further study procedures were aborted. The 296 uterine temperature correlated with body temperature (r = 0.45, p < 0.001). The uterine temperature 297 did not significantly differ between unstimulated women (36.94 ± 0.27oC) and stimulated women 298 (36.99 ± 0.15oC) (ΔT = 0.05 , p = 0.64). 299 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 11 Uterine pH 300 We determined the pH in the uterine cavity of 53 participants (46 unst imulated and 7 stimulated 301 women; Table 1 and Figure 1b). The mean pH in the human uterine cavity on the third day following a 302 positive LH test or ovum pick -up was pH 6.76 ± 0.22 (pH range: 6.31 to 7.34). Five participants felt 303 discomfort during temperature or pH measurement and therefore pH was not determined. The uterine 304 pH did not significantly differ between unstimulated women (pH 6.75 ± 0.22) and stimulated women 305 (pH 6.82 ± 0.18) (ΔpH = 0.07, p = 0.42). 306 The composition of uterine fluid 307 We aspirated uterine fluid from 50 participants: 43 unstimulated and 7 stimulated women (Table 1 308 and Figure 1b). For practical reasons, the first available twenty (two) samples of aspirated uterine fluid 309 were used to determine the mean concentrations of thirty -seven components of uterine fluid 310 aspirated on the third day following a positive LH test (n = 15) or ovum pick-up (n = 7) (Table 2). This 311 subset of components aligns with the thirty -seven components previously analysed in fifteen 312 commercial human embryo cultur e media (Tarahomi et al. 2019). Important energy sources for 313 preimplantation embryos were present in the following mean concentrations: 0.08 ± 0.02 mM 314 pyruvate, 5.1 ± 1.78 mM glucose and 6.60 ± 1.12 mM lactate. The amino acids glutamic acid, glycine 315 and alanine were present at much higher concentrations than the concentrations of other amino acids 316 determined in the aspirated uterine fluid samples. Glutamic acid alone represented 23% of the total 317 amino acid concentration in human uterine fluid. Citrulline, asparagine, tryptophan and methionine 318 were present at the lowest concentrations: approximately 45 -fold lower than glutamic acid. 319 Furthermore, immunoglobulins were present in different concentrations, with IgG being the most 320 predominant. 321

322 In vivo evidence-based optimization of the in vitro embryo culture environment may improve human 323 preimplantation embryo development and quality, and hold the potential to increase cumulative live 324 birth rates and improve child outcomes after IVF/ICSI. However, the in vivo evidence on the natural 325 environment of human preimplantation embryos is scarce. Better understanding of the in vivo 326 characteristics of the preimplantation embryo environment in humans is essential to enable mimicking 327 of this natural environment in an in vitro human embryo culture system. We aimed to gain 328 fundamental knowledge on the human uterine conditions during the physiologically relevant period of 329 the menstrual cycle. We measured the temperature (36.94 ± 0.26oC) and pH (6.76 ± 0.22) in the uterus 330 of healthy women of reproductive age with n ormal ovulation and uterine function on the third day 331 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 12 following a positive LH test or ovum pick -up, and determined the concentrations of thirty -seven 332 components of uterine fluid aspirated at the same time in the menstrual cycle. 333 The strength of this study lies in the aim to analyse the characteristics of the human uterine cavity 334 specifically at the physiologically relevant period of the menstrual cycle – when a preimplantation 335 embryo normally resides and develops within the human reproductive tract. Although some previous 336 studies have stated that in vivo measurement results throughout the menstrual cycle do not 337 significantly differ from each other (Kermack, et al., 2015, Utsunomiya, et al., 2022), other studies have 338 shown that uterine characteristics could change during the menstrual cycle (Feo, 1955, Yedwab, et al., 339 1976, Gardner, et al., 1996), possibly in response to shifting hormone levels, endometrial changes, or 340 other factors. Since we want to use in vivo insights for optimization of in vitro culture environment for 341 preimplantation embryos, it is important to ensure that the examined environment represents the 342 natural preimplantation embryo environment as close as possible at the time of the measurements . 343 We therefore timed the uterine measurements and uterine fluid aspiration in unstimulated women 344 specifically on the third day following a positive LH test. However, the LH test yields a positive result 345 at the onset of the LH surge, serving as an indicator of the impending ovulation rather than confirming 346 its occurrence. Ovulation is anticipated 24 -48 hours after a positive LH test result and therefore our 347 analyses of uterine characte ristics were conducted approximately 1.5 days post -ovulation. At this 348 moment in the menstrual cycle, a zygote or preimplantation embryo could be hypothesised to still be 349 in one of the oviducts. The time of arrival of an human embryo in the uterus is not established exactly, 350 but generally assumed to be around the third day after fertilization, although it could be earlier or 351 later. Regarding our timing, we may have identified an environment pa rticularly suitable to cleavage 352 stage embryos, which now could be considered a limitation. However, the measurements on the third 353 day following ovum pick-up, approximately 3-3.5 days after ovulation in stimulated women – when an 354 embryo is expected to reside in the uterus – , did not clearly differ from the results in unstimulated 355 women. We also consider measurements in the uterus relevant for determining conditions for in vitro 356 cleavage stage culture, because the timing of embryos entering the uterus has not exactly been 357 determined yet, as we mentioned earlier, but mostly since measuring oviduct conditions comes with 358 difficulties that challenge the results. Measuring oviductal pH is particularly difficult due to the 359 narrowness of the oviducts, complicating access and accurate measurements. The use of CO 2 for 360 insufflation during surgeries alters the pH, a change that also occurs in ex vivo measurements following 361 ovariectomy. Additionally, women scheduled for surgeries like ovariectomy often have reproductive 362 health issues that may modify the oviductal environment. 363 Other strengths of this study include the extensive number of uterine measurements conducted at one 364 specific point in the menstrual cycle. T he reliability of the measurements is ensured by the use of 365 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 13 catheters designed for internal t emperature and pH measurements. Furthermore, we identified the 366 concentrations of the largest number of components in each uterine fluid sample to date. Additionally, 367 the reliable techniques used for determining the concentrations of the uterine fluid components add 368 robustness to the results. 369

mainly regard the implications of the results. Firstly, a general and inevitable limitation of 370 identifying the in vivo preimplantation embryo environment is the absence of a preimplantation 371 embryo, which normally may have influenced its surroundings. Secondly, our study was confined to a 372 single centre and a specific population of women. The potential influence of genetic or nutritional 373 factors possibly limit the generalizability of the findings . Thirdly, uterine fluid likely consists of more 374 components than the thirty -seven that we analys ed. Fourthly, e xact mimicking of all known 375 characteristics of the in vivo environment in vitro may not provide an optimal human preimplantation 376 embryo environment in vitro. Follow-up research on the effect of in vivo characteristics in an in vitro 377 culture system for human preimplantation embryo culture will be necessary. 378 Temperature 379 The mean uterine temperature we determined, 36.94 ± 0.26oC, was similar to the average temperature 380 Yedwab et al. found in the uterine cavity of ten healthy women during their estimated ovulatory phase: 381 37.08 ± 0.18oC (Yedwab, et al., 1976). In absence of other in vivo studies, our findings add confidence 382 to the suggestion that a temperature of 37.0oC is optimal for human embryo culture. The standard 383 temperature of 37.0oC used in laboratory practice for human embryo culture in IVF matches these 384 results. 385 pH 386 The mean uterine pH we identified ( pH 6.76 ± 0.22) slightly differed from the mean pH reported by 387 two studies that previously investigated uterine pH (Feo, 1955, Sedlis, et al., 1967, Yedwab, et al., 388 1976). The early secretory pH values measured by Feo et al. (mean pH: 6.55, n=4; pH range 6.5- 6.6) 389 and early secretory and mid-secretory pH values measured by Sedlis et al. (mean pH: 6. 5, n=17; pH 390 range: 6.2-7.0) were a little lower, the mean pH reported by Yedwab et al. (pH 7.12, n=40) was higher. 391 Despite several limitations, the results from our study and these other in vivo studies indicate that the 392 pH of the human preimplantation embryo environment in vivo is lower than the pH currently 393 considered optimal and widely used for in vitro human embryo culture (pH 7.2-7.4). These findings 394 suggest that decreasing the pH of the in vitro human embryo culture system may im prove human 395 embryo development. Based on our study, we propose that in vitro embryo culture at a pH of 6.8 ± 0.1 396 may be more beneficial for human preimplantation embryos than the current ly standard used pH of 397 7.3 ± 0.1. Future research should be conducted to confirm this. 398 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 14 Uterine fluid composition 399 The a mino acid concentrations we determined in the aspirated uterine fluid samples were fairly 400 comparable to the concentrations of the 18 amino acids previously determined in 56 samples of human 401 uterine fluid, although the latter were predominantly aspirated from subfertile women (Kermack, et 402 al., 2015) . Interestingly, amino acid concentrations that were recently identified in 28 samples of 403 human midcycle and luteal oviduct fluid from infertile women were also similar (Utsunomiya, et al., 404 2022). Other components identified in these oviduct fluid samples , such as ions and energy sources 405 (pyruvate, glucose and lactate), showed slightly diverse results with the concentrations of the 406 components in our uterine fluid samples. For example, the average pyruvate concentration was higher 407 (0.18 mM ± 1. 48 mM) and the average lactate concentration was lower (4.66 ± 2.78 mM) than we 408 determined in uterine fluid , but not significantly different . The average glucose concentration in 409 midcycle oviduct fluid was lower (3.41 ± 1.61 mM) and in luteal oviduct fluid higher (4.16 ± 2.01 mM) 410 than what we found in uterine fluid. Interestingly, the average m agnesium concentration in oviduct 411 fluid (0.53 ± 0.25 mM) appeared to be significantly lower than in uterine fluid. This difference seems 412 relevant for embryo culture medium development, where two approaches (low or high magnesium 413 concentration) have been used (Morbeck 2017). 414 One of the two small and older studies that had previously analysed uterine fluid composition, 415 determined proliferative, midcycle and luteal phase uterine glucose, calcium, chloride, potassium, 416 sodium, urea and fructose concentrations (Casslen and Nilsson, 1984). The midcycle and luteal phase 417 mean glucose concentrations of respectively 5.7 mM and 5.2 mM (in total n = 6), align with the mean 418 glucose concentration of 5.1 ± 1.78 mM in our samples . The mean concentrations of the analysed 419 inorganic ions showed a similar pattern to our results, but were higher (potassium 25.8 mM and 420 chloride 110 mM) or lower (sodium 110 mM) in absolute numbers . The other study, from 1996, 421 investigated uterine concentrations of the metabolites pyruvate, lactate and glucose throughout the 422 menstrual cycle (Gardner, et al., 1996) . The mean glucose concentration (in total n = 15) was lower 423 than 5 mM, namely 3.15 ± 0.31 mM. The mean concentrations of pyruvate (0.10 ± 0.05 mM) and lactate 424 (5.87 ± 1.19 mM) were similar to our results. 425 In conclusion, o ur study contributes the most comprehensive analysis of human in vivo uterine 426 temperature (n=58), pH (n = 53) and uterine fluid composition to date (37 components in 22 samples). 427 We gained novel fundamental insights into the natural in vivo environment of human preimplantation 428 embryos, valuable for assessment and optimization of the in vitro embryo culture system. The mean 429 uterine temperature we found confirmed the use of 37.0 oC for human embryo culture to be optimal 430 for human preimplantation embryos. However, we observed a significantly lower pH (pH 6.76 ± 0.23) 431 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 15 compared to the current widely adopted pH range of 7.2 -7.4 for human embryo culture in IVF 432 laboratories. Our results suggest that decreasing the pH of human embryo culture medium may be 433 beneficial for the development and quality of human preimplantation embryos and potentially leads 434 to a higher (cumulative) live birth rate. We therefore propose to test the effects of culturing human 435 embryos in a pH of 6.8 ± 0.1. Furthermore, we provided a comprehensive overview of the 436 concentrations of 37 components of uterine fluid, which can also be used for in vivo evidence-based 437 embryo culture medium development. 438 Data availability 439 The data underlying this article are available in the article. 440 Authors’ roles 441 M.T., S.R. and S.M. designed the study. M.T., S.Z., S.F., A.M., Z.F., H.S and F.F. contributed substantially 442 to data collection. M.T., J.v.S., F.S. and F.V. performed the uterine fluid analyses. M.T., M.Z. and M.v.W. 443 analyzed and interpreted the data. M.Z., M.T., G.H. and S.M. wrote the final manuscript. All authors 444 revised the manuscript and approved publication of the last version. 445 Funding 446 This research was supported by ZonMw ( https://www.zonmw.nl/en) Programme Translational 447 Research 2 [project number 446002003] for manuscript preparation. 448 Conflict of interest 449 The authors used these in vivo data to develop a human embryo culture medium based on the in vivo 450 embryo environment. 451 452 Table 1. Participants' characteristics, including measured uterine temperature and pH 453 In the last row we presented the mean ± SD for age, BMI, vaginal temperature, uterine temperature 454 and uterine pH. The vaginal temperature of participants 12 and 23 was not included in the calculation 455 of the mean ± SD. 456 Figure 1. Human uterine temperature and pH 457 a. Human uterine temperature on the third day after a positive LH test (unstimulated participants, 458 n=51; in blue) or on the third day after ovum pick-up (stimulated participants, n=7; in orange). 459 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 16 b. Human uterine pH on the third day after a positive LH test (unstimulated participants, n=46; in blue) 460 or on the third day after ovum pick-up (stimulated participants, n=7; in orange). 461 Table 2. Concentrations of thirty -seven components of human uterine fluid aspirated on the third 462 day after a positive LH test (n=15) or on the third day after ovum pick-up (n=7). 463 Concentrations are presented as the mean ± SEM. 464 465

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The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 20 Tables and Figures 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 1 33 23,8 0 Az oospermia No 3rd day following + LH test 8.2 C 37,3 37,1 6.71 ± 0.02 2 24 29,4 0 Olig ospermia No 3rd day following + LH test 6,7 C 37,3 36,9 6.39 ± 0.09 3 29 25,4 0 Azoospermia No 3rd day following + LH test 7,1 C 37,1 37,1 6.58 ± 0.05 4 31 24,0 2 Normospermia No 3rd day following + LH test 7,1 C 37,5 37,2 6.81 ± 0.05 5 33 24,8 0 Azoospermia No 3rd day following + LH test 11,8 B 37,1 36,9 6.75 ± 0.11 6 33 24,3 0 Azoospermia No 3rd day following + LH test 8,2 C 37,1 36,9 6.72 ± 0.04 7 27 24,7 0 Azoospermia No 3rd day following + LH test 8,2 C 37,1 36,6 6.80 ± 0.09 8 31 20,2 0 Azoospermia No 3rd day following + LH test 6,3 C 37,1 37,3 6.72 ± 0.18 9 33 23,4 0 Azoospermia No 3rd day following + LH test 12,6 B 37,0 36,8 6.59 ± 0.05 10 29 27,6 0 Azoospermia No 3rd day following + LH test 8,8 B 37,3 37,0 n.a. 11 31 30,4 0 Azoospermia No 3rd day following + LH test 8,3 B 37,0 37,1 6.83 ± 0.07 12 30 24,0 0 Azoospermia No 3rd day following + LH test 10,3 B 37,9 n.a. n.a. 13 29 25,2 0 Azoospermia No 3rd day following + LH test 7,7 B 36,9 36,6 n.a. 14 30 22,5 0 Azoospermia Yes (OHSS) 3rd day following ovum pick-up 9,4 A 37,0 37,0 7.03 ± 0.07 15 25 26,4 0 Oligospermia No 3rd day following + LH test 8,4 B 37,1 36,8 6.72 ± 0.16 16 35 25,2 1 Normospermia Yes (elective freeze-all) 3rd day following ovum pick-up 8,4 B 37,1 37,0 6.95 ± 0.06 17 31 27,1 0 Oligospermia No 3rd day following + LH test 6,8 B 37,4 37,1 6.65 ± 0.07 18 25 20,7 0 Azoospermia Yes (OHSS) 3rd day following ovum pick-up 9,1 A 36,9 37,0 6.73 ± 0.08 19 37 27,5 2 Normospermia No 3rd day following + LH test 8,8 C 36,8 36,9 6.70 ± 0.03 20 32 28,5 0 Azoospermia Yes (OHSS) 3rd day following ovum pick-up 8,1 A 37,0 37,0 6.74 ± 0.17 21 35 32,8 2 Normospermia Yes (OHSS) 3rd day following ovum pick-up 10,5 B 37,0 37,0 6.79 ± 0.03 22 33 21,3 0 Azoospermia Yes (OHSS) 3rd day following ovum pick-up 8,8 B 37,3 37,2 7.00 ± 0.07 23 23 26,1 0 Azoospermia No 3rd day following + LH test 7,6 C 37,6 n.a. n.a. 24 38 29,7 0 Azoospermia No 3rd day following + LH test 7,4 C 36,9 36,6 6.86 ± 0.06 25 27 26,4 0 Azoospermia No 3rd day following + LH test 10,1 C 37,3 37,0 n.a. 26 26 31,9 0 Azoospermia Yes (OHSS) 3rd day following ovum pick-up 6,2 A 37,2 36,7 6.53 ± 0.05 27 24 21,2 0 Azoospermia No 3rd day following + LH test 12,1 B 37,2 36,8 6.70 ± 0.00 28 23 20,2 0 Azoospermia No 3rd day following + LH test 8,2 C 37,2 36,8 6.95 ± 0.07 29 34 24,6 0 Azoospermia No 3rd day following + LH test 7 C 36,7 36,8 6.75 ± 0.13 30 27 25,0 0 Oligospermia No 3rd day following + LH test 6 C 36,8 36,8 6.60 ± 0.14 31 28 21,0 0 Oligospermia No 3rd day following + LH test 8 C 36,7 36,8 n.a. 32 31 26,0 0 Oligospermia No 3rd day following + LH test 10 B 37,3 37,3 6.97 ± 0.10 33 31 20,6 0 Oligospermia No 3rd day following + LH test 9 B 37,2 37,3 6.66 ± 0.27 34 35 27,8 0 Azoospermia Yes (OHSS) 3rd day following ovum pick-up 5,3 C 37,1 n.a. n.a. 35 28 29,8 0 Azoospermia No 3r d day following + LH test 10,5 B 37,2 37,1 6.86 ± 0.15 36 24 22,6 0 Azoospermia No 3rd day following + LH test 6,7 B 36,3 36,6 6.59 ± 0.03 37 27 17,2 0 Azoospermia No 3rd day following + LH test 9,6 B 36,7 37,3 6.60 ± 0.03 38 29 27,1 0 Azoospermia No 3rd day following + LH test 8,8 A 36,7 37,1 n.a. 39 32 21,1 0 Azoospermia No 3rd day following + LH test 6,1 C 37,0 36,4 7.08 ± 0.04 40 29 28,6 0 Azoospermia No 3rd day following + LH test 7,3 C 37,0 36,8 6.76 ± 0.12 41 34 23,5 2 Normospermia No 3rd day following + LH test 11,7 C 37,1 37,1 6.50 ± 0.03 42 35 23,4 0 Oligospermia No 3rd day following + LH test 5,8 C 36,8 37,0 6.44 ± 0.05 43 29 21,4 0 Azoospermia No 3rd day following + LH test 13,2 B 37,0 36,9 6.99 ± 0.03 44 40 24,0 0 Azoospermia No 3rd day following + LH test 11 C 37,0 36,9 6.78 ± 0.04 45 27 28,0 0 Oligospermia No 3rd day following + LH test 12 B 36,9 36,8 6.72 ± 0.04 46 21 22,8 0 Azoospermia No 3rd day following + LH test 10,2 A 37,4 37,0 6.60 ± 0.00 47 24 22,0 0 Azoospermia No 3rd day following + LH test 7 C 37,0 36,9 7.25 ± 0.09 48 21 25,3 0 Oligospermia No 3rd day following + LH test 6,6 C 37,0 37,0 6.48 ± 0.06 49 34 17,9 0 Azoospermia No 3rd day following + LH test 11,9 C 36,9 36,8 6.93 ± 0.08 50 36 21,1 0 Azoospermia No 3rd day following + LH test 5,9 C 37,5 37,1 6.73 ± 0.11 51 36 27,2 0 Azoospermia No 3rd day following + LH test 7,9 C 36,9 37,1 7.21 ± 0.11 52 34 25,7 0 Azoospermia No 3rd day following + LH test 5,6 C 37,1 37,0 7.05 ± 0.05 53 31 27,7 1 Azoospermia No 3rd day following + LH test - - 36,7 36,9 6.83 ± 0.12 54 28 24,0 0 Azoospermia No 3rd day following + LH test 7,6 A 37,0 37,0 6.87 ± 0.11 55 28 19,5 0 Azoospermia No 3rd day following + LH test 9,4 C 37,0 36,7 6.31 ± 0.04 56 20 24,5 0 Azoospermia No 3rd day following + LH test 10 C 36,9 36,0 6.91 ± 0.19 57 30 28,7 1 Normospermia No 3rd day following + LH test 10 C 37,2 36,9 6.59 ± 0.03 58 34 28,4 1 Normospermia No 3rd day following + LH test 9 C 36,9 36,6 6.50 ± 0.02 59 29 22,0 0 Azoospermia No 3rd day following + LH test 6,7 B 37,0 37,0 7.34 ± 0.06 60 30 25,6 1 Normospermia No 3rd day following + LH test 12,2 B 37,4 37,6 6.39 ± 0.11 61 32 24,4 0 Azoospermia No 3rd day following + LH test 8,2 B 37,5 37,6 6.81 ± 0.06 Mean ± SD 29.9 ± 4.4 24.8 ± 3.3 37.05 ± 0.23 36.94 ± 0.26 6.76 ± 0.22 Table 1. Participants' characteristics, including measured uterine temperature and pH. In the last row we presented the mean ± SD for age, BMI, vaginal temperature, uterine temperature and uterine pH. The vaginal temperature of participants 12 and 23 was not included in the calculation of the mean ± SD. Participant study number Age BMI Semen analysis

partner Previous gravidity Hormonal stimulation for IVF treatment Timing of uterine measurements and uterine fluid aspiration Endometrial thickness (mm) Endometrial pattern (layers) Core body temperature (oC) Uterine temperature (oC) Uterine pH preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 21 636 637 Figure 1. Human uterine temperature and pH b. Human uterine pH on the third day after a positive LH test (unstimulated participants, n=46; in blue) or on the third day after ovum pick-up (stimulated participants, n=7; in orange). a. Human uterine temperature on the third day after a positive LH test (unstimulated participants, n=51; in blue) or on the third day after ovum pick-up (stimulated participants, n=7; in orange). 36 36,2 36,4 36,6 36,8 37 37,2 37,4 37,6 37,8 0 1 2 3 4 5 6 7 8 9 10111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596061 Human uterine temperature (oC) Participants Human uterine temperature 6,2 6,3 6,4 6,5 6,6 6,7 6,8 6,9 7 7,1 7,2 7,3 7,4 0 1 2 3 4 5 6 7 8 9 10111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596061 Human uterine pH Participants Human uterine pH preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint 22 Calcium 1.55 ± 0.18 Chloride 82.34 ± 3.62 Histidine 97 ± 17 Magnesium 1.83 ± 0.17 Isoleucine 114 ± 14 Phosphate 3.30 ± 0.59 Leucine 193 ± 31 Potassium 17.22 ± 2.07 Lysine 298 ± 53 Sodium 143.81 ± 8.02 Methionine 38 ± 9 Phenylalanine 100 ± 15 Threonine 117 ± 20 Glucose 5.1 ± 1.78 Tryptophan 37 ± 4 L actate 6.60 ± 1.12 Valine 190 ± 26 Pyruvate 0.08 ± 0.02 Alanine 513 ± 82 Albumin 12.02 ± 1.23 Arginine 136 ± 40 Total protein 41.93 ± 4.24 Asparagine 33 ± 7 Aspartic acid 230 ± 29 Citrulline 26 ± 3 IgA 0.42 ± 0.11 Glutamic acid 1162 ± 183 IgG 3.49 ± 0.44 Glutamine 227 ± 40 IgM 0.43 ± 0.07 Glycine 955 ± 156 Ornithine 125 ± 10 Other Proline 221 ± 33 Iron (µM) 115.11 ± 31.76 Serine 220 ± 46 Uric acid (mM) 0.14 ± 0.03 Tyrosine 86 ± 15 Immunoglobulins (g/L) Proteins (g/L) Energy sources (mM) Table 2. Concentrations of thirty-seven components of human uterine fluid aspirated on the third day after a positive LH test (n=15) or on the third day after ovum pick-up (n=7). Concentrations are presented as the mean ± SEM. Inorganic ions (mM) Amino acids (µM) Essential amino acids Non-essential amino acids 638 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 19, 2024. ; https://doi.org/10.1101/2024.11.18.623470doi: bioRxiv preprint