Skip to main content
NCBI home page
Reproduction & Fertility logoLink to Reproduction & Fertility
. 2026 Sep 21;7(3):RAF250192. doi: 10.1530/RAF-25-0192

Ovarian puncture alters genes but not follicle growth in cyclophosphamide mice model

Camila Stefane Ferreira 1, Luiza Aparecida Ansaloni Chagas Pereira 1, Stella Pollyanne de Oliveira 1, João Pedro Lobão Gomes de Castro 1, Vanessa Neves Guimarães 1, Patrick de Abreu Barbosa 1, Jade Carceroni de Sousa Carvalho 2, Ana Maria Alvarenga Fagundes 1, Antonio Sergio Varela Junior 3, Carine Dahl Corcini 4, Erika Cristina Jorge 2, Paulo Henrique Almeida Campos-Junior 1,✉
PMCID: PMC13615863  PMID: 42677808

Abstract

Graphical Abstract

graphic file with name RAF-25-0192inf1.jpg

Abstract

Cyclophosphamide administration often causes infertility. Ovarian puncture (OP), a component of in vitro fertilization, is performed to obtain oocytes. Our previous study showed that OP stimulated follicular activation in mice. However, its effects on folliculogenesis in cyclophosphamide-treated animals remain unclear. This study aimed to evaluate the impact of OP on follicular growth and gene expression. Female C57BL/06J mice (n = 52) were divided into three groups (control, cyclo, and cyclo + Pc). Cyclophosphamide was administered in four doses to the cyclo and cyclo + Pc groups (150 mg/kg), and OP was performed three days after the last dose and 96 h before euthanasia in the cyclo + Pc group. There was no difference in body weight; however, ovarian weight was higher in cyclo + Pc compared with cyclo. The number of all follicle classes was similar between cyclo + Pc and cyclo. Caspase-3-positive granulosa cells (%) in antral follicles were increased in cyclo compared with control and reduced in cyclo + Pc compared with control. Amh expression was increased in the cyclo + Pc group compared with the cyclo group. Pten expression was reduced in cyclo + Pc, whereas Kitl expression was increased. Bax expression was reduced, and there was no change in Casp3 expression due to OP. Moreover, collagen deposition was increased in cyclo + Pc compared with the control and cyclo groups. However, the treatments did not change the ovulation response, oocytes, or follicular morphometrics. Our experimental study demonstrates, for the first time, that OP is a safe procedure that can be performed during cyclophosphamide treatment in mice.

Lay summary

Infertility is a concern for cancer patients who wish to conceive after completing chemotherapy treatment. Puncturing the ovary is part of the IVF procedure, aimed at collecting eggs to fertilize and transferring them to the uterus. We decided to study whether the inflammation and tissue damage caused by puncturing the ovaries could stimulate the collection of healthy eggs in patients who have gone through chemotherapy. To this end, we used female mice that were treated with a frequently used treatment for breast cancer, along with ovarian puncture. We discovered the alterations did not affect follicle growth or egg retrieval. With these findings, we demonstrated, for the first time, the ability of ovarian puncture to bring about healthy egg retrieval, improving quality of life after cancer.

Keywords: fertility, chemotherapy, folliculogenesis, cancer, ovarian puncture

Introduction

Ovarian follicles are the functional units of the ovary, comprising an oocyte surrounded by granulosa cells. These somatic cells proliferate and interact closely with the oocyte, supporting its growth and maturation until ovulation (Zhou et al. 2019). Ovarian follicle activation depends on the proper regulation of early folliculogenesis, a topic that remains underexplored in the literature (Chen et al. 2021). Throughout life, however, follicles are progressively depleted through atresia (Ingole et al. 2025), and disruptions in the balance between follicular activation and atresia may compromise female fertility.

This delicate balance is particularly vulnerable to external factors. Chemotherapy and radiotherapy are well-known causes of ovarian damage and may lead to infertility (McClam & Xiao 2022). Among the available chemotherapeutic agents, cyclophosphamide, widely used in the treatment of ovarian and breast cancer, is especially gonadotoxic. One of its main side effects is the premature activation of primordial follicles, which ultimately leads to depletion of the ovarian reserve. In addition, cyclophosphamide treatment has been associated with increased ovarian fibrosis, oxidative stress, and damage to stromal and vascular compartments (Meirow et al. 2010, McClam & Xiao 2022).

To preserve fertility in cancer patients, several strategies have been developed, including ovarian transposition and the cryopreservation of oocytes, embryos, or ovarian tissue (Lee et al. 2021). Nevertheless, although there have been advances in fertility preservation, premature ovarian failure (POF) remains a well-established consequence of chemotherapy (Mauri et al. 2020). In this context, ovarian puncture, a key step in in vitro fertilization, represents an additional approach that may influence follicular growth. This procedure involves the ultrasound-guided aspiration of oocytes from antral follicles and is routinely performed (Wang et al. 2019). Experimental evidence further suggests that ovarian puncture may directly affect folliculogenesis. For instance, Olesen et al. (2023) demonstrated that repeated ovarian puncture increased follicular count and reduced follicular atresia. In line with these findings, our group recently established a murine model of ovarian puncture (Pereira et al. 2024), in which the procedure induced local inflammation and altered follicular growth and atresia. Taken together, these findings indicate that folliculogenesis is sensitive to mechanical and immunological disturbances induced by ovarian puncture, highlighting its potential as an experimental tool for modulating and investigating follicular activation, growth, and atresia.

In this scenario, we hypothesize that ovarian puncture modulates follicular growth in cyclophosphamide-treated ovaries. Therefore, this study aimed to evaluate the effect of ovarian puncture in cyclophosphamide-treated animals on follicular growth, ovulation rate, and the expression of genes related to folliculogenesis and atresia. These data should help enhance our understanding of the fundamental aspects of ovarian function in oncological patients undergoing oocyte collection.

Materials and methods

Animals

Female C57BL/06J mice (four weeks old – pubertal; n = 52) provided by the Animal Facility of the Federal University of São João del-Rei were divided into three experimental groups (control, cyclo, and cyclo + Pc) with n = 17, 18, and 17, respectively, and were maintained under a 12 h light:12 h darkness cycle at a controlled temperature (22–24°C), receiving water and food ad libitum. The cyclophosphamide treatment started in puberty (Nelson et al. 1982, Caligioni 2009, Brust et al. 2015) because the experimental period was quite long (4 weeks). The procedures were approved by the Animal Use Ethics Committee of the Federal University of São João del Rei (CEUA No. 9348070622), and the experimental design is shown in Fig. 1A.

Figure 1.

Figure 1

Open in a new tab

Experimental design and biometric parameters of the animals. (A) Experimental design related to the chemotherapy treatment and ovarian puncture assay. The cyclo and cyclo + Pc groups were treated once a week with cyclophosphamide, while the control group received PBS. During the 4-week administration period, the body weight was evaluated. Ovarian puncture was performed 96 h before euthanasia, at the end of the treatment. The ovaries were used for histology, immunohistochemistry, and qPCR analyses. PND23, postnatal day 23; PND47, postnatal day 47; PND51, postnatal day 51. (B and C) Cyclophosphamide and ovarian puncture did not alter biometric parameters: body weight was similar in the control, cyclo, and puncture groups at the beginning and end of the treatment (P > 0.05). (D) Ovarian weight increased in the cyclo + Pc group compared with the cyclo group (P = 0.0271). (E) The gonadosomatic index was not altered by the treatments (P > 0.05).

Body, ovarian weight, and gonadosomatic index

Throughout the period the animals were maintained, their body weight (g) was assessed twice a week. Absolute ovarian weight (mg) was obtained after euthanasia (Shimadzu Philippines Manufacturing Inc., Philippines). Gonadosomatic index (relative ovarian weight) was calculated as gonad weight divided by body weight, multiplied by 100 (Zheng et al. 2024).

Induction of premature ovarian failure

Animals from cyclo and cyclo + Pc were treated intraperitoneally with cyclophosphamide (Sigma-Aldrich Brasil Ltda – PHR1404; 150 mg/kg) diluted in phosphate-buffered saline (monobasic sodium phosphate/Cinética-Itapevi SP, sodium phosphate dibasic/Êxodo Científica-Sumaré SP, and sodium chloride/Êxodo Científica-Sumaré SP) according to one of the protocols described by Luan et al. (2019). The control group received only the vehicle (filtered PBS). To mimic chronic chemotherapy, cyclophosphamide was administered once weekly for 4 weeks (Luan et al. 2019), always on Mondays at 9 am. So, when the treatment started, the animals were 4 weeks old. On the second dose, they were 5 weeks old; on the third, 6 weeks old; and in the last one, 7 weeks old.

Ovarian puncture surgery

An experimental model standardized by our research group (Pereira et al. 2024) was used to mimic ovarian puncture. Briefly, the animals from the cyclo + Pc group, after four doses of cyclophosphamide and 96 h before euthanasia, at 7 weeks of age, were anesthetized by inhalation with isoflurane, trichotomized, and aseptically prepared with 10% povidone-iodine and 70% ethanol. The animals from the cyclo and control groups did not undergo any surgical procedure. They were positioned in right and left lateral decubitus to identify the incision site in the flank region. Both ovaries were exposed, punctured 10 times with an acupuncture needle (0.25 mm gauge – ClassicPlus Medisave UK Ltd), and immediately repositioned to their original anatomical regions. The procedure was conducted 96 h before euthanasia, corresponding to an entire estrous cycle (4 days). This time point (96 h) was chosen based on our previous study (Pereira et al. 2024), which reported pronounced follicle activation at that time.

Histological processing

After euthanasia of the animals by an overdose of the combination of ketamine and xylazine, as described by Campos-Junior et al. (2011), the ovaries were collected and fixed in 4% paraformaldehyde, dehydrated in increasing concentrations of alcohol (ACS Científica, Brazil), immersed in xylene (X09709RA – Êxodo Científica, Brazil), embedded in Paraplast (Sigma-Aldrich, San Louis, Missouri, USA) using an automated vacuum tissue processor (Leica, Germany, ASP 200), and sectioned using a microtome (Leica, Germany, RM2255 RTS – 5 μm). For follicle quantification, the whole ovary was sectioned, and every fifth section was stained with hematoxylin-eosin (Sigma-Aldrich, San Louis, Missouri, USA). The slides were mounted with enthelan (Sigma-Aldrich 1.07961) and coverslips. Subsequently, they were analyzed under an optical microscope at 100× and 400× magnification to image and analyze the ovaries (Olympus CX23 microscope, quad HD resolution, Japan).

Follicular quantification and morphometry

The follicular population was quantified using the analysis described by Pereira et al. (2024). Follicles were classified as primordial, transitional, primary, secondary, antral, and atretic. The follicular atresia rate was calculated as the number of atretic follicles divided by the total number of follicles, then multiplied by 100 (Campos-Junior et al. 2012).

To evaluate oocyte and follicle morphometrics, 20 healthy random follicles of each class (primordial, transitional, primary, secondary, and antral) from each group (control, cyclo, and cyclo + Pc) with an apparent nucleus were analyzed using an Olympus Soft Imaging Solutions GmbH system attached to an optical microscope (Olympus CX23 microscope, quad HD resolution, Japan) according to Nascimento et al. (2024). Two diameters of the oocytes and follicles were measured. The boundaries were defined as the basal membrane for the follicle and the zona pellucida for the oocyte. Two measurements of each structure were recorded to obtain the average diameter.

Collagen deposition

For collagen I and III analysis, ovaries from three animals in all groups (control, cyclo, and cyclo + Pc) were treated as previously mentioned and were evaluated by Picro-Sirius Red (PSR) (EP1120011A – EasyPath Diagnosis, Brazil). Middle sections of the treated ovaries were stained according to the manufacturer’s instructions. The PSR-stained area was evaluated using ImageJ software, as described by Mara et al. (2020), Umehara et al. (2022), and Duncan & Pritchard (2024). These data are expressed as the percentage of PSR area in pixels/μm2.

Immunohistochemistry

All procedures were conducted according to Nascimento et al. (2024). Eight sections from each female in all groups were used, following the same fixation, dehydration, inclusion, and sectioning protocol. After the standard hydration protocol, the slides were placed in tap water for 5 min. Then, the slides were incubated with citrate buffer (Cinética Itapevi – SP, Brazil) and placed in the microwave for 5 min after boiling for antigen retrieval. Sections were incubated in hydrogen peroxide (0.6%) to inhibit endogenous peroxidase activity. After washing, sections were incubated with 2.5% Normal Horse Serum solution (30022 – Vector Laboratories, USA) for 30 min.

Then, a primary antibody was added to the sections (anti-Caspase-3 1:1,000 – Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb IHC Formulation – Cell Signaling Technology, USA) and incubated overnight, while the negative control was incubated with the antibody dilution solution (10% horse serum). On the second day, after PBS washing, sections were incubated with the biotinylated secondary antibody (Vector Laboratories, 30082, USA). The sections were then incubated with VECTASTAIN Elite ABC (Vector Laboratories, 30083, USA) for 30 min. Finally, ImmPact DAB Eqn 1:1 (Vector Laboratories – 30147; 30149, USA) fresh solution was used for approximately 1 min and 20 s. Sections were washed in tap water for 5 min and counterstained with hematoxylin (Renylab Química e Farmaceutica Ltda, Brazil) for 3 s.

Subsequently, the slides were analyzed under an optical microscope (Olympus CX23, quadHD resolution, Japan), and images of the follicles were taken at 100× and 400× magnification. The sections were analyzed using ImageJ software, counting the Casp3-positive and -negative granulosa cells from primordial, transitional, primary, secondary, and antral follicles. Then, the percentage of Casp3-positive cells was calculated per follicle (active Casp3-positive cells/total number of granulosa cells).

Gene expression analysis

Five animals were used, and both ovaries from each animal were used. For gene expression, three biological replicates (pools) were performed to minimize individual variation, with three ovaries (Byers et al. 2012) per replicate. Then, to make the pools, the ovaries from different animals were mixed. Ten ovaries were available per treatment; however, nine ovaries were used, and the tenth was discarded.

For RNA isolation, the TriReagent kit (T9424, Sigma-Aldrich) was used according to the manufacturer’s protocol. The primer sequences are listed in Supplementary Table 1 (see section on Supplementary materials given at the end of the article) and were designed based on Mus musculus gene sequences. The target genes were chosen based on their importance to ovarian reserve (Amh), follicle activation, and folliculogenesis (Kitl, Pten), and also because Amh, Pten, and KitL have important roles in folliculogenesis and follicular activation/inhibition (Zhang et al. 2021, Lv et al. 2025). Apoptosis was also evaluated in the ovaries using Casp3 and Bax (Nascimento et al. 2023, Pereira et al. 2024) and the endogenous control gene beta-actin (Actb) (Nascimento et al. 2023). One microgram of RNA was converted into cDNA using RevertAid First Strand cDNA Synthesis Kit (containing Oligo (dT) 18 primer, water, nuclease-free, 5X Reaction Buffer, RiboLock RNase Inhibitor (20 U/μL), 10 mM dNTP Mix, RevertAid M-MuLV RT (200 U/μL), and Thermo Fisher Scientific Baltics UAB|V.A. Graiciuno 8, LT-02241 Vilnius, Lithuania).

Relative quantification was performed in triplicate. For RT-qPCR, 5 μL of iTAq™ Universal SYBR Green Supermix (Bio-Rad, USA), 0.4 μM of each primer, 1 μL of cDNA (1:10), and 3.2 μL of nuclease-free water were used in a final volume of 10 μL. The Corbett Rotor Gene 3000 Machine (Switzerland-Qiagen) was used with the following settings: 50°C for 2 min, 95°C for 2 min, followed by 40 cycles of 94°C for 15 s, 60°C for 30 s, and 72°C for 20 s, according to the manufacturer. After amplification, the melting temperature of each primer was measured by dissociation. REST 2009 software was used for relative expression. Ovaries from the control group were used to calibrate the transcriptions, and the results are expressed as n-fold differences relative to the calibrator. Nascimento et al. (2023), Nascimento et al. (2024), and Pereira et al. (2024) have already performed and described all these procedures.

Superovulation and oocyte retrieval

Female C57BL/06 mice (n = 5 for each group – control, cyclo, and cyclo + Pc), 4 weeks old, were used to analyze the effect of the treatment on the ovulatory response. Animals were treated as previously described. However, for the superovulation assay, animals followed a different timeline from that used for the other experimental analyses. Rather than being euthanized according to the main experimental timeline, animals from this assay were maintained alive to undergo hormonal stimulation, and euthanasia was performed only after the superovulation protocol. After the cyclophosphamide treatment and ovarian puncture surgery, at 8 weeks of age, superovulation was conducted using a standard protocol (Nascimento et al. 2023). One week after the last dose of cyclophosphamide, 20 IU of equine chorionic gonadotropin (eCG) (Novormon, Zoetis, Argentina) was administered intraperitoneally; 48 h later, 20 IU of human chorionic gonadotropin (hCG) (Fertcor, Ceva, Brazil) was administered to induce ovulation. After 14 h of the hCG pulse, and consequently 230 h after the last cyclophosphamide pulse, animals were euthanized, and all ovaries and oviducts were collected and kept in a Petri dish with human serum albumin (HSA) solution (Life Global – GHSA-005, 5 mL, Europe). The experimental design is shown in Fig. 6A.

Figure 6.

Figure 6

Open in a new tab

(A) Experimental design of the superovulation assay. Animals were maintained beyond the experimental endpoint used for the other analyses to undergo hormonal stimulation. One week after the last cyclophosphamide administration, 20 IU of equine chorionic gonadotropin (eCG) was administered intraperitoneally to stimulate follicular growth. After 48 h, 20 IU of human chorionic gonadotropin (hCG) was administered to induce ovulation. After 14 h of hCG administration, the animals were euthanized, and the ovaries, oviducts, and oocytes were collected. (B, C, D, E, F) There was no difference in the numbers of viable, total, and degenerated oocytes, corpora lutea, and corpora hemorrhagica between the groups (P > 0.05).

The oocytes were collected by holding the ovary with a clamp and rupturing the oviduct ampulla with a syringe (28-gauge needle), then they were transferred to a drop of hyaluronidase (INGASE, 80 IU/mL – Ingámed, Brazil) and kept at 37°C for 8 min for denudation. The denuded oocytes were evaluated using a Leica S6E stereomicroscope (Leica Microsystems, Germany). Oocytes with homogeneous, translucent cytoplasm were considered viable, and those with cytoplasmic granules were considered nonviable (Mara et al. 2020).

Statistical analysis

Data were analyzed using GraphPad Software, Inc. 8.0.2 (USA) and are presented as mean ± standard error of the mean. The means obtained were analyzed for normality (D’Agostino & Pearson and Shapiro–Wilk tests). Parametric data (body weight; ovarian weight; gonadosomatic index; number of primordial, transitional, primary, secondary, and antral follicles; caspase-3-positive cells in antral follicles; total number of oocytes, number of corpus luteum, and number of corpora hemorrhagica; oocyte diameter of primordial, transitional, primary, secondary, and antral follicles; follicular diameter of primordial, transitional, primary, and secondary; and oocyte–follicular ratio of primordial, primary, and secondary follicles) were tested by analysis of variance, followed by the Newman–Keuls test.

Nonparametric data (number of healthy follicles; quantification of caspase-3-positive cells in primordial, transitional, primary, and secondary follicles; follicular atresia rate; number of viable oocytes; number of degenerated oocytes; follicular diameter of antral follicles; and oocyte–follicular ratio of transitional and antral follicles) were analyzed using the Kruskal–Wallis and Dunn’s tests. The significance level used was P < 0.05.

Results

Ovarian puncture increased ovarian weight in cyclophosphamide-treated animals

Ovarian puncture significantly increased absolute ovarian weight in the cyclo + Pc group compared with the cyclo group (P = 0.0271) (0.98 ± 0.12 control, 0.75 ± 0.09 cyclo, and 1.22 ± 0.13 cyclo + Pc) (Fig. 1D). Ovarian puncture and cyclophosphamide treatment did not alter body weight at the beginning (first day of treatment – PND23) (16.0 ± 2.0 control, 13.33 ± 0.33 cyclo, and 15.57 ± 0.29 cyclo + Pc) (Fig. 1B) and end (last day of treatment – PND51) (18.00 ± 2.00 control, 16.33 ± 0.33 cyclo, and 15.71 ± 0.47 cyclo + Pc) (Fig. 1C) (P > 0.05) of the experimental period. There were no significant differences in the gonadosomatic index (relative ovarian weight) (P > 0.05) among the groups (0.01 ± 0.00 control; 0.01 ± 0.00 cyclo; and 0.01 ± 0.00 cyclo + Pc) (Fig. 1E).

Ovarian puncture did not alter the number of healthy follicles in cyclophosphamide-treated animals

As expected, there was a reduction in the total number of healthy follicles in the ovaries of the cyclo group compared with the control group (Fig. 2A, B, C, D, E, F). In cyclo + Pc, the numbers of primordial, primary, and antral follicles were lower than in control. However, the total number of healthy follicles across all classes was not altered in cyclo + Pc compared with cyclo (P > 0.05). The middle sections of the ovaries qualitatively confirmed these findings (Fig. 2G). In addition, the oocyte and follicular diameter did not differ between the groups (P > 0.05) (Supplementary Fig. 1A and B).

Figure 2.

Figure 2

Open in a new tab

Follicular population in the ovaries of mice. (A) The number of primordial follicles was reduced in the cyclo and cyclo + Pc groups compared with the control group (P = 0.0001; P < 0.0001). (B) The number of transitional follicles was reduced in the cyclo and cyclo + Pc groups compared with the control group (P = 0.0041; P = 0.0024). (C) The number of primary follicles was reduced in the cyclo and cyclo + Pc groups compared with the control group (P < 0.0001; P = 0.0127). (D) The number of secondary follicles was reduced in the cyclo group compared with the control group (P = 0.0007). (E) The number of antral follicles was reduced in the cyclo and cyclo + Pc groups compared with the control group (P = 0.0175; P = 0.0316). (F) There was a reduction in the total number of healthy follicles in the cyclo group compared with the control group (P = 0.0018), but no difference between the cyclo + Pc and control groups (P > 0.05). (G) Representative histological sections. The red arrows in the control group indicate follicles in different classes. In the cyclo group, the arrows indicate only atretic follicles. In the cyclo + Pc group, the arrows indicate follicles in different stages of folliculogenesis. Scale bars: 100 μm and 200 μm.

Collagen deposition was increased in punctured ovaries

Ovarian puncture increased collagen deposition in the ovaries of cyclo + Pc animals compared with the control (P < 0.0001) and to the cyclo (P = 0.0006) (Fig. 3) (2.97 ± 1.34 control; 1.98 ± 0.59 cyclo; and 78.35 ± 3.53 cyclo + Pc). These findings can be demonstrated by the histological sections of ovaries stained with PSR (Fig. 3B, C, D).

Figure 3.

Figure 3

Open in a new tab

Collagen I and III deposition was analyzed through the positive percentage area of PSR. (A) Percentage of PSR staining in control, cyclo, and cyclo + Pc groups. (B) Representative picture of PSR staining in control ovaries. (C) Representative picture of PSR staining in cyclo ovaries. (D) Representative picture of PSR staining in cyclo + Pc ovaries.

Ovarian puncture alleviated the follicular atresia rate and altered the percentage of caspase-3-positive granulosa cells in antral follicles

There was no difference between the cyclo + Pc and control groups (P > 0.05). This percentage was higher in the cyclo group (P = 0.0289) than in the control group (Fig. 4A). In addition, regarding the quantification of caspase-3-positive cells per follicle class (Fig. 4B, C, D, E, F), ovarian puncture reduced the percentage of caspase-3-positive cells in the antral follicles (Fig. 4F) of the punctured ovaries compared with the cyclo group (P < 0.0001) (22.64 ± 4.14 control, 50.82 ± 5.92 cyclo, 21.61 ± 2.52 cyclo + Pc). These findings can be qualitatively demonstrated in Supplementary Fig. 2.

Figure 4.

Figure 4

Open in a new tab

Quantitative evaluation of follicular atresia and caspase-3-positive granulosa cells. (A) The follicular atresia rate was altered only in the cyclo group compared with the control group (P = 0.0289). (B, C, D) There was no significant difference between the control, cyclo, and cyclo + Pc groups (P > 0.05). (E) The number of caspase-3-positive granulosa cells was reduced in the cyclo + Pc group compared with the control group (P = 0.0160). (F) There was a reduction in the number of caspase-3-positive cells in the cyclo and cyclo + Pc groups compared with the control group (P < 0.0001; P = 0.0002).

Ovarian puncture altered the expression of genes related to ovarian reserve, follicular growth, and atresia

Amh expression in the cyclo + Pc group was significantly higher than in the cyclo group (P = 0.005) (Fig. 5A). Pten expression was significantly downregulated in cyclo + Pc ovaries compared with the control (P < 0.0001) (Fig. 5B). In addition, Kitl expression was increased in the cyclo + Pc group compared with the control group (P = 0.001), but did not differ significantly from the cyclo group (P > 0.05) (Fig. 5C).

Figure 5.

Figure 5

Open in a new tab

Ovarian puncture and cyclophosphamide treatment altered gene expression. (A) Amh was downregulated in the cyclo and cyclo + Pc groups compared with the control group, but it was upregulated in the cyclo + Pc group compared with the cyclo group. (B) Pten was downregulated in the cyclo + Pc group compared with the control group. (C) Kitl was upregulated in the cyclo and cyclo + Pc groups compared with the control group. (D) Bax was downregulated in the cyclo and cyclo + Pc groups compared with the control group. (E) Casp3 was downregulated in the cyclo group compared with the control group.

For Bax, expression in the cyclo + Pc group did not differ significantly from that in the cyclo group (P > 0.05), but both groups showed lower expression than the control group (cyclo: P < 0.0001; cyclo + Pc: P = 0.018) (Fig. 5D). Finally, Casp3 expression in the cyclo + Pc group did not differ from either the cyclo group or the control group (P > 0.05), whereas the cyclo group showed reduced expression compared with the control group (P = 0.043) (Fig. 5E).

The number of ovulated oocytes was not affected by ovarian puncture in cyclophosphamide-treated animals

The number of viable ovulated oocytes after superstimulation was not affected by ovarian puncture in cyclophosphamide-treated or control animals (P > 0.05; 9.00 ± 3.85 control, 3.36 ± 1.95 cyclo, 4.27 ± 2.17 cyclo + Pc) (Fig. 6B). The total number of oocytes recovered (P > 0.05–10.00 ± 4.21 control, 3.90 ± 2.33 cyclo, 4.81 ± 2.43 cyclo + Pc) (Fig. 6C), degenerated oocytes (P > 0.05–1.00 ± 0.55 control, 0.54 ± 0.39 cyclo, 0.54 ± 0.28 cyclo + Pc) (Fig. 6D), hemorrhagic corpora (P > 0.05–5.00 ± 1.27 control, 5.09 ± 1.52 cyclo, 6.63 ± 1.10 cyclo + Pc) (Fig. 6E), and corpus luteum (P > 0.05–6.2 ± 0.77 control, 5.00 ± 1.13 cyclo, 4.72 ± 0.97 cyclo + Pc) (Fig. 6F) were not affected by the treatment.

Discussion

In a previous study from our group, we demonstrated that ovarian puncture promotes inflammation, follicular activation, and atresia in healthy ovaries (Pereira et al. 2024). We established a model of cyclophosphamide treatment followed by ovarian puncture in mice; this model could help us evaluate the safety of ovarian puncture at the morphological and molecular levels. In the present study, we showed that ovarian puncture does not affect biometric parameters in cyclophosphamide-treated females; however, it alters the expression of some key genes, affects collagen deposition, and, most importantly, maintains follicular survival.

The use of chemotherapeutics can significantly reduce the body weight of animals. In a study involving cyclophosphamide, the drug reduced body and ovarian weights in animals in a dose-dependent manner (Salian et al. 2024). We did not observe significant differences in body weight and the gonadosomatic index (relative ovarian weight), findings that demonstrate post-surgery recovery and indicate the safety of our experimental procedure. However, absolute ovarian weight was significantly higher in cyclo + Pc than in cyclo, likely reflecting an increased number of follicles in the late stages of folliculogenesis (Shareghi-Oskoue et al. 2021), as evidenced by our morphological and gene expression data. Because chemotherapy treatment can reduce the weight and volume of the treated ovaries (Yoshida et al. 2022, Jiang et al. 2023, Shuyuan et al. 2023) and ovarian puncture may have promoted an increase in vascular perfusion and follicular growth and survival (Pereira et al. 2024), it could explain the increase in ovarian weight. Although Song et al. (2024) demonstrated that 100–200 mg/kg cyclophosphamide can cause mortality, none of the animals in our study died, indicating the safety and reproducibility of our experimental procedure.

Folliculogenesis typically requires approximately 2–3 weeks in mice (Chen et al. 2021). We punctured ovaries 96 h before euthanasia, so we hypothesized that only follicles that have already been activated benefited from ovarian puncture. The reduced percentage of caspase-3-positive granulosa cells in antral follicles and the upregulation of Amh in the cyclo + Pc group support these data. Possibly, ovarian puncture ameliorated the microenvironment by promoting extracellular matrix remodeling and vascularization, thereby improving follicular survival, as previously reported (Henry et al. 2015, Francés-Herrero et al. 2024, Li et al. 2025). Therefore, this study offers new perspectives for further research into other aspects of physical alterations in punctured ovaries.

In this study, we observed that cyclophosphamide treatment and ovarian puncture increased collagen deposition in the ovaries. Fibrosis is a common process in aged ovaries and a common side effect of chemotherapy treatments (Gao et al. 2023, Nie et al. 2025), and it is associated with decreased ovulation (Umehara et al. 2022). Pereira et al. (2024) reported an increased presence of inflammatory cells in the healthy ovaries of mice following ovarian puncture, and was associated with enhanced follicular activation. In line with these findings, our results demonstrate that this response may persist even in the context of chemotherapy-induced damage. Wei et al. (2026) reported that administering recombinant type III collagen to an aged ovary increased granulosa cell proliferation and reduced mitochondrial dysfunction. In another study, melatonin-treated ovaries showed increased collagen I deposition, which was associated with improved fertility parameters, including cellular proliferation and follicular quantification, and reduced apoptosis (Shiroma et al. 2021). This stromal remodeling could contribute to changes in the ovarian microenvironment, potentially influencing follicular activation through mechanical and biochemical cues. Previous studies have also demonstrated that exosomes derived from mesenchymal stem cells attenuate cyclophosphamide damage by reducing collagen deposition (Park et al. 2024).

It is also well established that the aging ovary undergoes repeated cycles of ovulation throughout reproductive life, which causes accumulation of injured tissue and creates an inflammatory microenvironment, characterized by high levels of cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), and inflammatory cells such as neutrophils, macrophages, and T lymphocytes which are also a trigger for collagen deposition and matrix stiffness (Balough et al. 2024, Edepli & Yaba 2026). Therefore, it is possible that ovarian puncture in this study caused a temporary extracellular matrix remodeling response driven by inflammation, which led to follicle activation, as we observed in our previous study mimicking ovarian puncture without cyclophosphamide treatment (Pereira et al. 2024), and this could explain why collagen deposition was higher in these ovaries. However, further studies are required to determine whether these changes represent a temporary reparative response or contribute to long-term alterations in ovarian biology.

The PI3K/AKT pathway can be modulated by chemotherapeutic agents such as cyclophosphamide or cisplatin (Spears et al. 2019), which reduce the expression of Pten, its negative regulator. Depletion of Pten activates the pathway and promotes follicular hyperactivation and atresia because these germ cells are more prone to apoptosis (Jagarlamudi et al. 2009), which is consistent with the higher levels of atresia seen in cyclophosphamide-treated animals in this study. In the present study, cyclophosphamide treatment and ovarian puncture led to Pten downregulation. This gene regulates cell proliferation, negatively regulates the PI3K/AKT pathway, inhibits follicular activation, and preserves ovarian reserve (Jagarlamudi et al. 2009). It dephosphorylates PIP3 into PIP2, which is a pivotal initiator of the pathway (Kawamura et al. 2016, Namlı Kalem et al. 2023). Pten knockout mice show dysregulation of primordial follicular activation (Jagarlamudi et al. 2009). Consistent with these findings, ex vivo culture with PTEN inhibitors promoted follicular activation in human ovarian tissue (Ghezelayagh et al. 2022). The low Pten levels observed in this study corroborate this evidence, so these follicles may have been activated by the PI3K/AKT pathway, potentially triggered by inflammation, extracellular matrix remodeling, and downregulation of Pten caused by ovarian puncture.

Follicular atresia can be influenced by several factors, including signaling pathways, proteins, and genetic components (Stringer et al. 2023). Ovarian injury caused by puncture alters the number of healthy follicles, likely due to tissue remodeling (Pereira et al. 2024), a phenomenon similar to that observed in other settings (Mara et al. 2020, Umehara et al. 2022). The reduced number of caspase-3-positive granulosa cells in antral follicles of animals subjected to ovarian puncture suggests that this procedure ameliorates cyclophosphamide-induced follicular atresia. These findings, along with the histological findings, increased ovarian weight, and gene expression profile, support the idea that ovarian puncture reduced cyclophosphamide-induced follicular damage and favored follicular survival. Pereira et al. (2024) reported an increase in the inflammatory response that did not affect female fertility. The mechanical damage caused by ovarian puncture possibly aided in the remodeling of the ovarian microenvironment and, in the POF context, alleviated some of the deleterious effects of chemotherapy.

CASPASE-3 and BAX are well-established markers of apoptosis (Almeida et al. 2021). The downregulation of these genes in the cyclo group could indicate that cyclophosphamide treatment induces additional mechanisms of cell death that are independent of Bax and Casp3, as previously reported in other contexts (Greenfeld et al. 2007, Kaur & Kurokawa 2023). Xi et al. (2025) reported that autophagy, ferroptosis, and pyroptosis can trigger ovarian follicular atresia. Berkel (2024) described pyroptosis as a reactive oxygen species (ROS)-induced mechanism of cell death in granulosa cells (Pimenta et al. 2024). Given that cyclophosphamide induces ROS, other cell death pathways are probably activated.

Previous studies have reported that chemotherapy adversely affects oocyte quality, even when fertility is preserved. Yang et al. (2021) described epigenetic changes in maternal germ cells exposed to cyclophosphamide. Although the toxic effects of chemotherapy were attenuated over time, the remaining follicles were of a lower quality. Specifically, cyclophosphamide impaired meiotic progression, reduced mitochondrial function, and affected embryonic development. Our findings suggest that the ovarian puncture procedure itself does not compromise oocyte morphological quality and support the safety of this experimental model and its applicability in other contexts. We did not perform other assays to analyze oocyte competence, but this study opens new perspectives for oocyte recovery from a cyclophosphamide context associated with ovarian puncture in a mouse model and its potential to generate developmentally competent oocytes.

In conclusion, we demonstrated experimentally that ovarian puncture in cyclophosphamide-treated ovaries induces gene dysregulation, reduces the percentage of apoptotic granulosa cells in antral follicles, and increases collagen deposition. Overall, this study opens new possibilities for investigating the modulation of follicular growth following follicular depletion, as well as the molecular mechanisms underlying follicular activation and atresia in injured ovaries.

Supplementary materials

Declaration of interest

The authors declare no known conflicts of interest associated with this publication and that no significant financial support for this work has influenced its outcome.

Funding

This work was supported by the National Council for Scientific and Technological Development, Brazil (406600, 2021), Minas Gerais State Research Foundation, Brazil (00638, 2022 and 29675, 2022), and Coordination for the Improvement of Higher Education Personnel, Brazil. CSF, LAACP, SPDO, JCDSC, and AMAF received scholarships from FAPEMIG, CNPq, or CAPES.

Author contribution statement

PHACJ designed and supervised the experiments and revised the manuscript. CSF performed the experiments, analyzed all data, and prepared the manuscript. LAACP and SPDO contributed to the experiments, analyzed the data, and prepared the manuscript. JPLGDC, VNG, and PDAB performed the superovulation assay. JCDSC, AMAF, and ECJ contributed to the gene expression analysis. ASVJ and CDC contributed to the histological processing and data interpretation.

Availability of data and material (data transparency)

The datasets generated and analyzed during the current study are available from the corresponding author (paulohenrique@ufsj.edu.br) upon reasonable request.

Ethics approval

All procedures were approved by the Ethics Committee in the Use of Animals of the Federal University of São João Del Rei (protocol #9348070622).

Acknowledgments

The authors gratefully acknowledge the Animal Facility (NUCAL) for providing the experimental model.

References

  1. Almeida JZ, Lima LF, Vieira LA, et al. 2021. 5-Fluorouracil disrupts ovarian preantral follicles in young C57BL6J mice. Cancer Chemother Pharmacol 87 567–578. ( 10.1007/s00280-020-04217-7) [DOI] [PubMed] [Google Scholar]
  2. Balough JL, Dipali SS, Velez K, et al. 2024. Hallmarks of female reproductive aging in physiologic aging mice. Nat Aging 4 1711–1730. ( 10.1038/s43587-024-00769-y) [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berkel C 2024. Inducers and inhibitors of pyroptotic death of granulosa cells in models of premature ovarian insufficiency and polycystic ovary syndrome. Reprod Sci 31 2972–2992. ( 10.1007/s43032-024-01643-3) [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brust V, Schindler PM & Lewejohann L. 2015. Lifetime development of behavioural phenotype in the house mouse (Mus musculus). Front Zool 12 (Supplement 1) S17. ( 10.1186/1742-9994-12-s1-s17) [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Byers SL, Wiles MV, Dunn SL, et al. 2012. Mouse estrous cycle identification tool and images. PLoS One 7 e35538. ( 10.1371/journal.pone.0035538) [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Caligioni CS 2009. Assessing reproductive status/stages in mice. Curr Protoc Neurosci 48 A4I.1–A.4I.8. ( 10.1002/0471142301.nsa04is48) [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Campos-Junior PH, Silva CA, Grazia JG, et al. 2011. Use of ultrasound biomicroscopy to evaluate induced ovarian follicular growth and ovulation in mice. Lab Anim 45 254–258. ( 10.1258/la.2011.011031) [DOI] [PubMed] [Google Scholar]
  8. Campos-Junior PH, Assuncao CM, Carvalho BC, et al. 2012. Follicular populations, recruitment and atresia in the ovaries of different strains of mice. Reprod Biol 12 41–55. ( 10.1016/s1642-431x(12)60076-x) [DOI] [PubMed] [Google Scholar]
  9. Chen Y, Liu Q, Liu R, et al. 2021. A prepubertal mice model to study the growth pattern of early ovarian follicles. Int J Mol Sci 22 5130. ( 10.3390/ijms22105130) [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duncan FE & Pritchard MT. 2024. Picrosirius Red (PSR) Staining and Quantification in Mouse Ovaries. Protocols.io. ( 10.17504/protocols.io.4r3l295o4v1y/v1) [DOI] [Google Scholar]
  11. Edepli BG & Yaba A. 2026. Molecular mechanisms of ovarian fibrosis. Mol Hum Reprod 32 gaaf058. ( 10.1093/molehr/gaaf058) [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Francés-Herrero E, Bueno-Fernandez C, Rodríguez-Eguren A, et al. 2024. Growth factor-loaded ovarian extracellular matrix hydrogels promote in vivo ovarian niche regeneration and enhance fertility in premature ovarian insufficiency preclinical models. Acta Biomater 186 125–140. ( 10.1016/j.actbio.2024.07.056) [DOI] [PubMed] [Google Scholar]
  13. Gao Y, Wu T, Tang X, et al. 2023. Increased cellular senescence in doxorubicin-induced murine ovarian injury: effect of senolytics. Geroscience 45 1775–1790. ( 10.1007/s11357-023-00728-2) [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ghezelayagh Z, Abtahi NS, Rezazadeh Valojerdi M, et al. 2022. The effect of mTOR activation and PTEN inhibition on human primordial follicle activation in ovarian tissue culture. J Assist Reprod Genet 39 1739–1747. ( 10.1007/s10815-022-02537-6) [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Greenfeld CR, Babus JK, Furth PA, et al. 2007. BAX is involved in regulating follicular growth, but is dispensable for follicle atresia in adult mouse ovaries. Reproduction 133 107–116. ( 10.1530/rep-06-0144) [DOI] [PubMed] [Google Scholar]
  16. Henry L, Labied S, Fransolet M, et al. 2015. Isoform 165 of vascular endothelial growth factor in collagen matrix improves ovine cryopreserved ovarian tissue revascularisation after xenotransplantation in mice. Reprod Biol Endocrinol 13 12. ( 10.1186/s12958-015-0015-2) [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ingole S, Khare K, Dwivedi V, et al. 2025. Decoding ovarian aging in women: cellular damage, signaling networks, and treatment frontiers. Reprod Biol 25 101075. ( 10.1016/j.repbio.2025.101075) [DOI] [PubMed] [Google Scholar]
  18. Jagarlamudi K, Liu L, Adhikari D, et al. 2009. Oocyte-specific deletion of Pten in mice reveals a stage-specific function of PTEN/PI3K signaling in oocytes in controlling follicular activation. PLoS One 4 e6186. ( 10.1371/journal.pone.0006186) [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jiang H, Dong R, Kim SY, et al. 2023. The gonadotoxic effects of conditioning chemotherapy regimens prior to hematopoietic stem cell transplantation. Obstet Gynecol 141 22S. ( 10.1097/01.aog.0000929988.78942.3b) [DOI] [Google Scholar]
  20. Kaur S & Kurokawa M. 2023. Regulation of oocyte apoptosis: a view from gene knockout mice. Int J Mol Sci 24 1345. ( 10.3390/ijms24021345) [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kawamura K, Kawamura N & Hsueh AJ. 2016. Activation of dormant follicles: a new treatment for premature ovarian failure? Curr Opin Obstet Gynecol 28 217–222. ( 10.1097/gco.0000000000000268) [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lee S, Ozkavukcu S & Ku SY. 2021. Current and future perspectives for improving ovarian tissue cryopreservation and transplantation outcomes for cancer patients. Reprod Sci 28 1746–1758. ( 10.1007/s43032-021-00517-2) [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li L, Yang J, Jia L, et al. 2025. A dual-drug strategy to enhance the function of cryopreserved ovaries by promoting revascularization and inhibiting follicle over-activation. Reprod Biol Endocrinol 23 95. ( 10.1186/s12958-025-01422-y) [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Luan Y, Edmonds ME, Woodruff TK, et al. 2019. Inhibitors of apoptosis protect the ovarian reserve from cyclophosphamide. J Endocrinol 240 243–256. ( 10.1530/joe-18-0370) [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lv M, Feng A, Cheng D, et al. 2025. The effect of AMH on folliculogenesis. Reprod Sci 32 3848–3860. ( 10.1007/s43032-025-01879-7) [DOI] [PubMed] [Google Scholar]
  26. Mara JN, Zhou LT, Larmore M, et al. 2020. Ovulation and ovarian wound healing are impaired with advanced reproductive age. Aging 12 9686–9713. ( 10.18632/aging.103237) [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mauri D, Gazouli I, Zarkavelis G, et al. 2020. Chemotherapy associated ovarian failure. Front Endocrinol 11 572388. ( 10.3389/fendo.2020.572388) [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McClam M & Xiao S. 2022. Preserving oocytes in oncofertility†. Biol Reprod 106 328–337. ( 10.1093/biolre/ioac008) [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meirow D, Biederman H, Anderson RA, et al. 2010. Toxicity of chemotherapy and radiation on female reproduction. Clin Obstet Gynecol 53 727–739. ( 10.1097/grf.0b013e3181f96b54) [DOI] [PubMed] [Google Scholar]
  30. Namlı Kalem M, Anadol E, Kalem Z, et al. 2023. A rat study on the PTEN expression in ovarian tissue in PCOS and folliculogenesis. Sci Rep 13 20774. ( 10.1038/s41598-023-47809-y) [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nascimento BR, de Freitas DS, Nogueira JM, et al. 2023. Drastic loss of antral follicles due to gene expression dysregulation occurs on the first day after subcutaneous ovarian transplantation. Reprod Sci 30 2524–2536. ( 10.1007/s43032-023-01184-1) [DOI] [PubMed] [Google Scholar]
  32. Nascimento BC, Ferreira CS, Oliveira SP, et al. 2024. Naproxen administration affects murine late folliculogenesis, reduces granulosa cell proliferation and the number of ovulated oocytes. Reprod Toxicol 124 108527. ( 10.1016/j.reprotox.2023.108527) [DOI] [PubMed] [Google Scholar]
  33. Nelson JF, Felicio LS, Randall PK, et al. 1982. A longitudinal study of estrous cyclicity in aging C57BL/6J mice: I. Cycle frequency, length and vaginal cytology. Biol Reprod 27 327–339. ( 10.1095/biolreprod27.2.327) [DOI] [PubMed] [Google Scholar]
  34. Nie P, Yao B, Zhang Z, et al. 2025. Metformin protects against cyclophosphamide-induced ovarian fibrosis by MIF/CD74-mediated macrophage polarization. J Transl Med 23 1273. ( 10.1186/s12967-025-07294-5) [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Olesen HØ, Pors SE, Adrados CS, et al. 2023. Effects of needle puncturing on re-vascularization and follicle survival in xenotransplanted human ovarian tissue. Reprod Biol Endocrinol 21 28. ( 10.1186/s12958-023-01081-x) [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Park HS, Seok J, Cetin E, et al. 2024. Fertility protection: a novel approach using pretreatment with mesenchymal stem cell exosomes to prevent chemotherapy-induced ovarian damage in a mouse model. Am J Obstet Gynecol 231 111.e1–111.e18. ( 10.1016/j.ajog.2024.02.023) [DOI] [PubMed] [Google Scholar]
  37. Pereira LAAC, Ferreira CS, Dias KSSA, et al. 2024. Ovarian puncture triggers an inflammatory response that did not affect late folliculogenesis, ovulation rate, and fertility. Reprod Sci 31 3202–3214. ( 10.1007/s43032-024-01654-0) [DOI] [PubMed] [Google Scholar]
  38. Pimenta GF, Awata WMC, Orlandin GG, et al. 2024. Melatonin prevents overproduction of reactive oxygen species and vascular dysfunction induced by cyclophosphamide. Life Sci 338 122361. ( 10.1016/j.lfs.2023.122361) [DOI] [PubMed] [Google Scholar]
  39. Salian SR, Daddangadi A, Predheepan D, et al. 2024. Comparison of large single and small multiple doses of cyclophosphamide exposure in mice during early prepubertal age on fertility outcome. Sci Rep 14 31042. ( 10.1038/s41598-024-82264-3) [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shareghi-Oskoue O, Aghebati-Maleki L & Yousefi M. 2021. Transplantation of human umbilical cord mesenchymal stem cells to treat premature ovarian failure. Stem Cell Res Ther 12 454. ( 10.1186/s13287-021-02529-w) [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shiroma ME, Damous LL, Cotrim FP, et al. 2021. Pretreatment with melatonin improves ovarian tissue cryopreservation for transplantation. Reprod Biol Endocrinol 19 17. ( 10.1186/s12958-021-00705-4) [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Shuyuan Y, Meimei W, Fenghua L, et al. 2023. hUMSC transplantation restores follicle development in ovary damaged mice via re-establish extracellular matrix (ECM) components. J Ovarian Res 16 172. ( 10.1186/s13048-023-01217-y) [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Song Y, Guo Z, Song L, et al. 2024. Role of DNA damage response in cyclophosphamide-induced premature ovarian failure in mice. J Obstet Gynaecol Res 50 1655–1666. ( 10.1111/jog.16004) [DOI] [PubMed] [Google Scholar]
  44. Spears N, Lopes F, Stefansdottir A, et al. 2019. Ovarian damage from chemotherapy and current approaches to its protection. Hum Reprod Update 25 673–693. ( 10.1093/humupd/dmz027) [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stringer JM, Alesi LR, Winship AL, et al. 2023. Beyond apoptosis: evidence of other regulated cell death pathways in the ovary throughout development and life. Hum Reprod Update 29 434–456. ( 10.1093/humupd/dmad005) [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Umehara T, Winstanley YE, Andreas E, et al. 2022. Female reproductive life span is extended by targeted removal of fibrotic collagen from the mouse ovary. Sci Adv 8 eabn4564. ( 10.1126/sciadv.abn4564) [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wang X, Wang W, Qu Q, et al. 2019. Effect of large follicle puncture on IVF-ET outcome in patients with unsynchronized follicle maturationcan. J Int Med Res 47 2056–2066. ( 10.1177/0300060519831178) [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wei Q, Liu S, Dong Z, et al. 2026. Recombinant humanized type III collagen improves ovarian function via ITGA2-mediated mitochondrial function restoration in granulosa cells and extracellular matrix remodeling. Regen Biomater 13 rbag046. ( 10.1093/rb/rbag046) [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Xi H, Chen X, Wang X, et al. 2025. Role of programmed cell death in mammalian ovarian follicular atresia. J Steroid Biochem Mol Biol 247 106667. ( 10.1016/j.jsbmb.2024.106667) [DOI] [PubMed] [Google Scholar]
  50. Yang W, Ma Y, Jin J, et al. 2021. Cyclophosphamide exposure causes long-term detrimental effect of oocytes developmental competence through affecting the epigenetic modification and maternal factors’ transcription during oocyte growth. Front Cell Dev Biol 9 682060. ( 10.3389/fcell.2021.682060) [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yoshida K, Erdenebayar O, Kadota Y, et al. 2022. Effect of intraperitoneal docetaxel on ovarian function in mice. J Obstet Gynaecol 42 3672–3678. ( 10.1080/01443615.2022.2153024) [DOI] [PubMed] [Google Scholar]
  52. Zhang Y, Zhou X, Zhu Y, et al. 2021. Current mechanisms of primordial follicle activation and new strategies for fertility preservation. Mol Hum Reprod 27 gaab005. ( 10.1093/molehr/gaab005) [DOI] [PubMed] [Google Scholar]
  53. Zheng B, Hu X, Hu Y, et al. 2024. Type III adenylyl cyclase is essential for follicular development in female mice and their reproductive lifespan. iScience 27 110293. ( 10.1016/j.isci.2024.110293) [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zhou J, Peng X & Mei S. 2019. Autophagy in ovarian follicular development and Atresia. Int J Biol Sci 15 726–737. ( 10.7150/ijbs.30369) [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

RAF-25-0192_supplementary_figure_1.pdf (256.9KB, pdf)
RAF-25-0192_supplementary_figure_2.pdf (309.7KB, pdf)
RAF-25-0192_supplementary_table_1.pdf (28.4KB, pdf)

Articles from Reproduction & Fertility are provided here courtesy of Bioscientifica Ltd.

RESOURCES