Reproductive senescence impairs the energy metabolism of human luteinized granulosa cells

Disusun Untuk Memenuhi Tugas Mata Kuliah Keperawatan Maternitas II

Dosen Pengampu Diyan Indriyani, S.Kp.,M.Kep.,Sp.Mat.

Disususn oleh :
Nama : Siti Nur Amanah
Nim : 1911011013
Kelas: 5A keperawatan

UNIVERSITAS MUHAMMADIYAH JEMBER

FAKULTAS ILMU KESEHATAN
PRODI S1 KEPERAWATAN
OKTOBER, 2021

KATA PENGANTAR

Puji syukur kehadirat Allah SWT yang telah melimpahkan rahmat serta hidayah-Nya sehingga saya
dapat menyelesaikan makalah ini dengan lancar. Tidak lupa juga sholawat serta salam kami panjatkan
kepada junjungan kita Nabi Muhammad SAW yang menjadi tauladan dalam menuntut ilmu.
Mata Kuliah “Keperawatan Maternitas II” yang kami susun dalam bentuk makalah judul “Journal
Reproductive senescence impairs the energy metabolism of human luteinized granulosa cells” dan dengan
selesainya penyusunan makalah ini, saya juga tidak lupa mengucapkan terimakasih kepada :
1. Ns. Sasmiyanto, S.Kep.,M.Kes. sebagai Dekan Fakultas Ilmu Kesehatan Universitas
Muhammadiyah Jember.
2. Ns. Yeni Suryaningsih, sebagai Wakil Dekan Fakultas Ilmu Kesehatan Universitas Muhammadiyah
Jember.
3. Dr. Dian Damayanti sebagai Kepala Prodi S1 Ilmu Keperawatan Universitas Muhammadiyah
Jember.
4. Ns. Cipto Susilo, S.Pd., S.Kep., M.Kep. sebagai Sekretaris Prodi S1 Ilmu Keperawatan Universitas
Muhammadiyah Jember.
5. Diyan Indriyani, S.Kp.,M.Kep.,Sp.Mat. Sebagai Dosen Pengampu Mata Kuliah Keperawatan
Maternitas II Universitas Muhammadiyah Jember.

Kami menyadari masih banyak kekurangan dalam penulisan makalah ini oleh karena itu kami sangat
senang dan terbuka untuk menerima kritik dan saran untuk perbaikan tugas makalah ini.

Jember, 05 Oktober 2021

DAFTAR ISI

COVER

KATA PENGANTAR...............................................................................................................I

DAFTAR ISI............................................................................................................................II

BAB I PENDAHULUAN.........................................................................................................4

1.1 LATAR BELAKANG...................................................................................................4

1.2 TUJUAN PENULISAN.................................................................................................5

BAB II CRITICAL APPRAICAL..........................................................................................6

2.1 RINGKASAN ARTIKEL ......................................................................................................6

2.2 CRITICAL APPRAICAL...........................................................................................................8
BAB III PEMBAHASAN.......................................................................................................17

3.1 INTERPRETASI HASIL.....................................................................................................17

3.2 KETERBATASAN PENELITIAN.........................................................................................18
3.3 IMPLIKASI TERHADAP PELAYANAN KESEHATAN..........................................................19

BAB IV PENUTUP................................................................................................................20

4.1 KESIMPULAN...............................................................................................................20

4.2 SARAN............................................................................................................................20

DAFTAR PUSTAKA.............................................................................................................21

LAMPIRAN JURNAL...........................................................................................................22

BAB I

Pendahuluan

A. Latar belakang
Kesehatan reproduksi menurut WHO terdiri dari proses reproduksi, fungsi dan sistem reproduksi
pada semua tahap kehidupan. Tahun 1984 pada Konferensi Internasional tentang Kependudukan dan
Pembangunan (International Conference on Population and Development) di Kairo, Mesir,
menyepakati adanya perubahan paradigma dalam pengelolaan masalah kependudukan dan
pembangunan dari pendekatan fertilitas (keluarga berencana) menjadi pendekatan yang terfokus pada
kesehatan reproduksi serta upaya pemenuhan hak reproduksi. Salah satu ruang lingkup kesehatan
reproduksi adalah pencegahan dan penanganan infertilitas (Lestari et al, 2013). Infertilitas merupakan
masalah pada sistem reproduksi ditandai dengan tidak bisa mencapai kehamilan dalam waktu 12
bulan atau lebih, setelah melakukan hubungan seksual yang teratur tanpa kontrasepsi (Hochschild et
al, 2009). Tahun 2010 diperkirakan ada sekitar 48,5 juta pasangan yang infertil atau jumlahnya
sekitar 15% dari seluruh populasi dunia (Mascarenhas et al, 2012). Di Indonesia angka infertilitas
diperkirakan kurang lebih 10% (WHO, 2004). Infertilitas dapat mengenai pria atau wanita. Banyak
faktor yang menyebabkan terjadinya infertilitas pada berbagai macam populasi dunia. Secara umum,
10% sampai 15% disebabkan karena anovulasi, 10% - 15% adanya abnormalitas penetrasi sperma ke
mukus seviks, 30% - 40% adanya masalah pada pelvis seperti endometriosis, infeksi dan oklusi tuba
serta 30%-40% karena abnormalitas sistem reproduksi pria (Daniel dan Mishell, 2001).
Motilitas spermatozoa merupakan salah satu fungsi sperma yang penting yaitu untuk berpindah
dan menembus mukus seviks pada organ reproduksi wanita, banyak faktor yang mempengaruhi
motilitas spermatozoa salah satunya yaitu gangguan pada semen seperti gagalnya semen menjadi cair,
konsistensi semen yang kental (viskositas semen yang tinggi) atau pH yang rendah (Gonzales, 2001).
Tingginya viskositas semen menunjukkan efek negatif pada motilitas spermatozoa dan kualitas
 semen. Hiperviskositas berdampak berkurangnya motilitas sperma, kemungkinan karena trapping
effect (efek penjebakan) yang mencegah pergerakan progresif sperma pada traktus genitalia wanita
(Elzanaty et al, 2004). Motilitas sperma pada pria dengan hiperviskositas semen lebih rendah dari
pada pria yang mempunyai viskositas semen yang normal dengan perbedaan 4% (Esfandiari et al,
2008). Pria yang mempunyai viskositas semen yang tinggi memiliki motilitas progresif hanya 20%
(Elia et al, 2009).
B. Tujuan penulisan
1. Tujuan umum
Untuk mengetahui keterkaitan pengaruh penuaan reproduksi yang dapat merusak
energi metabolisme granulosa luteinisasi terhadap sel manusia.
2. Tujuan khusus
a. Mengidentifikasi pengaruh penuaan sistem reproduksi pada wanita.
b. Untuk mengetahui kerusakan energi metabolisme granulosa luteinisasi terhadap sel manusia.
c. Untuk mengetahui faktor-faktor kerusakan penuaan reproduksi terhadap energi metabolisme.
 BAB II

Critical Appraisal

A. Ringkasan Artikel Ilmiah

B. Critical Appraisal
 Reproductive senescence impairs the energy metabolism of human luteinized granulosa cells
1. Abstrak
Pertanyaan penelitian: Usia wanita adalah satu-satunya faktor terbesar yang mempengaruhi kinerja
reproduksi dan sel granulosa dianggap sebagai biomarker potensial kualitas oosit. Apakah ada efek
usia pada metabolisme energi manusia? sel granulosa mural?
Desain: Kohort prospektif observasional dan studi eksperimental termasuk 127 wanita yang telah
menjalani siklus IVF. Wanita dialokasikan ke dalam dua kelompok: kelompok pasien infertil berusia
di atas 38 tahun dan kelompok kontrol kelompok yang terdiri dari donor oosit berusia kurang dari 35
tahun. Individu dengan patologi yang dapat mengganggu kesuburan dikeluarkan dari kedua
kelompok. Setelah pengambilan oosit, sel kumulus dan granulosa diisolasi dan sifat bioenergi
(parameter fosforilasi oksidatif, laju glikolisis aerobik dan nukleotida adenin konsentrasi) dianalisis
dan dibandingkan.
Hasil: Sel granulosa dan sel kumulus mural manusia menunjukkan tingkat glikolisis aerobik yang
tinggi yang tidak dapat ditingkatkan lebih lanjut ketika sintesis ATP mitokondria dihambat.
Penambahan cairan folikel ke dalam percobaan media diperlukan untuk mencapai kapasitas
pernapasan penuh sel. Sel granulosa dari wanita lanjut usia hadir lebih rendah respirasi mitokondria
(12,8 ± 1,6 versus 11,2 ± 1,6 pmol O2/menit/mg; P = 0,046), meskipun massa mitokondria tidak
menurun, dan glikolisis aerobik lebih rendah, dibandingkan dengan donor muda (12,9 ± 1,3 versus
10,9 ± 0,5 mpH/menit/mg; P = 0,009). Penurunan bersamaan dalam dua jalur pasokan energi
menyebabkan penurunan energi seluler pengisian (0,87 ± 0,01 versus 0,83 ± 0,02; P < 0,001).
Kesimpulan: Sel granulosa luteinized mural manusia menunjukkan penurunan metabolisme energi
mereka seiring bertambahnya usia wanita yang mungkin mempengaruhi potensi reproduksi wanita.
2. Ringkasan Artikel
usia wanita adalah yang terbesar faktor yang mempengaruhi reproduksi kinerja pasangan menjalani
perawatan infertilitas (Igarashi dkk., 2015 ). Final 2016 laporan dari Society for Assisted Teknologi
Reproduksi (SART, https:// www.cdc.gov/art/pdf/2016-report/ART- 2016-National-Ringkasan-
Report.pdf) menunjukkan bahwa hampir setengah dari pasien di bawah 35 tahun menjalani siklus
IVF akan mencapai hidup melahirkan menggunakan telurnya sendiri, sedangkan lebih sedikit dari
26% wanita berusia di atas 37 tahun akan. Ini menjadi lebih dramatis di atas 42 tahun, dengan
kumulatif hidup tingkat kelahiran serendah 3,7% (Pusat untuk Pengendalian dan Pencegahan
Penyakit, 2017). Kompetensi oosit yang buruk adalah yang utama penyebab kemerosotan terkait usia
kapasitas reproduksi (Navot et al., 1991). Memang, siklus donasi oosit mempertahankan tingkat
kelahiran hidup 50% terlepas dari usia wanita (Pusat Pengendalian Penyakit) dan Pencegahan, 2017).
Penelitian tentang biologi mitokondria dan bioenergi seluler telah diperoleh meningkatkan perhatian
dalam beberapa tahun terakhir, memberikan peluang baru dalam berbagai bidang kedokteran (Picard
et al., 2016). Dengan demikian, peran mitokondria dalam seluler penuaan dan penuaan manusia telah
meningkat minat yang cukup besar (Bratic dan Larsson, 2013; Sun dkk., 2016; Ziegler dkk., 2015).
Diketahui bahwa mitokondria disfungsi mendasari beberapa kesuburan cacat pada manusia dan
mitokondria itu memainkan peran penting dalam kualitas oosit dan perkembangan embrio awal
(Cecchino dkk., 2018; Demain dkk., 2017; Kasapoglu dan Seli, 2020; Mei-Panloup dkk., 2007).
Faktanya, mitokondria memiliki telah disarankan sebagai biomarker yang menjanjikan untuk hasil
IVF ( Kim dan Seli, 2019 ; Legro, 2019 ). Selanjutnya, kemungkinan mengatasi disfungsi
mitokondria untuk meningkatkan kualitas oosit dan infertilitas terkait usia telah menjadi tujuan dari
banyak studi ( Ben-Meir dkk., 2015, 2019; Bentov dkk., 2010; Cagnone dkk., 2016; Labarta dkk.,
2019). Bioenergi ovarium termasuk sinergisme metabolik yang canggih antara oosit dan sel
granulosa, yang sangat penting untuk pematangan oosit selama pertumbuhan folikel (Canipari, 2000;
Cinco dkk., 2016). Namun, sedikit yang diketahui tentang energi metabolisme sel granulosa manusia
dan dampaknya pada hasil IVF. Di dalam bidang reproduksi berbantuan teknologi (ART), studi
sebelumnya
yang dimaksudkan untuk menjelaskan cacat pada meiosis dan proses seluler lainnya mempengaruhi
pematangan oosit, fertilisasi atau perkembangan embrio sering difokuskan baik pada analisis DNA
mitokondria (mtDNA) sebagai indikator kandungan mitokondria/fungsi (Cecchino dan Garcia-
Velasco, 2019; Cecchino dkk., 2018; MayPanloup dkk., 2007; Reynier dkk.,2001) atau pada
konsentrasi ATP sebagai indikator status energi seluler (Dalton et al., 2014; Downs, 1995; Hsu dkk.,
2014; Pasquariello dkk., 2019; Zeng dkk., 2007; Zhao dan Li, 2012). Bioenergi menyediakan alat
untuk menganalisis faktor-faktor yang dapat menyebabkan perubahan dalam tingkat energi seluler.
Karena itu, penelitian ini bertujuan untuk mengkarakterisasi profil bioenergi manusia luteinized sel
granulosa untuk mendeteksi dampak potensial penuaan pada energi metabolisme. Ini menunjukkan
bahwa usia sel menunjukkan penurunan yang signifikan dalam dua jalur suplai ATP utama, yang
memimpin untuk kekurangan muatan energi seluler.
a. Metode
Desain studi dan populasi Kohort prospektif observasional termasuk 127 wanita yang telah
menjalani IVF dan injeksi sperma intracytoplasmic setelah stimulasi ovarium (TABEL 1).
pasien dialokasikan untuk dua kelompok umur: kelompok kontrol terdiri dari 84 oosit donor
berusia di bawah 35 tahun, sedangkan kelompok usia reproduksi lanjut (ARA) termasuk 43
wanita infertil berusia lebih dari 38 tahun. Untuk kedua kelompok, kriteria eksklusi berikut
diadopsi: penyakit sistemik yang relevan, alkohol atau penyalahgunaan obat, diwariskan atau
kromosom gangguan, dikenal mitokondria disfungsi, cacat anatomis sistem reproduksi, formal
kontraindikasi untuk kehamilan atau ovarium stimulasi, paparan radiasi sebelumnya atau
kemoterapi dan indeks massa tubuh (BMI) 30 kg/m2. Diantara penyakit yang berpotensi
mengganggu kesuburan dan fungsi mitokondria, sebagai berikut: dikeluarkan pada kedua
kelompok: endokrin kelainan, keguguran berulang, neurodegeneratif dan jantung penyakit,
sindrom ovarium polikistik, endometriosis, menular dan seksual penyakit menular, dan
neoplasma. Dengan demikian, kelompok wanita berusia di atas 38 tahun pada dasarnya terdiri
dari wanita tidak subur karena faktor pria atau infertilitas yang tidak dapat dijelaskan di mana
penyebab yang diduga adalah usia lanjut.
b. Hasil
Karakterisasi bioenergi profil granulosa manusia dan sel kumulus untuk mengkarakterisasi
bioenergi sifat dari kedua mural luteinized sel granulosa dan sel kumulus, sebuah kohort dari
donor oosit berusia di bawah 35 tahun adalah digunakan. Karakterisasi dilakukan menggunakan
XF24 Extracellular Flux Analyzer. Eksperimen menggunakan teknologi ini harus dirancang
dalam media kulturdilengkapi dengan satu set substrat untuk memahami spesifik kebutuhan
metabolisme sel. NS suplemen yang lebih umum adalah glukosa, piruvat atau glutamin.
Bergantung kepada pengaturan eksperimental, sel akan ditampilkan perbedaan dalam profil
bioenergi mereka dan parameter terkait seperti kapasitas pernapasan atau keseimbangan antara
glikolisis aerobik dan oksidatif fosforilasi (OXPHOS).
c. Pembahasan
Analisis bioenergi sifat mural manusia luteinized sel granulosa dan sel kumulus disajikan di sini
telah memungkinkan lebih lanjut pemahaman tentang metabolisme ini dua jenis sel folikel, yang
 dikenal mempengaruhi pematangan oosit dan, pada akhirnya, hasil IVF (Liu et al., 2017; Shufaro
dkk., 2012). Sel-sel ini diyakini sebagai biomarker potensial kualitas oosit (Anderson et al., 2018;
Dumesic et al., 2015), dan telah mengusulkan bahwa perubahan energi mereka metabolisme dapat
menyebabkan infertilitas (Dong dkk., 2016; Hsu dkk., 2014; Liu dkk., 2017; Shufaro dkk., 2012).
Hasil penelitian ini menunjukkan (i) profil glikolitik tinggi dari kedua sel ini jenis, (ii) bahwa
kapasitas pernapasan bergantung pada keberadaan folikel fluida dalam media percobaan, dan (iii)
sel granulosa yang mengalami luteinisasi dari wanita yang lebih tua menunjukkan pernapasan
yang lebih rendah dan glikolisis aerobik, yang mengarah ke penurunan konsentrasi ATP seluler.
Metabolisme energi yang berubah ini dapat memberikan penjelasan mekanistik untuk penurunan
potensi reproduksi ketika usia wanita.
d. Kesimpulan
meskipun banyak laporan telah menunjuk ke bermain mitokondria peran penting dalam kualitas
oosit dan Hasil IVF (Cecchino et al., 2018; Demain dkk., 2017; Kasapoglu dan Seli, 2020; May-
Panloup et al., 2007), ini studi menunjukkan untuk pertama kalinya bahwa keduanya OXPHOS
dan glikolisis menurun dalam sel granulosa luteinisasi mural selama penuaan reproduksi dan
bahwa keduanya acara bersama menyebabkan penurunan dalam konsentrasi ATP. Selain itu, data
saat ini mengungkapkan bahwa mitokondria massa tidak berkurang seiring bertambahnya usia
karena sel granulosa mural mempertahankan kapasitas pernapasan penuh mereka.

3. Critical Appraisal

CRITICAL
POINT CRITICAL APPRAISAL YA TIDA HASILKRITISIJURNAL
APPRAISAL K
Pada jurnal yang kami kritisi, peneliti sudah
menampilkan abstrak dihalaman pertama.
Dalam abtrak tersebut peneliti juga telah
Apakah penelitian menjelaskan tentang introduction,methods,
mencantumkan abtrak dalam  results,discussion, refences. Jumlah kata dala
jurnal? abtrak sebanyak 230 dan hal ini melebihi sya
dari abtrak terdapat 150 kata dalam suatu
ABSTRAK
abstrak. Abstrak pada jurnal yang dipilih dal
bahas Inggris

Pada jurnal tidak di jelaskan tentang tujuan

 penelitian tersebut
Apakah tujuan
penelitian disebutkan?

Kata di dalam judul tersebut telah memenuh

Apakah judul memenuhi kaidah
persyaratan penulisan judul penelitian,dima
penulisan judul?
syarat-syarat judul penelitian yaitu:
JUDUL
  Diketik dengan huruf kapital,
pada jurnal ini judul diketik
menggunakan huruf kapital
  Menggunakan huruf Times
NewRoman,
pada judul huruf menggunakan time
new roman
 Ukuran huruf minimal 12,
ukuran sesuai dengan ketentuan
penulisan judul

CRITICAL
POINT CRITICAL APPRAISAL YA TIDA HASILKRITISIJURNAL
PPRAISAL K

 Format ketikan harus dalam bentuk

piramida terbalik,penulisan judul tidak
sesuai yaitu judul seperti piramidater
balik
 Menggunakan spasi2 jika lebih dari
satu baris dan spasi1 jika lebih dari
dua baris, judul sesuai dengan syarat
penelitian
 Jumlah kata pada judul penelitian
antara 12-20 kata, jumlah judul sesuai
dengan syarat yaitu berjumlah 20 kata.
 Tidak boleh disingkat dan ditulis pada
bagian tengah ,judul telah sesuai
dengan syarat yaitu tidak ada kata yang
disingkat
Apakah penulisan judul Pada penulisan judul jurnal yang telah dipilih

menggunakantandabaca(?) (!)atau tidak menggunakan tanda baca(!),(?), atau pun
tanda hubung(-) tanda hubung(-)
Apakah nama penulis di Namapenelitisudahtercantum dalam jurnal
 dihalaman pertama yaitu terdiri dari Gustavo
cantumkan?
Nardini Cecchino1,2,3, Juan Antonio García-
PENULIS Velasco2,3, dan Eduardo Rial4,

Apakah asal institusi penulis Jurnal penelitian yang telah dipilih

mencantumkan asal institusi

CRITICAL
POINTCRITICALAPPRAISAL YA TIDA HASILKRITISIJURNAL
APPRAISAL K
dicantumkan? 
tepat dibiografi peneliti yaitu spesialis
 kesuburan di Mater Prime Clinic
(Brasil). Dia telah menyelesaikan
program fellowship dua tahun di IVI-
Madrid (Spanyol) dan saat ini sedang
menyelesaikan PhD-nya dipenuaan
ovarium dan metabolisme energi.

Apakah asal institusi penulis Berdasarkan jurnal yang kami critical,

 institusi penulis sesuai dengan topik peneli
sesuai dengan topik penelitian?
dimana berlatar belakang kesehatan y
terletak di PHD penuaan ovarium
metabolisme energi Mulai dari latar belak
bidang keperawatan dan penelitian nya.
Apakah bidang ilmu penelitian Bidang ilmu peneliti dalam jurnal ini sudah
 sesuai dengan judul risetnya peneliti
sesuai dengan judul penelitian?
mengambil jurusan keperawatan sesuai
dengan topikrisetnya yang meneliti dalam
BIDANG ILMU
bidang ilmu keperawatan yaitu“Reproducti
senescence impairs the energy metabolism
human luteinized granulosa cells"

Apakah peneliti mencantumkan Pada jurnal yang telah dicritical peneliti ti

LITERATUR literatur review dalam
 mencantumkan beberapa literatur review
penelitiannya?
REVIEW
untuk memperkuat hasil dari penelitianny
Apakah penelitimenampilkan Didalam jurnal, peneliti telah

kerangka konsep dalam menampilkan kerangka konsep
KERANGKA
penelitiannya? sehingga membuat pembaca pada
KONSEP
saat awal membaca dapat
memahami konsep jurnal

CRITICAL
POINTCRITICALAPPRAISAL YA TIDAK HASILKRITISIJURNAL
APPRAISAL
Apakah peneliti mencantumkan Definisi operasional juga tidak di cantum

definisi operasional pada pada jurnal yang berjudul“Reproduct
penelitian nya? senescence impairs the energy metabolism
human luteinized granulosa cells. Seharus
Definisi operasional dicantumkan sehin
DEFINISI
pembaca mampu memahami terkait parame
OPERASIONAL
hasil ukur, skaladari penelitian yang diangk
Akan tetapi pada jurnal hanya menyantum
alat ukur dari masing-masing variabel saja.
 Apakah desain penelitian sesuai Penelitian ini merupakan penelitian kuantita

dengan model penelitian? dengan desain Reproductive senescence
impairs the energy metabolism of human
METODE
luteinized granulosa cells sehingga
PENELITIAN
pengumpulan data sesuai dengan model
penelitian nya.
Apakah sesuai level of evidence Dari desain penelitian pada jurnal yang dikr
 yaitu dengan Reproductive senescence imp
(fakta) dari desain penelitian?
the energy metabolism of human luteinized
granulosa cells,dengan derajat.

CRITICAL
POINTCRITICALAPPRAISAL YA TIDAK HASILKRITISIJURNAL
APPRAISAL
Apakah sesuai pemilihan sampel Pemilihan sempel pada penelitian tersebut ya
 43 responden. Responden di tempat kan p
dalam penelitian tersebut?
kelompok eksperimen.

Apakah peneliti menggunakan Peneliti pada analisis menggunakan t2 samp

analisa data yang tepat atau tidak?
ANALISA
DATA Apakahpenelitimencantumkan
jenis uji statistik yang digunakan?
 bebas dimana Tingkat signifikansi ditetapka
sebagai nilai P
Jenis uji statistik yang dipakai dalam penelit
 yang telah dikritik yaitu menggunakan uji M
Whitney

Dalam bentuk apa hasilbpenelitian

Peneliti menyajikan hasil penelitian dalam

disajikan? bentuk keterangan atau hasil dari penelitiann
HASIL
Apakah hasil penelitian dapat Hasil penelitian yang didapatkan dapat
PENELITIAN
di implementasikan di  diimplementasikan dikeperawatan, misalny
dapat dilakukan pada pasien yang mengalam
penuaan gangguan metabolisme

CRITICAL
POINT CRITICAL APPRAISAL YA TIDAK HASILKRITISIJURNAL
APPRAISAL
keperawatan? Pada sistem reproduksi.
Apakah ada rekomendasi khusus  Pada hasil dari penelitian,dalam jurnal tid
terdapat beberapa
terkait hasil penelitian?
rekomendasi dari peneliti.
 Apakah daftar pustaka yang  Dalam penelitian ini terdapat 48 daftar
digunakan up to date? pustaka dengan referensi tahun terbaru ya
2020
Apakah daftar pustaka yang  Daftar pustaka yang dipakai dalam jurnal
tersebut sangat sesuai dengan topik yang
DAFTAR digunakan sesuai?
dibahas yaitu pada lingkup dunia kesehatan
PUSTAKA Terutama pada gangguan sistem reproduks
Apakah daftar pustaka yang Berhubungan dengan penelitian ini daftar

digunakan dari sumber yang pustaka yang digunakan termasuk jurnal d
terpercaya? buku yang sesuai dengan bidang ilmunya.
Kesimpulan pada penelitian ini di lampirka
oleh peneliti, yaitu: Human mural luteinize
granulosa cells exhibit a reduction in their

energy metabolism as women age that is lik
KESIMPULAN
to influence female reproductive potential (
granulosa luteinisasi mural manusia
menunjukkan pengurangan metabolisme en
mereka seiring bertambahnya usia wanita y
mungkin mempengaruhi potensi reproduks
wanita).

SARAN  Tidak ada saran pada penelitian ini.

BAB III
Pembahasan

Bab ini menjelaskan makna hasil penelitian tentang “penuaan reproduksi yang dapat merusak energi
metabolisme granulosa luteinisasi terhadap sel manusia.”. Beberapa hal yang akan dipaparkan meliputi
interpretasi hasil, keterbatasan penelitian dan implikasinya untuk keperawatan.

Interpretasi hasil membahas tentang teori yang ada di dalam tinjauan pustaka dengan fakta dan opini
dari peneliti serta didukung dengan literatur yang didapatkan. Keterbatasan penelitian membahas tentang
alasan–alasan rasional yang bersifat teknis. Implikasi pelayanan keperawatan menyampaikan tentang kaitan
hasil penelitian dengan tatanan layanan kesehatan umumnya dan layanan keperawatan khususnya.
 A. Intervensi hasil
Jalur lain yang menyediakan energi ke dalam sel adalah glikolisis. Data saat ini tunjukkan bahwa,
di bawah eksperimen ini kondisi, laju glikolisis muncul menjadi maksimal karena penghambatan
sintesis ATP mitokondria tidak menghasilkan peningkatan kompensasi dalam glikolisis dan,
akibatnya, penghambatan sintesis ATP mitokondria menyebabkan runtuhnya energi seluler
muatan. Penemuan-penemuan ini menekankan pentingnya keduanya jalur dalam mempertahankan
ATP yang memadai pasokan dan pembangunan berkelanjutan. NS penurunan baik OXPHOS dan
glikolisis bisa jadi hasil dari penurunan energi kebutuhan sel karena reproduksi penuaan. Jika hal
ini terjadi, muatan energi seluler harus tetap tidak berubah. Namun, data dari ini penelitian
dengan jelas menunjukkan bahwa penuaan merusak bioenergi seluler dan sebagai alhasil ada
penurunan yang signifikan dalam konsentrasi ATP . Dia membingungkan bahwa meskipun
energinya lebih rendah muatan yang diamati pada kelompok ARA, OXPHOS tidak ditingkatkan
untuk mencoba mengembalikan konsentrasi ATP yang optimal meskipun ada pernafasan
cadangan kapasitas. Dalam konteks ini, itu harus menunjukkan bahwa pernapasan maksimal
kapasitas tetap tidak berubah di keduanya kelompok eksperimen. Ini penting karena
mengungkapkan bahwa mitokondria massa (kapasitas pernapasan total) tidak tampaknya tidak
terpengaruh oleh penuaan. A studi terbaru yang menggunakan flow cytometry untuk
mengevaluasi massa mitokondria dan Kapasitas pernapasan sel kumulus gagal mendeteksi
perbedaan yang signifikan antara pasien yang lebih muda dan lebih tua dari 35 tahun (Anderson et
al., 2018). Oleh karena itu, dasar molekuler untuk metabolisme energi yang berkurang tidak dapat
saat ini dipertimbangkan. Namun, ada adalah laporan yang menunjukkan, misalnya, bahwa pada
defisiensi sel granulosa manusia dalam kompleks V (Liu et al., 2017) atau variasi dalam ekspresi
sirtuin (Tatone et al., 2018) bisa berada di belakang mitokondria disfungsi pada penuaan ovarium.
Bukti yang meyakinkan menunjukkan bahwa komposisi cairan folikel manusia memainkan peran
dalam hasil IVF (Cordeiro dkk., 2018; O'Gorman dkk., 2013; wen dkk., 2018); namun, cacat
pada metabolisme energi selama reproduksi penuaan hanya sebagian dijelaskan oleh
komposisinya. Dengan demikian, tingkat basal respirasi dan glikolisis di granulosa sel dari
kelompok kontrol adalah berkurang secara signifikan dengan adanya cairan folikel dari wanita
yang lebih tua, tetapi cairan folikel dari donor muda gagal meningkatkan bioenergi parameter
dalam kelompok ARA. Baru-baru ini, pematangan in-vitro babi model menunjukkan bahwa
melengkapi media pematangan dengan cairan folikel tidak hanya meningkatkan DNA
mitokondria nomor salinan dan konten ATP di oosit dan sel kumulus, tetapi juga kelangsungan
hidup sel kumulus dan tingkat blastulasi (Ogawa et al., 2018).
B. Keterbatasan Peneliti
Dalam pencarian jurnal dan pengerjaan karya ilmiah ini sedikit terkendala jaringan internet yang
kurang mendukung dalam efektifitas penggunaan waktu dalam penyelesaiannya.
Peneliti belum begitu familiar dalam menggunakan metode pencarian jurnal yang tepat dan baik.
C. Implikasi Terhadap Pelayanan Keperawatan

Pengetahuan dan sikap perawat mengenai perawatan paliatif sangat diperlukan dalam mengkaji dan
mengevaluasi keluhan pasien. Perawat dengan anggota tim berbagai keilmuan dapat mengembangkan dan
mengimplementasikan rencana perawatan secara menyeluruh untuk meningkatkan kualitas hidup pasien.
Perawat juga harus mengidentifikasikan pendekatan baru yang dikembangkan berdasarkan standar
perawatan di rumah sakit untuk melaksanakan tindakan. Diharapkan kepeda perawat agar dapat
meningkatkan pengetahuan dan sikap mengenai perawatan paliatif dengan mengikuti pendidikan atau
seminar serta pelatihan mengenai perawatan paliatif guna meningkatkan kualitas hidup pasien.

BAB IV

Penutup

A. Kesimpulan

meskipun banyak laporan telah menunjuk ke bermain mitokondria peran penting dalam kualitas oosit
dan Hasil IVF (Cecchino et al., 2018; Demain dkk., 2017; Kasapoglu dan Seli, 2020; May-Panloup
et al., 2007), ini studi menunjukkan untuk pertama kalinya bahwa keduanya OXPHOS dan glikolisis
menurun dalam sel granulosa luteinisasi mural selama penuaan reproduksi dan bahwa keduanya acara
bersama menyebabkan penurunan dalam konsentrasi ATP. Selain itu, data saat ini mengungkapkan
bahwa mitokondria massa tidak berkurang seiring bertambahnya usia karena sel granulosa mural
mempertahankan kapasitas pernapasan penuh mereka. Itu harus ditunjukkan bahwa semua sampel
adalah diperoleh dari wanita yang telah pergi melalui kriteria eksklusi yang ketat untuk memastikan
tidak ada yang mendasari patologi yang dapat mempengaruhi kesuburan. Oleh karena itu, tampaknya
masuk akal untuk menyimpulkan bahwa kekurangan yang diamati dalam metabolisme energi
berhubungan dengan sifat fungsional intrinsik karena penuaan reproduksi yang mungkin
mempengaruhi kinerja IVF secara keseluruhan. baru jendela kesempatan untuk diagnostik dan alat
terapi mungkin timbul dari: studi yang berfokus pada bioenergi sel granulosa, oosit, dan embrio

B. Saran
Permasalahan pada masa lansia atau yang menjelang masa menopause sering terabaikan, tidak hanya
di lingkungan keluarga sendiri, tetapi juga di lingkungan masyarakat bahkan pusat pelayanan
kesehatan. pengetahuan tentang permasalahan seksual pada wanita yang menjelang perimenopause
baik pria maupun wanita perlu sebarluaskan sejak dini, dan perlunya kerjasama yang optimal disetiap
instansi pemerintah dan masyarakat untuk mengatasi masalah ini agar mereka mendapatkan
kehidupan yang layak, dan harmonis sebagai manusia dan warga negara seutuhnya. Jadi sebagai
perempuan, kita harus senantiasa menjaga kesehatan reproduksi kita sejak dini. Sebagai bidan, kita
juga harus memberikan penyuluhan kesehatan reproduksi kepada kaum wanita, khususnya remaja
pada masa pubertas.
 Daftar pustaka

Dahlan, Sopiyudin. 2009. Besar Sampel dan Cara Pengambilan Sampel Ed. 3.
Salemba Medika. Jakarta.
Departemen Kesehatan Republik Indonesia. 2005. Terjadi Pergeseran Umur Menopause.

Kumalasari, Intan. 2012. Kesehatan Reproduksi. Salemba Medika. 194 Halaman. Jakarta.
Anderson, S.H., Glassner, M.J., Melnikov, A., Friedman, G., Orynbayeva, Z. Respirometric reserve capacity
of cumulus cell mitochondria correlates with oocyte maturity. J. Assist. Reprod. Genet. 2018; 35: 1821–1830
Lampiran Artikel Ilmiah
1 RBMO VOLUME 00 ISSUE 0 2021

ARTICLE

Reproductive senescence impairs the energy

metabolism of human luteinized granulosa
cells
BIOGRAPHY
Gustavo Cecchino is a fertility specialist at the Mater Prime Clinic (Brazil). He has completed a two-year
fellowship programme at IVI-Madrid (Spain) and is currently finishing his PhD in ovarian ageing and energy
metabolism. His current research interests include mitochondrial
bioenergetics in reproductive ageing and new technologies to improve reproductive outcomes.

Gustavo Nardini Cecchino1,2,3, Juan Antonio García-Velasco2,3, Eduardo Rial4,*

KEY MESSAGE
Luteinized granulosa cells of women of advanced reproductive age show lower respiration and aerobic glycolysis, which
lead to a decrease in cellular ATP levels. The observed reduction in energy metabolism is likely to influence
female reproductive potential.

ABSTRACT
Research question: Female age is the single greatest factor influencing reproductive performance and granulosa cells
are considered as potential biomarkers of oocyte quality. Is there an age effect on the energy metabolism of human
mural granulosa cells?
Design: Observational prospective cohort and experimental study including 127 women who had undergone IVF
cycles. Women were allocated to two groups: a group of infertile patients aged over 38 years and a control
group comprising oocyte donors aged less than 35 years. Individuals with pathologies that could impair fertility
were excluded from both groups. Following oocyte retrieval, cumulus and granulosa cells were isolated and their
bioenergetic properties (oxidative phosphorylation parameters, rate of aerobic glycolysis and adenine nucleotide
concentrations) were analysed and compared.
Results: Human mural luteinized granulosa and cumulus cells present high rates of aerobic glycolysis that cannot
be increased further when mitochondrial ATP synthesis is inhibited. Addition of follicular fluid to the experimental
media is necessary to reach the full respiratory capacity of the cells. Granulosa cells from aged women present lower
mitochondrial respiration (12.8 ± 1.6 versus 11.2 ± 1.6 pmol O2/min/mg; P = 0.046), although mitochondrial mass is not
decreased, and lower aerobic glycolysis, than those from young donors (12.9 ± 1.3 versus 10.9 ± 0.5 mpH/min/mg; P =
0.009). The concurrent decrease in the two energy supply pathways leads to a decrease in the cellular energy charge
(0.87 ± 0.01 versus 0.83 ± 0.02; P < 0.001).
Conclusions: Human mural luteinized granulosa cells exhibit a reduction in their energy metabolism as women age
that is likely to influence female reproductive potential.
1
Department of Gynecology, Federal University of São Paulo, São Paulo, Brazil
2
Department of Gynecology and Obstetrics, Rey Juan Carlos University, Alcorcón Madrid, Spain KEYWORDS
3
IVI-Madrid, Aravaca Madrid 28023, Spain Ageing
4
Department of Structural and Chemical Biology, Centro de Investigaciones Biológicas Margarita Salas, CSIC, Madrid, ATP concentrations
Spain Bioenergetics
Granulosa cells
© 2021 The Author(s). Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd. This is an open access article
IVF
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
*Corresponding author. E-mail address: rial@cib.csic.es (E. Rial). https://doi.org/10.1016/j.rbmo.2021.08.006 1472-6483/© 2021 The
Author(s). Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
Declaration: The authors report no financial or commercial conflicts of interest.
2 RBMO VOLUME 00 ISSUE 0 2021
INTRODUCTION
emale age is the single greatest factor
F
influencing the reproductive performance of
couples undergoing infertility treatments
(Igarashi et al., 2015). The 2016 final
report from the Society for Assisted
Reproductive Technology (SART, https://
www.cdc.gov/art/pdf/2016-report/ART- 2016-
National-Summary-Report.pdf) shows that
nearly half of patients under 35 years
undergoing an IVF cycle will achieve a live
birth using their own eggs, whereas less
than 26% of women aged over 37 years
will. This becomes even more dramatic
beyond 42 years old, with cumulative live
birth rates as low as 3.7% (Centers for
Disease Control and Prevention, 2017).
Poor oocyte competence is the primary
cause of age-related deterioration of
reproductive capacity (Navot et al., 1991).
Indeed, oocyte donation cycles sustain a
live birth rate of 50% irrespective of the
woman's age (Centers for Disease Control
and Prevention, 2017).
Research on mitochondrial biology and
cellular bioenergetics has gained
increasing attention in recent years,
providing new opportunities in different
fields of medicine (Picard et al., 2016).
Thus, the role of mitochondria in cellular al., 2007; Reynier et al., 2001) or on ATP
senescence and human ageing has raised
considerable interest (Bratic and Larsson, cellular energy status (Dalton et al.,
2013; Sun et al., 2016; Ziegler et al., 2015). It 2014; Downs, 1995; Hsu et al., 2014;
is known that mitochondrial dysfunction Pasquariello et al., 2019; Zeng et al., 2007;
underlies some of the fertility defects in
humans and that mitochondria play a
critical role in oocyte quality and early
embryo development (Cecchino
et al., 2018; Demain et al., 2017;
Kasapoglu and Seli, 2020; May-Panloup et bioenergetic profile of human luteinized
al., 2007). In fact, mitochondria have
TABLE 1 BASELINE CHARACTERISTICS, CYCLE PARAMETERS AND OVARIAN RESPONSE TO OVARIAN STIMULATION
Characteristic
Age (years)
BMI (kg/m2)
AMH (ng/ml)
AFC
Oestradiol concentration (pg/ml)
Days of stimulation
Gonadotrophin dose (IU)
Number of oocytes
Number of mature oocytes
Oocyte maturity rate
AFC = antral follicle count; AMH = anti-Müllerian hormone; ARA = advanced reproductive age; BMI = body mass index.
a
The AMH concentrations of egg donors are not routinely quantified at the study institution.
been suggested as a promising biomarker
for IVF outcomes (Kim and Seli, 2019;
Legro, 2019). Furthermore, the possibility of
overcoming mitochondrial dysfunction to
improve oocyte quality and age-
related infertility has become the goal of
numerous studies (Ben-Meir et al., 2015,
2019; Bentov et al., 2010; Cagnone et al.,
2016; Labarta et al., 2019).
Ovarian bioenergetics includes a
sophisticated metabolic synergism
between oocytes and granulosa cells,
which is crucial for oocyte maturation
during follicular growth (Canipari, 2000;
Cinco et al., 2016). However, little is
known about the energy metabolism
of human granulosa cells and its
impact on IVF outcomes. In the field
of assisted reproductive technology
(ART), previous studies that intended
to explain defects in meiosis and
other cellular processes
affecting oocyte maturation, fertilization or
embryo development have often
focused either on the analysis of
mitochondrial DNA (mtDNA) as an
indicator of mitochondrial content/
function (Cecchino and Garcia-Velasco,
2019; Cecchino et al., 2018; May- Panloup et
concentrations as an indicator of the
Zhao and Li, 2012). Bioenergetics provides
tools to analyse the factors that may lead
to alterations in cellular energy levels.
Therefore,
this study aimed to characterize the
granulosa cells in order to detect the Serum hormonal measurements Serum
potential impact of ageing on energy anti-Müllerian hormone (AMH)
metabolism. It demonstrates that aged quantification was performed prior to
Control group (n = 43) ARA group (n = 84)
24.1 ± 3.9 40.8 ± 2.0
22.4 ± 3.0 22.9 ± 2.7
a
Not determined 1.27 ± 0.95
19.0 ± 4.4 9.0 ± 4.2
2452 (288–9847) 1729 (285–5844)
11 (8–17) 10 (7–16)
2000 (775–4800) 2250 (1350–4275)
19 (9–54) 8 (2–25)
14 (5–41) 6 (1–23)
0.78 ± 0.13 0.80 ± 0.18
cells show a significant decrease in the
two main ATP supply pathways, leading to
deficient cellular energy charge.
MATERIALS AND METHODS
Study design and population Observational
prospective cohort including 127 women
who had undergone IVF and
intracytoplasmic sperm injection after
ovarian stimulation (TABLE 1). Patients were
allocated to two age groups: the
control group consisted of 84 oocyte
donors aged under 35, whereas the
advanced reproductive age (ARA) group
included 43 infertile women aged
over 38 years. For the two groups, the
following exclusion criteria were adopted:
relevant systemic diseases, alcohol or
drug abuse, heritable or chromosomal
disorders, known mitochondrial
dysfunction, anatomical defects of
the reproductive system, formal
contraindication to pregnancy or ovarian
stimulation, prior exposure to radiation or
chemotherapy and a body mass index
2
(BMI) ≥30 kg/m . Among the diseases
that could potentially impair fertility and
mitochondrial function, the following
were excluded in both groups: endocrine
abnormalities, recurrent miscarriages,
neurodegenerative and heart
diseases, polycystic ovary syndrome,
endometriosis, infectious and sexually
transmitted disorders, and neoplasms.
Thus, the group of women aged over 38
basically comprised infertile women due to
male factor or unexplained infertility in
which the presumed cause was the
advanced age.
P-value
<0.001
0.323
–
<0.001
0.080
0.711
<0.001
<0.001
<0.001
0.260
RBMO VOLUME 00 ISSUE 0 2021 3

ovarian stimulation. Blood samples were the cell suspension was centrifuged at incubator before loading the plate into
obtained by venipuncture. The blood 500g for 5 min. Subsequently, cells were the analyser. The buffering power of the
+
samples collected were allowed to clot for incubated for 2 min at room temperature medium was 0.036 mpH units/pmol H ,
20 min and were then centrifuged for 10 min in the presence of red blood cell lysis which was determined by adding known
at 6000g. Serum samples were analysed by buffer (Miltenyi Biotec Inc. Merck, amounts of HCl to the medium and
chemiluminescence using a cobas e411 Darmstadt, Germany) according to the recording the changes using a pH meter
analyser (Roche Diagnostics, Sussex, UK). The manufacturer's instructions. Finally, (Mookerjee et al., 2015).
analytical sensitivity of the AMH assay was cumulus cells and purified granulosa cells
0.01 ng/ml and the coefficient of variation were washed with PBS and resuspended Control experiments were performed
was below 5%. in standard culture media. All the with A549 lung adenocarcinoma cells that
The analytical sensitivity of the oestradiol centrifugations were performed at room were obtained from the American Type
assay was <20 pg/ml and the coefficient of temperature. Patient samples were Culture Collection (ATCC). Cells were
variation was below 6%, and for occasionally pooled in order to reach the cultured at 37°C in a humidified 5% CO2
progesterone it was <0.05 ng/ml and minimum cell number needed to perform atmosphere in Gibco Dulbecco's Modified
below 7%, respectively. the experiments, but always granulosa Eagle's Medium (DMEM) supplemented
cells within the same group of patients with 10% heat-inactivated FBS (Gibco,
Ovarian stimulation protocol and ovum (control with control; ARA with ARA). Life Technologies, Paisley, UK ), 2 mmol/l
retrieval The same procedure was followed, when glutamine, and 100 IU/ml of penicillin/
In all cases an antagonist protocol was used necessary, with cumulus cells. A scheme streptomycin (Gibco, Life Technologies,
to carry out ovarian stimulation. with the complete experimental protocol is Paisley, UK) (ATCC, LGC Ltd, Teddington,
Individualized doses of gonadotrophins were shown in Supplementary Figure 1. UK). A549 cells were seeded in XF24-well
4
determined by an experienced specialist microplates at a concentration of 3 × 10
physician. Transvaginal ultrasound for When collecting and preparing samples, cells/well and experimental medium
cycle monitoring was performed every 2 aliquots of pooled follicular aspirates was XF-DMEM medium with 2% FBS, 5
days starting on stimulation day 5. Fixed from each group of patients were mmol/l glucose, 2 mmol/l glutamine,
daily doses of centrifuged at 800g for 20 min to and 5 mmol/l HEPES pH 7.4.
0.25 mg of gonadotrophin-releasing remove cells. The supernatant consisted of
hormone (GnRH) antagonist (Orgalutran, purified follicular fluid which was The experimental protocol to determine the
Cetrotide, Merck Serono Europe Ltd, sterile-filtered using a 0.22 µm pore size bioenergetics parameters was
London, UK; or Fyremadel, Ferring, Malmö, membrane filter (Minisart®; Sartorius essentially as described in Brand and
Sweden) were started when the leading Stedim Biotech SA, Madrid, Spain), Nicholls (2011), which has often been
follicle reached a mean diameter of 13–14 aliquoted and stored at –80°C. called the ‘mitochondrial stress test’.
mm. All patients received Basal OCR and ECAR were determined
GnRH agonist 0.2 mg (Decapeptyl, Ipsen Assessment of bioenergetic properties by performing four measurements
PharmaParis, France) to achieve final oocyte Characterization of the bioenergetic before the addition of the inhibitors or
maturation when the mean size of at least properties of mural granulosa cells and activators. Subsequently, ATP turnover
two follicles was 18 mm. Oocyte retrieval cumulus cells was performed using was assessed from the decrease in
was performed 36 h later under ultrasound an XF24 Extracellular Flux Analyser oxygen consumption after the addition
guidance. (Agilent Technologies, Santa Clara, of the ATPase inhibitor oligomycin
CA, USA). The analyser performs (2 µmol/l). The ECAR value after the
Sample collection automatic measurements of the oxygen inhibition of mitochondrial ATP synthesis
Following oocyte retrieval, cumulus cells were consumption rate (OCR) and the was considered to be the maximal ECAR.
mechanically stripped from each oocyte using extracellular acidification rate (ECAR) in The maximal respiratory capacity and the
fine needles. Mural luteinized granulosa cells real time, the latter being a proxy spare respiratory capacity were
were obtained from pooled follicular aspirate of for lactate formation. Fresh purified established from the increase in the rate of
follicles of at least granulosa cells were seeded in XF24- respiration elicited by the uncoupler
14 mm mean diameter, as previously well microplates (Agilent Technologies) at carbonyl cyanide p-(trifluoromethoxy)-
5
described (Ferrero et al., 2012) with minor a concentration of 3.5 × 10 cells/ well phenylhydrazone (FCCP) after two
5
modifications. Briefly, follicular fluid was slowly and 3 × 10 cells/well for cumulus cells. consecutive additions (0.5 and
layered on a 3:1 Ficoll gradient Cells were maintained for 24 h 0.3 µmol/l). Finally, 1 µmol/l rotenone
(Histopaque®-1077, Sigma-Aldrich Merck, at 37°C and 5% CO2. One hour prior to (complex I inhibitor) and 1 µmol/l
Darmstadt, Germany) and centrifuged at 400g OCR and ECAR measurements, the antimycin A (complex III inhibitor) were
for 20 min. Follicular-derived cells were supernatant was carefully removed, wells added to quantify the non-mitochondrial
collected from the middle layer and washed washed with 1 ml of assay medium and, respiration. ECAR values were corrected
twice with phosphate-buffered saline (PBS). finally, 500 µl of assay medium were estimating the CO2 contribution to
Cellular aggregates from both cell lines were added. The assay medium consisted the ECAR signal from the correlation
dissociated using 0.5 ml of a 0.05% trypsin– of XF-DMEM medium supplemented with between the decreases in the OCR
EDTA solution at 37°C for 5 min; 4.5 ml of 2% FBS, 5 mmol/l glucose, and the ECAR upon the addition of 30
standard culture medium (M-199 5 mmol/l 4-(2-hydroxyethyl)-1-piperazine mmol/l 2-deoxyglucose (2-DOG).
supplemented with 10% heat-inactivated fetal ethanesulfonic acid (HEPES), 2 mmol/l In order to normalize OCR and ECAR
bovine serum [FBS] and 100 IU/ml of glutamine, and 6% follicular fluid (see data to take into account differences in
penicillin/streptomycin) was added to stop Results section) pH 7.4. Cells were cell content, once the experiments were
trypsin digestion, and incubated at 37°C for 1 h in a CO2-free finished, wells were washed with PBS and
4 RBMO VOLUME 00 ISSUE 0 2021

the protein concentration in each well

variables were reported as mean ± SD (Dumesic et al., 2015; Fortune, 1994;
was determined by bicinchoninic acid
or SEM and compared using the t-test O'Gorman et al., 2013). Therefore, the
assay using bovine serum albumin as
or analysis of variance, accordingly. current study tested whether the
standard. The protein concentration of
The parameters expressed as medians addition of follicular fluid to the assay
cumulus cells could not be determined
with ranges presented a non-normal medium would improve the respiratory
because the PBS wash led to a partial
distribution and were compared by the capacity and/or the bioenergetic
detachment of the cells and, thus,
Mann–Whitney U-test. As appropriate, the properties of granulosa and cumulus
measurements were unreliable.
categorical variables were analysed by cells. Interestingly, the addition of low
either the chi-squared test or Fisher's concentrations of follicular fluid (2–6%
Adenine nucleotide measurement AMP,
exact test and described as percentages. v/v) markedly increased the response to
ADP and ATP concentrations were
Statistical significance was set at a two- FCCP in the two cell types (FIGURE 1B
determined by reverse-phase high
tailed P-value of <0.05. and Supplementary Figure 2). Higher
performance liquid chromatography
follicular fluid concentrations (10%)
(HPLC) essentially as described in
RESULTS compromised the adherence of the cells
De Korte et al. (1985). A minimum of
6 to the plates. Therefore, the standard
4 × 10 purified granulosa cells were Characterization of the bioenergetic medium for the subsequent bioenergetic
suspended in 10 ml of the same assay profile of human granulosa and cumulus studies always contained the Seahorse
medium described in the previous cells XF-DMEM medium supplemented with 6%
section and incubated at 37°C for 1 h. follicular fluid, 5 mmol/l glucose,
In order to characterize the bioenergetic
Next, they were centrifuged at 8000g for properties of both mural luteinized
5 mmol/l HEPES, 2 mmol/l glutamine
30 s, homogenized in 660 mmol/l HClO4 granulosa cells and cumulus cells, a cohort and 2% FBS. Under these
plus 10 mmol/l theophylline and kept on of oocyte donors aged under 35 was conditions, the stimulation of
ice. The homogenates were centrifuged used. The characterization was performed respiration by FCCP was 107 ± 6.1%
once more at 16,000g for 15 min at 4°C. using the XF24 Extracellular Flux Analyser. for granulosa cells and 146 ± 5.9% for
The supernatants were neutralized with cumulus cells. The rate of
Experiments using this technology
2.8 mol/l K3PO4 until a pH between 6 ought to be designed in culture media glycolysis, as determined from the
and 7 was reached and stored at –80°C. supplemented with a set of substrates extracellular acidification rate, reveals
Having completed the collection, samples in order to understand the specific the high glycolytic activity of the two
were thawed and centrifuged at 16,000g cell types. Remarkably, ECAR could
metabolic requirements of the cells. The
for 15 min at 4°C. The supernatants more common supplements are glucose, not be further stimulated when
were passed through a 0.45 µm filter and pyruvate or glutamine. Depending on the mitochondrial ATP production was
analysed with a Shimadzu Prominence experimental set-up, cells will display inhibited by oligomycin (FIGURE 2). This
chromatograph (Canby, Oregon, USA) differences in their bioenergetic profile was observed in both cell types and
using a C18 column (Mediterranea and associated parameters such as the it was not influenced by the presence
SEA18, Teknokroma, Spain). Peaks were respiratory capacity or the balance of follicular fluid. Their glycolytic
identified according to the retention between aerobic glycolysis and oxidative profile is also reflected in the
times of standard adenine nucleotides. OCR/ECAR ratio, which was 1.00 ±
phosphorylation (OXPHOS). The standard
Chromatograms were analysed using conditions commonly used to grow both 0.036 pmol O2/mpH unit for granulosa
PeakFit software (version 4.12, Systat granulosa and cumulus cells include high cells and 2.25 ± 0.12 pmol O2/ mpH
Software Inc., London, UK). Peak glucose M-199 media with 10% FBS [34– unit for cumulus cells in the
assignment was confirmed using samples 36]. However, experiments with the XF24 presence of 6% follicular fluid.
treated with 2 µmol/l oligomycin plus were modified to accommodate standard Although there is a statistically
30 mmol/l 2-DOG in which the AMP assay conditions including the use of the significant difference between the two
and ADP peaks markedly increase. The Seahorse XF-DMEM medium buffered types of cells (P < 0.001), both values
cellular energy charge was calculated with 5 mmol/l HEPES and containing 2% correspond to
using the equation (ATP+ADP/2)/ FBS and 2 mmol/l glutamine. Additionally, highly glycolytic cells (Zhang et al., 2012).
(ATP+ADP+AMP) (Atkinson, 1968). 5 mmol/l glucose was used to approximate
normoglycemic concentrations. The bioenergetic profile of mural
Ethical approval granulosa cells and cumulus cells
The Ethics Committee of the University Initial experiments on the XF24 revealed an presented an intriguingly high component of
Hospital Puerta de Hierro (Majadahonda, unusual profile in which the addition of non-mitochondrial respiration, which
Spain) approved the final version of the the uncoupler FCCP caused a modest and should be due to oxygen-consuming
study protocol (identification code 1512- transient stimulation of respiration that processes like those catalysed by
MAD-064-JG, 24 September was followed by a decline in the cyclooxygenases, lipoxygenases or
2018), which also complied with Spanish respiratory rate (FIGURE 1A). The presence NADPH oxidases (NOX). In most cellular
and European legislation on ART. All of glutamine and/or pyruvate did not systems the non-mitochondrial OCR
participants signed an informed consent. alter the profile significantly (data generally accounts for less than 10% of
not shown). Inside the pre-ovulatory the basal OCR (Brand and Nicholls, 2011)
Statistical analysis follicles, cells are immersed in a complex (the bioenergetic profile of A549 cells
Data analysis and graphing were medium, the follicular fluid, containing is shown in Supplementary Figure 3A for
performed using SigmaPlot v14 (Systat comparison). There are important
a variety of metabolites, hormones and
Software Inc.) and SPSS v24 (SPSS Inc., other protein factors that are essential exceptions, such as the activity of non-
Chicago, IL, USA). The continuous for oocyte developmental competence mitochondrial NOX in macrophages,
which can contribute significantly to
cellular oxygen uptake. Under the
experimental conditions in this
study,
RBMO VOLUME 00 ISSUE 0 2021 5

FIGURE 1 Influence of follicular fluid (FF) on the bioenergetics of luteinized granulosa cells (GC) and cumulus cells (CC). (A) Oxygen consumption rates
(OCR) of granulosa cells in the absence (black circles) or in the presence (red circles) of 6% FF in the experimental medium. Additions
are indicated with the arrows: ‘Oligo’, 1 µmol/l oligomycin; ‘FCCP’, two consecutive additions of 0.5 and 0.3 µmol/l carbonyl cyanide p-
(trifluoromethoxy)-phenylhydrazone; ‘Rot+AA’, 1 µmol/l rotenone plus 1 µmol/l antimycin A (see Supplementary Figure 2 for further details). (B)
Dependence of the maximum respiratory capacity on the presence of FF in GC (red circles) and CC (blue circles). The results are presented as
mean ± SEM of six (GC + no FF), four (GC + 2% FF), four (GC + 4% FF), 11 (GC + 6% FF), four (CC + no FF), four (CC + 2% FF), six (CC +
4% FF) and three (CC + 6% FF) independent experiments.

the non-mitochondrial respiration in

changes in the energy metabolism of two experimental groups allowed the
granulosa cells accounted for 56 ± 1.1% of
luteinized granulosa cells from a group of effect of age-related infertility on the
the basal OCR. The NOX inhibitors
patients of ARA. The study was restricted to bioenergetics of human granulosa cells
apocynin and VAS2870 had no effect on the
mural granulosa cells because the yield of to be explored. The mean concentration
non-mitochondrial respiration of granulosa
cells from each patient was significantly of anti-Müllerian hormone (AMH) in
cells, thus ruling out the involvement of
higher and, as previously stated, the the ARA group was 1.27 ± 0.95 ng/ml.
NOX (Supplementary Figure 3B).
attachment of the cumulus cells to AMH concentrations of egg donors are
the XF24 plate was weaker. Patient not routinely quantified in the study
Effects of senescence on the energy
characteristics, cycle parameters and institution, however the antral follicle
metabolism of granulosa cells Because
ovarian response to ovarian stimulation are count (AFC) was remarkably higher in
female fertility and IVF
shown in TABLE 1. The mean age of the ARA this group (19.0 ± 4.4 versus 9.0 ± 4.2;
outcomes dramatically decline with age,
group was 40.8 ± 2.0 while P < 0.001). The ARA group required
a study was designed to explore potential
in the control group it was 24.1 ± 3.9. higher gonadotrophin doses [2000 IU
Therefore, the difference between the (775–4800) versus 2250 IU (1350–4275);

FIGURE 2 The rate of glycolysis (extracellular acidification rate, ECAR) in luteinized granulosa (GC) and cumulus cells (CC). (A) Effect of
oligomycin on the rate of lactate formation (ECAR) of GC in the presence of 6% follicular fluid (FF). Data points represent the mean ± SEM of six
independent experiments. (B) Change in ECAR in response to the addition of oligomycin in GC and CC both in the presence and absence of
FF. Data are expressed as the per cent change with respect to basal values. (C) Bioenergetic profile of GC and CC expressed as the OCR/ECAR ratio
in the presence and absence of FF. Bars represent the mean ± SEM of 11 (GC + 6% FF), six (GC + no FF), three (CC + 6% FF) and four (CC no
FF) independent experiments. ***P < 0.001 between the indicated groups (analysis of variance test).
 6 RBMO VOLUME 00 ISSUE 0 2021

FIGURE 3 Bioenergetic parameters of mural luteinized granulosa cells (GC) from young donors (empty bars) and advanced reproductive age (ARA)
women (grey bars) and influence of the follicular fluid (FF) origin: DFF = FF from donors; AFF = FF from ARA group. (A) Basal rate of mitochondrial
respiration. OCR = oxygen consumption rate. (B) Basal rate of glycolysis as determined from the rate of lactate formation (extracellular
acidification rate, ECAR) value. (C) Bioenergetic profile expressed as the OCR/ECAR ratio. (D) Maximum respiratory capacity. Bars represent
the mean ± SEM of 11 (donor DFF and ARA DFF) or nine (donor AFF, ARA AFF) independent experiments. FIGURE 3A, *P = 0.046 and FIGURE
3B,
*P = 0.026, **P = 0.009, between the indicated groups (analysis of variance test).

P < 0.001]. As expected, the number of

conditioned by the cells, the composition of bioenergetic profile as estimated by the
oocytes retrieved was significantly higher
the follicular fluid or a combination OCR/ECAR ratio (FIGURE 3C). Notably, the
in the egg donor group [19 (9–54) versus
of the two factors. FIGURE 3 shows a decrease in the basal respiration in the
8 (2–25); P < 0.001], as was the
summary of the bioenergetic properties ARA group is not reflected in the
number
of granulosa cells from the control maximal respiratory capacity of the cells,
of mature oocytes [14 (5–41) versus 6
donors and the ARA group. It is apparent which remains unchanged (FIGURE 3D).
(1–23); P < 0.001)].
that the basal rate of respiration and
the ECAR are significantly reduced in The influence of the origin of the
Knowing the influence of the follicular
the ARA group, thus indicating a global follicular fluid used to supplement the
fluid on granulosa cell bioenergetics,
decrease in the energy metabolism media was investigated by performing
the assay media for the control and
as women age (FIGURE 3A, *P = 0.046 experiments in which the donor follicular
ARA patients were supplemented with
and FIGURE 3B, *P = 0.026, **P = 0.009, fluid was used to supplement the media
follicular fluid isolated from individuals
between the indicated groups). As for the ARA group and vice versa.
belonging to their respective groups. It
expected, the parallel decrease in the FIGURE 3 reveals that the highest rates of
might be expected that the observed
two parameters does not modify the respiration and glycolysis were observed
bioenergetic properties could be
RBMO VOLUME 00 ISSUE 0 2021 7

FIGURE 4 Effect of reproductive senescence on the adenine nucleotide pool in mural luteinized granulosa cells (GC) in the presence of 6%
follicular fluid (FF). Empty bars, control donors. Grey bars, ARA group. Hatched bars in each group are the values obtained in the presence of
oligomycin. Black bar is the value in the presence of oligomycin and 2-deoxyglucose (O+D). (A) Energy charge calculated using the equation
(ATP+ADP/2)/(ATP+ADP+AMP). (B) ATP/ADP ratio. (C) ATP/AMP ratio. Bars represent the mean ± SEM of 14 (donor), nine (ARA), seven
(donor + oligomycin), five (ARA + oligomycin), seven (donor + oligomycin + deoxyglucose) independent determinations. ***P < 0.001 between the
indicated groups (analysis of variance test).

in the donor group with medium

expected, the combination of oligomycin competence and quality (Dumesic et al.,
supplemented with the donor follicular
and 2-DOG totally collapsed the energy 2015; Freitas et al., 2017; Hennet and
fluid. The remaining combinations led to a
levels. Combelles, 2012). The main components
decrease in the energy metabolism
are steroid hormones, metabolites
although under no conditions was the
DISCUSSION (amino acids, glucose, fatty acids, etc.),
maximal respiratory capacity affected.
polysaccharides, proteins and growth
The analysis of the bioenergetic factors, among others (Dumesic et al.,
Adenine nucleotide concentrations in
properties of human mural luteinized 2015; Fortune, 1994; O'Gorman et al., 2013).
human luteinized granulosa cells
granulosa cells and cumulus cells The presence of enzymatic and non-
To investigate whether the decreased
presented here has allowed further enzymatic antioxidants is essential to
metabolism is due to senescence and
understanding of the metabolism of these maintain homeostatic concentrations of
causes a reduction in the cellular
two follicular cell types, which are known to reactive oxygen species that are
energy charge, the adenine nucleotide
influence the maturation of the oocyte potentially harmful but also necessary for
concentrations (ATP, ADP and AMP) of the
and, ultimately, IVF outcomes (Liu et al., oocyte growth (Da Broi et al., 2018;
granulosa cells in the two experimental
2017; Shufaro et al., 2012). These cells are Freitas et al., 2017). Although the current
groups were determined.
believed to be potential biomarkers of data have revealed that the follicular
The incubation media was the same as in
oocyte quality (Anderson et al., 2018; fluid is a fundamental element to attain
the XF24 experiments, supplementing
Dumesic et al., 2015), and it has been the full respiratory capacity of mural
the XF-DMEM medium with 6% follicular fluid
proposed that alterations in their energy granulosa cells and cumulus cells, it
of the respective experimental group. FIGURE
metabolism could lead to infertility was beyond the scope of the current
4 summarizes the changes in the adenine
(Dong et al., 2016; Hsu et al., 2014; work to elucidate which are the critical
nucleotide pool and reveals that granulosa
Liu et al., 2017; Shufaro et al., 2012). The components. The respiratory capacity of
cells from the ARA group exhibited a marked
results of this study show (i) the high the cells was determined by adding the
decrease in the cellular energy charge (P <
glycolytic profile of these two cell types, uncoupling agent FCCP and, therefore, its
0.001), which is also reflected in the ATP/
(ii) that the respiratory capacity relies on physiological relevance should be
ADP (P < 0.001) and ATP/AMP ratios
the presence of follicular fluid in the interpreted with caution. However, if this
(P < 0.001). Because experiments on the
experimental media, and bioenergetic parameter reflects the
XF24 analyser had shown that inhibition of
(iii) that luteinized granulosa cells from OXPHOS capacity of the oocyte and/or
mitochondrial ATP synthesis did
older women present lower respiration the supporting cells, then follicular fluid
not cause a compensatory increase in
and aerobic glycolysis, leading to a components could significantly affect the
glycolysis (ECAR), the effect of
decrease in cellular ATP concentrations. outcome of the energy-demanding
oligomycin on the adenine nucleotide pool
This altered energy metabolism could processes that take place upon
was studied. Results in FIGURE 4
provide a mechanistic explanation for the fertilization.
(hatched bars) show that this inhibitor
decrease in reproductive potential when
markedly decreased ATP concentrations in
women age. The other pathway that provides energy
both groups (P < 0.001) confirming that,
to the cell is glycolysis. The current data
because glycolysis cannot be further
The follicular fluid is a complex medium show that, under these experimental
stimulated, it cannot compensate for the loss
produced by granulosa and thecal cells, conditions, the rate of glycolysis appears to
of mitochondrial ATP synthesis. As
and is also enriched with biomolecules be at its maximum because the
from plasma, which influences oocyte inhibition of mitochondrial ATP synthesis
8 RBMO VOLUME 00 ISSUE 0 2021
did not result in a compensatory increase
in glycolysis (FIGURE 2) and, consequently,
inhibition of mitochondrial ATP synthesis
led to a collapse in the cellular energy
charge (FIGURE 4). These findings
emphasize the importance of the two
pathways in maintaining an adequate ATP
supply and sustaining development. The
decrease in both OXPHOS and glycolysis
could be the result of a decreased energy
demand of the cell due to reproductive
senescence. If this were to be the case,
the cellular energy charge should remain
unchanged. However, the data from this
study clearly show that ageing impairs
cellular bioenergetics (FIGURE 3) and as
a result there is a significant decrease
in ATP concentrations (FIGURE 4). It is
puzzling that despite the lower energy
charge observed in the ARA group,
OXPHOS is not increased to attempt to
restore the optimal ATP concentrations
despite the existence of spare respiratory
capacity. In this context, it must be
pointed out that the maximal respiratory
capacity remained unchanged in the two
experimental groups. This is important
because it reveals that the mitochondrial
mass (total respiratory capacity) does
not appear to be affected by ageing. A
recent study that used flow cytometry
to evaluate mitochondrial mass and
respiratory capacity of cumulus cells
failed to detect significant differences
between patients younger and older than 35
years of age (Anderson et al., 2018).
Therefore, the molecular basis for the
diminished energy metabolism cannot
currently be envisaged. However, there
are reports showing, for example, that
in human granulosa cells deficiencies in
complex V (Liu et al., 2017) or variations
in sirtuin expression (Tatone et al.,
2018) could be behind mitochondrial
dysfunction in ovarian ageing.
Compelling evidence suggests that the
composition of the human follicular fluid
plays a role in IVF outcomes (Cordeiro
et al., 2018; O'Gorman et al., 2013; Wen et
al., 2018); however, the defects in
energy metabolism during reproductive
senescence are only partially explained by
its composition. Thus, basal rates of
respiration and glycolysis in granulosa
cells from the control group were
significantly reduced in the presence of
follicular fluid from older women, but
follicular fluid from the young donors
failed to improve the bioenergetic
parameters in the ARA group (FIGURE 3).
Recently, a porcine in-vitro maturation
model showed that supplementing the
maturation medium with follicular fluid
not only increased mitochondrial DNA
copy number and the ATP content in
both oocytes and cumulus cells, but also
the survival of cumulus cells and the
blastulation rate (Ogawa et al., 2018).
In conclusion, although many reports
have pointed to mitochondria playing a
critical role in oocyte quality and
IVF outcome (Cecchino et al., 2018;
Demain et al., 2017; Kasapoglu and Seli,
2020; May-Panloup et al., 2007), this study
shows for the first time that both
OXPHOS and glycolysis are decreased in
mural luteinized granulosa cells during
reproductive ageing and that these two
concerted events lead to a decrease
in ATP concentrations. Moreover, the
current data reveal that mitochondrial
mass does not decrease with ageing
because mural granulosa cells retain
their full respiratory capacity. It must
be pointed out that all samples were
obtained from women that had gone
through stringent exclusion criteria
to ensure there were no underlying
pathologies that could influence fertility.
Therefore, it seems reasonable to
conclude that the observed deficiencies
in energy metabolism are related to
intrinsic functional properties due to
reproductive ageing that are likely to
influence overall IVF performance. A new
window of opportunity for diagnostic
and therapeutic tools may arise from
studies focusing on the bioenergetics of
granulosa cells, oocytes and embryos.
DATA AVAILABILITY STATEMENT
The data underlying this article will be
shared on reasonable request to the
corresponding author.
ACKNOWLEDGEMENTS
This work was supported by IVI-Madrid.
GNC received grants from the Capes
Foundation, Brazil/PDSE/process number
88881.132905/2016-01.
SUPPLEMENTARY MATERIALS
Supplementary material associated
with this article can be found, in
the online version, at doi:10.1016/j.
rbmo.2021.08.006.
REFERENCES
Anderson, S.H., Glassner, M.J., Melnikov, A.,
Friedman, G., Orynbayeva, Z. Respirometric reserve
capacity of cumulus cell mitochondria correlates
with oocyte maturity. J. Assist.
Reprod. Genet. 2018; 35: 1821–1830
Atkinson, D.E. The energy charge of the
adenylate pool as a regulatory parameter.
Interaction with feedback modifiers.
Biochemistry 1968; 7: 4030–4034
Ben-Meir, A., Burstein, E., Borrego-Alvarez, A.,
Chong, J., Wong, E., Yavorska, T., Naranian, T.,
Chi, M., Wang, Y., Bentov, Y., Alexis, J.,
Meriano, J., Sung, H.-K., Gasser, D.L., Moley,
K.H., Hekimi, S., Casper, R.F., Jurisicova, A.
Coenzyme Q10 restores oocyte mitochondrial
function and fertility during reproductive
aging. Aging Cell 2015; 14: 887–895
Ben-Meir, A., Kim, K., McQuaid, R., Esfandiari,
N., Bentov, Y., Casper, R.F., Jurisicova, A.
Co-enzyme q10 supplementation rescues
cumulus cells dysfunction in a maternal aging
model. Antioxidants 2019; 8: 58
Bentov, Y., Esfandiari, N., Burstein, E., Casper, R.F.
The use of mitochondrial nutrients to improve the
outcome of infertility treatment in older patients.
Fertil. Steril. 2010; 93: 272–275
Brand, M.D., Nicholls, D.G. Assessing
mitochondrial dysfunction in cells. Biochem. J.
2011; 435: 297–312
Bratic, A., Larsson, N.G. The role of mitochondria in
aging. J. Clin. Invest. 2013; 123: 951–957
Cagnone, G.L.M., Tsai, T.S., Makanji, Y., Matthews, P.,
Gould, J., Bonkowski, M.S., Elgass, K.D.,
Wong, A.S.A., Wu, L.E., McKenzie, M.,
Sinclair, D.A., St John, J.C. Restoration of
normal embryogenesis by mitochondrial
supplementation in pig oocytes exhibiting
mitochondrial DNA deficiency. Sci. Rep. 2016; 6:
23229
Canipari, R. Oocyte–granulosa cell interactions.
Hum. Reprod. Update 2000; 6: 279–289 Cecchino,
G.N., Garcia-Velasco, J.A.
Mitochondrial DNA copy number as a
predictor of embryo viability. Fertil. Steril.
2019; 111: 205–211
Cecchino, G.N., Seli, E., Alves da Motta, E.L.,
García-Velasco, J.A. The role of mitochondrial activity
in female fertility and assisted
reproductive technologies: overview and current
insights. Reprod. Biomed. Online 2018; 36: 686–
697
Centers for Disease Control and Prevention,
American Society for Reproductive Medicine,
Society for Assisted Reproductive Technology. 2015
Assisted Reproductive Technology National
Summary Report. Atlanta: US Dept of Health and
Human Services; 2017. https://www.
cdc.gov/art/pdf/2015-report/ART-2015-National-
Summary-Report.pdf
Cinco, R., Digman, M.A., Gratton, E., Luderer,
U. Spatial characterization of bioenergetics and
metabolism of primordial to preovulatory
follicles in whole ex vivo murine ovary. Biol.
Reprod. 2016; 95
Cordeiro., F.B., Montani, D.A., Pilau, E.J., Gozzo,
F.C., Fraietta, R., Lo Turco, E.G. Ovarian
environment aging: follicular fluid lipidomic and
related metabolic pathways. J. Assist.
Reprod. Genet. 2018; 35: 1385–1393
Da Broi, M.G., Giorgi, V.S.I., Wang, F., Keefe,
D.L., Albertini, D., Navarro, P.A. Influence of
follicular fluid and cumulus cells on oocyte
RBMO VOLUME 00 ISSUE 0 2021 9
quality: clinical implications. J. Assist. Reprod. Genet. 2018; 35: 735–751
Dalton, C.M., Szabadkai, G., Carroll, J.
Measurement of ATP in single oocytes: impact of maturation and cumulus cells on levels
and consumption. J. Cell. Physiol. 2014; 229:
353–361
De Korte, D., Haverkort, W.A., van Gennip, A.H., Roos, D. Nucleotide profiles of normal human blood cells determined by high-performance liquid
chromatography. Anal. Biochem. 1985; 147: 197–209
Demain, L.A.M., Conway, G.S., Newman, W.G.
Genetics of mitochondrial dysfunction and infertility. Clin. Genet. 2017; 91: 199–207
Dong, Z., Huang, M., Liu, Z., Xie, P., Dong, Y., Wu,
X., Qu, Z., Shen, B., Huang, X., Zhang, T., Li,
J., Liu, J., Yanase, T., Zhou, C., Xu, Y. Focused screening of mitochondrial metabolism reveals a crucial role for a tumor suppressor Hbp1 in
ovarian reserve. Cell Death Differ. 2016; 23: 1602–1614
Downs, S.M. The influence of glucose, cumulus cells, and metabolic coupling on ATP levels and meiotic control in the isolated mouse
oocyte. Dev. Biol. 1995; 167: 502–512
Dumesic, D.A., Meldrum, D.R., Katz-Jaffe,
M.G., Krisher, R.L., Schoolcraft, W.B. Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health. Fertil. Steril.
2015; 103: 303–316
Ferrero, H., Delgado-Rosas, F., Garcia-Pascual, C.M., Monterde, M., Zimmermann, R.C., Simon, C., Pellicer, A., Gomez, R. Efficiency and
purity provided by the existing methods for the isolation of luteinized granulosa cells: a comparative study. Hum. Reprod. 2012; 27: 1781–1789
Fortune, J.E. Ovarian follicular growth and development in mammals. Biol. Reprod. 1994; 50: 225–232
Freitas, C., Neto, A.C., Matos, L., Silva, E., Ribeiro, Â., Silva-Carvalho, J.L., Almeida, H. Follicular fluid redox involvement for ovarian follicle growth. J.
Ovarian Res. 2017; 10: 44
Hennet, M.L., Combelles, C.M.H. The antral
follicle: a microenvironment for oocyte differentiation. Int. J. Dev. Biol. 2012; 56: 819–831
Hsu, A.L., Townsend, P.M., Oehninger, S., Castora,
F.J. Endometriosis may be associated with mitochondrial dysfunction in cumulus cells from subjects undergoing in vitro fertilization-
intracytoplasmic sperm injection, as reflected by decreased adenosine triphosphate production. Fertil. Steril. 2014; 103: 347–352
Igarashi, H., Takahashi, T., Nagase, S. Oocyte aging underlies female reproductive aging: biological mechanisms and therapeutic strategies.
Reprod. Med. Biol. 2015; 14: 159–169
Kasapoglu, I., Seli, E. Mitochondrial dysfunction
and ovarian aging. Endocrinology 2020; 161 Kim, J., Seli, E. Mitochondria as a biomarker
for IVF outcome. Reproduction 2019; 157: R235–R242
Labarta, E., de los Santos, M.J., Escribá, M.J., Pellicer, A., Herraiz, S. Mitochondria as a tool for oocyte rejuvenation. Fertil. Steril. 2019; 111:
219–226
Legro, R.S. Introduction: stimulate the
mitochondria! A Bonington approach to improving assisted reproductive technology outcomes or a call to action? Fertil. Steril.
2019; 111: 195–196
Liu, Y., Han, M., Li, X., Wang, H., Ma, M., Zhang,
S., Guo, Y., Wang, S., Wang, Y., Duan, N., Xu., B., Yin, J., Yao, Y. Age-related changes in the mitochondria of human mural granulosa cells.
Hum. Reprod. 2017; 32: 2465–2473
May-Panloup, P., Chretien, M.F., Malthiery, Y., Reynier, P. Mitochondrial DNA in the oocyte and the developing embryo. Curr. Top. Dev. Biol.
2007; 77: 51–83
Mookerjee, S.A., Goncalves, R.L.S., Gerencser, A.A., Nicholls, D.G., Brand, M.D. The contributions of respiration and glycolysis to
extracellular acid production. Biochim. Biophys. Acta Bioenerg. 2015; 1847: 171–181
Navot, D., Bergh, R.A., Williams, M.A., Garrisim, G.J., Guzman, I., Sandler, B., Grunfeld, L. Poor
oocyte quality rather than implantation failure as a cause of age-related decline in female fertility. Lancet 1991; 337: 1375–1377
O'Gorman, A., Wallace, M., Cottell, E., Gibney,
M.J., McAuliffe, F.M., Wingfield, M., Brennan,
L. Metabolic profiling of human follicular fluid identifies potential biomarkers of oocyte developmental competence. Reproduction 2013;
146: 389–395
Ogawa, K., Itami, N., Ueda, M., Kansaku, K., Shirasuna, K., Kuwayama, T., Iwata, H. Non-
esterified fatty acid-associated ability of follicular fluid to support porcine oocyte maturation and development. Reprod. Med.
Biol. 2018; 17: 155–163
Pasquariello, R., Ermisch, A.F., Silva, E., McCormick, S., Logsdon, D., Barfield, J.P., Schoolcraft, W.B., Krisher, R.L. Alterations in oocyte
mitochondrial number and function are related to spindle defects and occur with maternal aging in mice and humans. Biol.
Reprod. 2019; 100: 971–981
Picard, M., Wallace, D.C., Burelle, Y. The rise of mitochondria in medicine. Mitochondrion 2016; 30: 105–116
Reynier, P., May-Panloup, P., Chrétien, M.F., Morgan, C.J., Jean, M., Savagner, F., Barrière, P., Malthièry, Y. Mitochondrial DNA content
affects the fertilizability of human oocytes. Mol. Hum. Reprod. 2001; 7: 425–429
Shufaro, Y., Lebovich, M., Aizenman, E., Miller,
C., Simon, A., Laufer, N., Saada, A. Human granulosa luteal cell oxidative phosphorylation function is not affected by age or ovarian
response. Fertil. Steril. 2012; 98: 166–172
Sun, N., Youle, R.J., Finkel, T. The mitochondrial basis of aging. Mol. Cell 2016; 61: 654–666 Tatone, C., Di Emidio, G., Barbonetti, A.,
Carta, G., Luciano, A.M., Falone, S., Amicarelli, F.
Sirtuins in gamete biology and reproductive physiology: emerging roles and therapeutic potential in female and male infertility. Hum.
Reprod. Update 2018; 24: 267–289
Wen, X., Kuang, Y., Zhou, L., Yu, B., Chen, Q.,
Fu, Y., Yan, Z., Guo, H., Lyu, Q., Xie, J., Chai,
W.. Lipidomic components alterations of human follicular fluid reveal the relevance of improving clinical outcomes in women using
progestin-primed ovarian stimulation compared to short-term protocol. Med. Sci. Monit. 2018; 24: 3357–3365
Zeng, H.T., Ren, Z., Yeung, W.S.B., Shu, Y.M.,
Xu, Y.W., Zhuang, G.L., Liang, X.Y. Low mitochondrial DNA and ATP contents contribute to the absence of birefringent spindle
imaged with PolScope in in vitro matured human oocytes. Hum. Reprod. 2007; 22: 1681–1686
Zhang, J., Nuebel, E., Wisidagama, D.R.R., Setoguchi, K., Hong, J.S., van Horn, C.M.,
Imam, S.S., Vergnes, L., Malone, C.S.,
Koehler, C.M., Teitell, M.A. Measuring energy metabolism in cultured cells, including human pluripotent stem cells and differentiated cells.
Nat. Protoc. 2012; 7: 1068–1085
Zhao, J., Li, Y. Adenosine triphosphate content in human unfertilized oocytes, undivided zygotes and embryos unsuitable for transfer or
cryopreservation. J. Int. Med. Res. 2012; 40: 734–739
Ziegler, D.V., Wiley, C.D., Velarde, M.C. Mitochondrial effectors of cellular senescence: beyond the free radical theory of aging. Aging
Cell 2015; 14: 1–7

Received 7 April 2021; received in revised form 23

July 2021; accepted 5 August 2021.