Chronic gestational hypoxia accelerates ovarian aging and lowers ovarian reserve in next-generation adult rats

: 58 Chronic fetal hypoxia is a common complications observed in human pregnancy, impacting 59 pregnancies across global contexts. Exposure to chronic intrauterine hypoxia has major short 60 and long-term consequences for offspring health. However, the impact of chronic gestational 61 hypoxia on female reproductive system development is unknown. We aimed to understand 62 the impact of exposure to chronic fetal hypoxia on the developing female reproductive 63 system. Wistar rat dams underwent normoxia (21%) or hypoxia (13%) during pregnancy. 64 Postnatally, all female offspring were maintained in normoxic conditions into early 65 adulthood. Females rats exposed to chronic gestational hypoxia (13%) during their 66 intrauterine development had decreased ovarian primordial follicular reserve compared to 67 controls (p<0.05). Adult females who had been exposed to chronic fetal hypoxia had 68 significantly reduced somatic ovarian telomere length (p<0.05), and reduced ovarian protein 69 expression of KU70, a critical component of the DNA-PK repair complex (p<0.01). Gene 70 expression of NOX2-mediated oxidative stress markers was increased (p<0.05). Exposure to 71 chronic hypoxia during fetal development leads to accelerated ageing of the somatic ovary 72 and decreased ovarian reserve in adulthood. Ovarian ageing is highly sensitive to gestational 73 hypoxia, with implications for future fertility in next-generation offspring of high-risk 74 pregnancies. 75


Introduction 80 81
Chronic gestational hypoxia is a common feature of a number of suboptimal intrauterine 82 environments, including placental insufficiency, preeclampsia, maternal smoking and 83 pregnancy at high altitude(1, 2) Exposure to chronic hypoxia during gestation adversely 84 influenced fetal and placental development, and is associated with adverse pregnancy 85 outcomes (2-6). The short term adverse effects of chronic gestational hypoxia include 86 increased risks of late miscarriage, fetal growth restriction, and low birth weight (3)(4)(5)(6)(7)(8). 87 Chronic gestational hypoxia also has long-term effects on the physiology of exposed 88 offspring, termed developmental programming. The effects of gestational hypoxia are best 89 characterized in the cardiovascular system, where the impact of low oxygen tension on the 90 developing heart and vasculature has been extensively studied in animal models (1, 3). The 91 consequences of a suboptimal fetal environment on long term reproductive health is an under 92 explored area in the field of developmental programming but an area of huge importance 93 given that the reproductive system is the mediator of information across generations. In 94 particular, the impact of chronic hypoxia on the development of the female reproductive 95 system is unknown. 96

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The developing female reproductive system is particularly vulnerable to the impact of a 98 suboptimal intrauterine environment because of the specific developmental windows during 99 which ovarian reserve is established. Ovarian reserve refers to the total finite number of 100 primordial follicles remaining in both ovaries at any point in life, and is the key determinant 101 of fertility potential in the female (4). Disruptions to the fetal environment during the crucial 102 phase of ovarian follicular endowment result in a decreased ovarian reserve in adult 103 reproductive life (5-9). In vitro evidence suggests that the ovarian follicle is particularly 104 sensitive to oxygen tension. Oocyte development within follicles in the adult ovary is 105 markedly influenced by the oxygen content of the follicular fluid (10), with hypoxic follicles 106 containing a higher percentage of oocytes with derangements of chromosomal organization. 107 Therefore, there is a strong rationale to hypothesise that follicular dynamics in the developing 108 follicles in the ovary in utero may be highly influenced by exposure to chronic gestational 109 hypoxia. In this study, we investigated whether ovarian reserve in the young adult female is 110 influenced by exposure to chronic hypoxia during gestation and determined underlying 111 mechanisms. 112 Wistar rat dams at 10-12 weeks of age (Charles River Ltd., Margate, UK) were housed in 120 individually ventilated cages (21% oxygen, 70-80 air changes/hour) under standard 121 conditions. All animals were fed a standard laboratory chow diet (20% protein) and fed ad 122 libitum with free access to water. After initial acclimatization (10 days) they were mated with 123 fertile male Wistar rats (n=14), and pregnancy confirmed through the observation of a 124 vaginal plug. The day of the plug was designated day 0 of pregnancy (full term 21-22 days). 125

Materials and Methods
Upon confirmation of pregnancy, dams were weighed and housed individually. On day 6 of 126 pregnancy, dams were randomly divided into two groups; control (21%) and hypoxic (13%) 127 pregnancy (n=8 per group). Pregnant rats assigned to the hypoxia group were placed inside a 128 chamber, which combined a PVC isolator with a nitrogen generator, as previously described 129 (11,12). Hypoxic pregnancies were maintained at a constant inspired fraction of oxygen of 130 13% from day 6 to 20 of gestation. This model of hypoxic pregnancy does not decrease 131 maternal food intake (11). All dams delivered under normoxic conditions. There were no 132 complete pregnancy losses in either group during the study. The respective litter sizes were 133 12.3±1.0 pups in the normoxia group compared to 9.3±1.2 pups in the hypoxia group 134 (p<0.05). Gestational length averaged 20±1 days in both normoxic and hypoxic groups. 135 Normoxia (21%) was maintained for all animals during lactation, weaning and thereafter. 136 Following determination of birth weight, litters were culled to 4 males and 4 females to 137 standardise nutritional access and maternal care during suckling (11, 12). All pups were 138 suckled by their own mothers. At four months of age, adult female pups underwent euthansia. 139 At post mortem, the reproductive tract tissues were harvested and weighed fresh, immediately 140 after dissection. One ovary from each animal was snap-frozen in liquid nitrogen and the other 141 fixed in formalin/paraldehyde. The fixed ovaries were sectioned and subjected to 142 haematoxylin and eosin (H&E). An equal distribution of estrous cycle stages in each group 143 was confirmed using the serial sections of whole H&E stained ovary prepared for primordial 144 follicle counting. However, the study was not powered for comparisons between estrous 145 cycle stages and thus parameters were selected to be non-varying with cycle stage. Sample 146 analysis was performed using project codes to blind the investigators to the experimental 147 groups. The adequacy of the sample size was determined via a power calculation based on 148 the effect sizes for ovarian primordial follicle counts in Wistar rats reported in our previous 149 studies (6, 13) using an alpha level of 0.05 to give power of 0.8. 150 151 Primordial follicle counts 152 Primordial follicle counts were performed as described previously (6, 13). Fixed ovaries were 153 processed for microscopy and the entire ovary sectioned at 8μm. Every 9 th section was 154 stained with H&E for morphometric analysis (72μm between analysed sections). Only 155 follicles with a visible oocyte nucleus were counted, in order to avoid repeat counts of the 156 same follicle (14). Primordial follicles were identified morphologically by the presence of a 157 single layer of flattened granulosa cells surrounding the oocyte (15)  Protein was extracted from whole tissue lysates of snap-frozen ovaries, as described 209 previously (18,22). To ensure equal protein loading, protein assays were performed on all 210 samples to ensure that each sample was diluted to the same protein concentration (1mg). 211 Protein (20μg) was loaded onto 10%, 12% or 15% polyacrylamide gels, dependent upon the 212 molecular weight of the protein to be measured. The samples were electrophoresed and  Maternal hypoxia effects were compared between groups using 2-tailed Student's T tests. In 235 order to correct for multiple hypothesis testing of gene expression levels, p values were 236 transformed to q values to take account of the false discovery rates using the p.adjust function 237 in R stats package (R Foundation for Statistical Computing, Vienna, Austria). This 238 adjustment was designed for this study in order to take account of the specific number of 239 genes that were tested within the initial screen (32) and therefore to ensure that the p values 240 were optimally transformed. Data are represented as means ± SEM. Where p values are 241 reported, an alpha level <0.05 was considered statistically significant. All data analysis was 242 conducted using the R statistical software package version 2.14.1 (R Foundation for 243 Statistical Computing, Vienna, Austria). Only ovaries of one female offspring per litter were 244 used for analysis to account for within litter variation. Therefore, in all cases, n refers to the 245 number of litters, and n=8 was used for all groups. 246

Results 248
There was no significant difference in the body weight of female rats at 4 months of age 249 exposed to gestational hypoxia compared to those that experienced normoxia, however there 250 was a trend towards a slightly lower body weight in the hypoxia group (p=0.06; Table 2). 251 Ovarian weight was not significantly different between the groups, whether expressed as 252 absolute organ weight or normalized to body weight (Table 2) (48.5-8.6kB, p<0.05) telomeres in the somatic ovarian tissue of gestational hypoxia-exposed 261 animals compared to the normoxic group ( Figure 2). Conversely, there was a higher 262 proportion of very short telomeres in the hypoxia exposed group animals (1.1-4.2kB, 263 p<0.05), strongly suggesting that telomere length maintenance is impaired in the somatic 264 ovary following the developmental challenge of hypoxia in utero. 265 266 One possible mechanism of accelerated telomere shortening is impaired recognition of DNA 267 damage. Accordingly, we measured the gene expression levels of a range of DNA-damage 268 sensing and repair proteins in the hypoxia-exposed animals. Expression of Ogg1 (p<0.05) 269 and Neil1 (p<0.05) was elevated in the hypoxia-exposed group compared to the normoxic 270 group (Figure 3), which is in keeping with an increased burden of DNA damage in the 271 hypoxia-exposed group. There was no difference in gene expression of either Nthl1 or Xrcc1 272 in either group (Figure 3). At the protein level, NTH1 was increased in the hypoxia-exposed 273 group compared to the controls (p<0.01), but there was no difference in the protein level of 274 OGG1 (Table 3). 275 276 Gene expression levels of the key functional subunit components of DNA-PK, Ku70 and 277 Ku80, primarily responsible for repairing double-stranded DNA breaks and hence playing a 278 role in maintaining telomere length, did not vary significantly between the hypoxia and 279 normoxia-exposed groups ( Figure 4A). However, at the protein level, there was a highly 280 significant reduction in KU70 in the animals exposed to gestational hypoxia (p<0.001), with no difference between groups in KU80 levels ( Figure 4B). Inability to repair double-stranded 282 DNA breaks, despite adequate detection, is consistent with the accelerated telomere 283 shortening observed in the somatic ovarian tissue of the group exposed to chronic gestational 284 hypoxia. There were no differences between the hypoxic and normoxic groups in the gene 285 expression of any other DNA damage-sensing or protection mechanisms that were assayed 286 (Pot1, Chk1, Chk2, Cdk4, Cdk6; Table 4). 287 288 Gene expression levels of p53 were significantly higher in the somatic ovarian tissue of 289 gestational hypoxia-exposed animals, than in normoxic controls (p<0.01; Table 4). There 290 were trends towards a similar increase in levels of p21 and p16ink, but these were not 291 significant after correction for multiple hypothesis testing (Table 4). At the protein level there 292 was a significant increase in both P53 (p<0.001) and P16 ink (p<0.05) in the hypoxia-exposed 293 group compared to the normoxia-exposed group ( Figure 5). 294 295 There was a significantly higher gene expression of Hif1α in the somatic ovary following 296 exposure to chronic gestational hypoxia than in the normoxic control group (p<0.05, Table  297 4), however there was no difference in expression levels of Nfkβ (Table 4). Various oxidative 298 stress markers were included in the initial gene expression screen (Table 3). There was a 299 specific up-regulation of oxidative stress markers Gp91 phox (p<0.05) and P22 phox (p<0.05) in 300 the hypoxia-exposed group at the gene expression level, but other markers (Xo,P47 phox ,301 P67 phox ,Nrf2,Hmox1,Gpx1) were not significantly different between experimental groups 302 (Table 4). At the protein level, there was an increase in GP91 phox expression in the hypoxia-303 exposed group but this did not reach statistical significance (p=0.08; Table 4). There was no 304 difference between groups at the protein level in expression of XO, P67 phox , or HIF1α (Table  305 3). 306

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In keeping with increased levels of oxidative stress in the somatic ovary in the hypoxia-308 exposed group, there was also a significantly higher gene expression of the cytoplasmic anti-309 oxidant CuZnsod (p<0.001, Table 4). There was no difference in the gene expression levels 310 of several other anti-oxidants included in the initial screen (Mnsod, Ecsod, Catalase; Table 4) 311 between the gestational hypoxia and normoxia-exposed groups. There were no differences in 312 anti-oxidant protein expression between the hypoxia-and normoxia-exposed groups, except 313 for Catalase, which was decreased in the hypoxia exposed group (p<0.05; Table 3). Gene 314 expression levels of markers of lipid peroxidation included in the initial screen (Alox12, Alox15; Table 4) were unchanged between the gestational hypoxia and normoxia-exposed We show that chronic gestational hypoxia leads to decreased ovarian reserve in female 338 offspring in early adulthood. Ovarian reserve is a key determinant of female fecundity and 339 hence a reduction in the number of primordial follicles available in early adulthood is highly 340 likely to be associated with an early decline in fertility (35). Fecundity in later life relates to 341 both oocyte quality and quantity, however there is, as yet, no well-established method of 342 reliably predicting oocyte quality (36), hence reliance on occyte quantity. Our results suggest 343 that accelerated ageing in the somatic ovary in response to early-life hypoxia may be the 344 result of a post-transcriptional reduction in expression of a component of the DNA-PK 345 complex, which in turn prevents telomere maintenance and leaves ovarian follicular cells 346 vulnerable to accumulating age-associated damage. It is thus highly plausible that accelerated 347 reproductive ageing is a key mechanism by which ovarian reserve in the next generation 348 female offspring is reduced following exposure to chronic gestational hypoxia.   (35,52) suggest that ovarian reserve 384 reflects age at menopause, which is the best available proxy in women for the point at which 385 unassisted conception becomes highly unlikely. Hence, our finding may translate into an 386 important functional deficit in fertility, particularly in the older mother, following exposure to 387 a suboptimal intrauterine environment. This is particular relevant in many populations where 388 age at first pregnancy is progressively increasing. A key advantage of the model used in this 389 study (13% oxygen) is that it closely reflects the oxygenation during human pregnancy at 390 altitude. At altitudes of 3000-3500m above sea level, maternal arterial oxygen tension can fall 391 to around 60% of the value at sea level (95mmHg at sea level v. 50mmHg (25)). The severity 392 of the hypoxia used in our study is approximately equivalent to women experiencing 393 pregnancy in the city of La Paz in Bolivia (3600m -4150m), where ~40,000 women give 394 birth annually (53). When considering high altitude populations, it is important to consider 395 population mobility and thus the impact not only of prenatal hypoxia, but also the postnatal 396 environment on ovarian reserve into adulthood. This is an important area for future study. 397 398 As immediate survival of high-risk pregnancies improves (54, 55), it becomes increasingly 399 important to understand the multitude of ways in which the health of survivors of adverse 400 intrauterine environments may be affected in the longer term (56-58). Our study provides 401 important novel evidence that fertility issues may also be among these programmed 402 complications. Advances in assisted reproductive technologies mean that fertility problems 403 are now often amenable to treatment, but this is much more likely to be successful if high-404 risk groups can be identified early in reproductive life (59). The finding that chronic fetal 405 hypoxia results in decreased ovarian reserve in adulthood is therefore an important 406 conceptual advance in understanding which future potential mothers are at high risk of 407 experiencing fertility problems in later life. Moreover, our results provide mechanistic insight 408 into how hypoxia-induced low ovarian reserve is associated with a specific defect in DNA 409 repair and telomere maintenance in the somatic ovarian tissue. Insight into such molecular 410 pathways is the first step towards developing effective interventions to protect ovarian 411 reserve in the female offspring of high-risk pregnancy.